KR102020144B1 - Auxenochlorella protothecoides MM0011 and use thereof - Google Patents

Abstract

The present application provides a novel oxeno having a lipid producing ability including high concentrations of polyunsaturated fatty acids and saturated fatty acids, a biomass producing ability including high concentrations of organics and proteins, excellent removal of all nitrogen and whole humans, and a high concentration of lutein production. Chlorella protothecoid MM0011 strain is disclosed. The strain according to the present invention can be effectively used in the development of biofuels, functional foods, natural pigments, medicinal substances, animal feed, and aquaculture feed.

Description

Oxenochlorella protothecoide MM0011 strain and its use {Auxenochlorella protothecoides MM0011 and use}

The present application relates to a novel microalgae oxenochlorella protothecoid MM0011 strain and its use.

Microalgae are photosynthetic organisms that synthesize oxygen and produce oxygen using carbon dioxide, water, and solar energy. These microalgae can fix carbon dioxide in the atmosphere and can be utilized as biomass for food, feed or fuel production because of high protein and lipid content. Specifically, lipids, sugars or proteins, which are the primary metabolites of microalgae, may be used as a source of various biofuels such as biodiesel, bioethanol or biogas and as raw materials of polyunsaturated fatty acids (PUFAs). In addition, high value-added substances such as vitamins, carotenoids, polysaccharides, and lutein, which are secondary metabolites of microalgae, may be used as functional foods, natural pigments, medicinal substances, animal feeds, aquaculture feeds, and the like.

Microalgae are recognized as optimal materials for bioenergy, unlike land plants, because they have excellent CO2 reduction effect, do not compete with farmland for producing food resources, and have high production yields.

Republic of Korea Patent No. 1525319 relates to a 'new strain of microtinium inner NLP-F014 strain and its use' discloses a new microalgae capable of producing biofuel.

Republic of Korea Patent No. 1639467 relates to 'new microalgae of aldon and its use,' it is disclosed a new microalgae with a high lipid content.

Since microalgae are photosynthetic, they have excellent carbon dioxide reduction effects, do not cause political and social problems related to food resources, and have high production yields, which are the best environmentally friendly materials for bioenergy, unlike land plants. Therefore, there is a need for the development of new microalgae with high production efficiency of materials that can be utilized as various useful resources.

The present application seeks to provide a novel oxenochlorella protothecoid MM0011 strain with unsaturated fatty acids, biofuels and antioxidant pigment production.

In one aspect the present application is rbc represented by the 18S rRNA (ribosomal riboucleic acid), ITS (internal transcribed spacer) and SEQ ID NO: 3 as shown in SEQ ID NO: 2 as shown in SEQ ID NO: 1 L (the large subunit of the ribulose-bisphosphate carboxylase It provides a microalgal oxenochlorella protothecoides MM0011 strain having a nucleotide sequence of the gene.

The sequences of SEQ ID NOs: 1, 2 and 3 are novel sequences that can be distinguished from other strains, which are characteristic of the microalgae according to the present application.

Strain according to the present embodiment is in the microalgae Chlorella oxide ginsenoside Teco protocol id (Auxenochlorella deposited as accession number KCTC13291BP protothecoides ) MM0011 strain.

The strain according to the present invention has a biooil production capacity including a high concentration of polyunsaturated fatty acids and saturated fatty acids, a biomass production capacity including a high concentration of organic substances and proteins, an excellent ability to remove all nitrogen and whole persons, and a high concentration of lutein production. .

In one embodiment the strains according to the invention can produce polyunsaturated fatty acids, in particular omega-3 and omega-6, in particular in an amount of 60% to 70% of the total lipid weight.

In another embodiment, the strains according to the invention can be produced as saturated fatty acids, in particular palmitic acid, in particular in an amount of about 10-15% of the total lipid weight.

In another embodiment, the strains according to the present disclosure can produce lutein, a high value added substance, at a high concentration of about 3.5 ± 0.1 mg · g ⁻¹ , based on the dry weight of the microalgae.

In another embodiment, the organic material included in the biomass of the strain according to the present application is at least 85% by weight, the protein is included at least 50% by weight.

In another embodiment, the strains according to the present disclosure have at least 40% and at least 27% removal of whole nitrogen and whole humans, respectively.

In another embodiment the microalgae according to the present invention have a NaCl resistance of concentration up to 1.5 M as well as culture temperature and salt-free of 5-35 ° C.

In another aspect, the present application also provides for at least one oxenochlorella prototyping selected from the group consisting of 18S rRNA represented by SEQ ID NO: 1, ITS represented by SEQ ID NO: 2, and rbc L represented by SEQ ID NO: 3, the new sequence identified herein Provided nucleic acid for detecting the Tecode MM0011 strain.

The sequences of SEQ ID NOs: 1, 2 and 3 are characteristic sequences that can distinguish the microalgae according to the present application from other strains, and for the detection, isolation or identification of the strains according to the present application, for example, from therein, After designing, synthesizing, and can be used for the detection, isolation or identification of the strain according to the present application using methods known in the art, such as PCR (polymerase chain reaction) or hybridization (hybridization).

In another aspect, the present application provides a microbial agent comprising a strain or a culture thereof disclosed herein, wherein the microbial agent is for biooil production, biomass production, lutein production, animal feed production, or removal of whole nitrogen and whole persons. It can be usefully used.

In another aspect, the present invention provides a method for producing a lipid comprising culturing the strains disclosed herein under appropriate conditions suitable for lipid production; And it provides a lipid production method comprising the step of separating the lipid from the strain.

In another aspect, the present application provides a method for removing nitrogen and phosphorus from wastewater, comprising culturing the strain disclosed herein with wastewater.

In another aspect, the present invention comprises the steps of culturing the strains disclosed herein; And extracting a pigment from the strain; And it provides a lutein production method comprising the step of separating lutein from the pigment.

The microalgae according to the present invention have a lipid producing ability including a high concentration of polyunsaturated fatty acids and saturated fatty acids, a biomass producing ability including a high concentration of organic substances and proteins, an excellent ability to remove all nitrogen and whole humans, and a high concentration of lutein production. It is a novel strain. Such a strain according to the present application can be effectively used in the development of biofuels, functional foods, natural pigments, medicinal substances, animal feed, and aquaculture feed.

Specifically, the microalgae of the present invention can biosynthesize polyunsaturated fatty acids such as omega-6 or omega-3 and can be used as an alternative resource of fish and / or plant-derived polyunsaturated fatty acid raw materials. Omega-3 polyunsaturated fatty acids, which are effective in preventing cardiovascular diseases, are known to be abundant in back blue fish, but are actually produced mainly by microalgae and stored in the body of fish consumed through the food chain. Microalgae omega-3 polyunsaturated fatty acids have no fishy smell and are unlikely to enrich heavy metals due to marine pollution. It is also suitable for biofuel production because it can biosynthesize saturated fatty acid that can be converted into biodiesel. The microalgae of the present application can biosynthesize high concentration of lutein, which is a natural carotenoid, and can be used as a raw material for food, medicine, and cosmetics. Lutein is an antioxidant carotenoid that forms the macular pigment in the retina and acts as a filter to protect the eyes from ultraviolet or blue light. Lutein is also incapable of biosynthesis, which is an essential ingredient for external consumption. Due to these lutein properties, lutein is one of the fastest growing carotenoid pigments, with an average annual growth rate of 3.6% and a market of about $ 309 million by 2018. have. Most of the supplements currently on the market use lutein extracted from marigold (marigold) flowers, but the lutein content is very low and it is difficult to separate during extraction.This microalgae is a functional raw material that can replace marigold. Expect Another feature of the microalgae of the present invention is that the amount of volatiles and total calorific value is higher than that of wood-based biomass, and thus the potential as a biofuel raw material is also high. In addition, the high protein content can be used as a good animal feed by the biomass itself, and can remove each of the high nitrogen and whole phosphorus from the culture medium in high proportion, so the isolated strain of the present invention can be effectively used for wastewater treatment.

Figure 1 shows an optical micrograph of the oxenochlorella protothecoide MM0011 according to an embodiment of the present application.
Figure 2 shows a field emission scanning electron micrograph of oxenochlorella protothecoide MM0011 according to an embodiment of the present application.
Figure 3A shows a transmission electron micrograph of the oxenochlorella protothecoide MM0011 according to an embodiment of the present application. CHL: chloroplast, M: mitochondion, N: nucleus.
Figure 3B shows an enlarged photograph of the cell wall structure of oxenochlorella protothecoide MM0011 according to an embodiment of the present application. Arrows indicate the outer wall of cells consisting of three layers.
Figure 4 shows the systematic relationship between oxenochlorella protothecoide MM0011 and related species interpreted from 18S rRNA sequence data.
Figure 5 shows the thermogravimetric analysis of the oxenochlorella protothecoide MM0011 according to an embodiment of the present application.

The present application is a novel unicellular green algae strain isolated and isolated from seawater, Auxenochlorella protothecoides ) based on MM0011 strain discovery.

The microalgae oxenochlorella protococcoids disclosed herein are facultative heterotrophic green algae belonging to the Trebouxiophyceae class. Analysis of the morphological, molecular, and biochemical properties of the microalgae disclosed herein revealed that it is an unrecorded species belonging to the oxenochlorella prototeoid, but has not been officially recorded in Korea. The strain was named, and it was deposited on July 13, 2017 to the Korean Collection for Type Cultures (KCTC13291BP).

In one aspect the present application relates to a novel microalgae oxenochlorella protothecoide MM0011 deposited with accession number KCTC13291BP.

Microalgae oxenochlorella protothecoide MM0011 according to the present invention has the 18S rRNA of SEQ ID NO: 1, the ITS of SEQ ID NO: 2, and the rbc L gene sequence of SEQ ID NO: 3.

Therefore, the sequence is a specific sequence existing only in the strain according to the present application, and can be effectively used for the detection of the strain according to the present application. In this aspect, the present application refers to the oxenochlorella prototheco of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 ID MM0011 relates to a nucleic acid for detection.

The microalgae according to the present invention have a lipid producing ability including a high concentration of polyunsaturated fatty acids and saturated fatty acids, a biomass producing ability including a high concentration of organic substances and proteins, an excellent ability to remove all nitrogen and whole humans, and a high concentration of lutein production. .

“Productivity” herein means an increase in the amount of total fatty acids per microalgal population or unit dry or wet weight, an increase in the amount of polyunsaturated fatty acids, an increase in the amount of omega-3 or omega-6, or polyunsaturated fatty acids in the produced fatty acids. Increase in the relative proportions of omega-3 and omega-6 in the total fatty acids or polyunsaturated fatty acids produced; Or an increase in the total amount of biomass per microalgal population or unit dry or wet weight, an increase in the amount of organics and proteins, or an increase in the relative proportions of organics and proteins in the produced biomass; Or an increase in the total amount of pigment per microalgal population or per unit dry or wet weight, an increase in the amount of lutein, or an increase in the relative proportion of lutein in the pigments produced. Any of the above may lead to an increase in the production of the desired useful material, for example, when the relative ratio is increased, the desired useful material may be increased, as well as the concentration may be easily separated.

The term "biomass" herein refers to an organic material from microalgae that can be used for the production of biofuels and useful materials. Such biomass may contain extracellular materials as well as cellular and / or intracellular materials. Extracellular materials include, but are not limited to, compounds secreted by cells.

As used herein, “biofuel” is a fuel obtained by converting biomass into a resource and converting it into energy, and includes solid biofuel, liquid biofuel (biooil, bioethanol / butanol, biodiesel) and gaseous biofuel. .

The microalgae according to the present invention contain high concentrations of lipids, sugars or proteins, which are primary metabolites, and can be used as a source of solid and liquid biofuels.

In one embodiment, the organic material contained in the biomass extracted from the microalgae according to the present invention is 85% by weight or more, and may be effectively used as a source of solid biofuel. In particular, the gross calorific value (GCV) was 20.3 MJ · kg ^{−1, which} was higher than the total calorific value range (17.0-20.0 MJ · kg ⁻¹ ) of terrestrial energy crops.

In another embodiment the microalgae according to the present disclosure also produce high concentrations of saturated fatty acids. Saturated fatty acids are fatty acids that do not have a double bond, and in one embodiment of the present disclosure, the microalgae according to the present disclosure are palmitic acid (C _{16: 0} ), which is a 16-carbon saturated fatty acid, at least about 10% of the total fatty acid weight, particularly 12.0. It can be used as a raw material for the production of liquid biofuels such as biodiesel through transesterification.

In addition, the strain according to the present application was found to have a high content of polyunsaturated fatty acids.

As used herein, “polyunsaturated fatty acid (PFUA)” refers to unsaturated fatty acids containing two or more double bonds (π) of 18 or more carbon atoms. Polyunsaturated fatty acids, such as omega-3 or omega-6 unsaturated fatty acids, have a very important function in the human body, but because they are not naturally synthesized in the human body, they should be consumed primarily through food.

In one embodiment, the microalgae according to the present invention include polyunsaturated fatty acids such as linoleic acid (C _{18: 2} ω6; Omega-6) and α-linolenic acid (C _{18: 3} ω3; Omega-3). Contains in high content. In one embodiment, the unsaturated fatty acid comprises about 60 to 70%, in particular 67.2% of the total lipid weight. Thus, the microalgae according to the present application can be used for the production of omega-3 and omega-6 unsaturated fatty acids, replacing existing vegetable oils, marine animal oils, fish oils and oilseeds. can do.

The microalgae according to the present invention may biosynthesize lutein in a high content as a secondary metabolite. The term “lutein” is referred to herein as a xanthophyll as a pigment present in the largest amount of xanthophylls of carotenoids. In one embodiment of the present disclosure, the microalgal MM0011 strain according to the present disclosure has a lutein content of 3.5 ± 0.1 mg · g ^{−1 on} a dry weight basis. The microalgae according to the present application can be used as a raw material of functional foods, natural pigments, medicinal substances and the like.

In addition, the microalgae according to the present invention can effectively remove total nitrogen and total phosphorus from the medium.

'All nitrogen' refers to the total amount of nitrogen compounds contained in the water. Total nitrogen (TN) is included in natural water during the natural nitrogen cycle, but increases with anthropogenic inflows such as domestic sewage, factory wastewater, and livestock wastewater. It is also used as an indicator of eutrophication in lakes and coasts along with phosphorus, and is often used for rivers and lakes in areas with high concentrations of population. Examples of the measurement methods include ultraviolet absorbance method, cadmium reduction method, reduced distillation-Kjeldahl method (summing method), and the like. Nitrogen is included as food waste in domestic sewage, and livestock wastewater contains very high concentrations of nitrogen.

The whole person refers to the total concentration of the phosphorus compound contained in the water. The whole person refers to the concentration of phosphorus contained in the water as one of the indicators of eutrophication of the lake, rivers and the like. Phosphorus, together with nitrogen, acts as a limiting substrate in closed water eutrophication such as lakes. Total phosphorus refers to a value obtained by measuring the total amount of phosphorus (P) present in water, such as particulate phosphorus, organic phosphorus, polyphosphate, and phosphate ions. The unit is expressed as mg · l ⁻¹ .

In one embodiment of the present application, the microalgae according to the present application can effectively remove whole nitrogen and whole phosphorus from the culture solution, and thus can be used for wastewater treatment. "Waste water" as used herein refers to wastewater of water that generally contains wash water, wash water, feces, urine and other liquid or semi-liquid waste. This includes not only municipal waste but also some form of sewage treated second. Also, 'sewage' is water in a state in which liquid or solid contaminated substances are mixed. As a by-product of material civilization activities in the society of consumer life and industrial activities, various wastes are continuously discharged, which refers to wastes that are discharged in the form of liquid and flow into the sewer. Wastewater that is generated after living in human daily life is called sewage and industrial wastewater is called industrial wastewater. The microalgae according to the present invention can remove 40.5% of all nitrogen and 27.9% of the whole phosphorus from the culture.

In addition, the microalgae according to the present application has a high protein content, and may be a raw material of animal feed. In one embodiment the biomass of the microalgae according to the present application has a high protein content of at least 50%, in particular 51.4%. Animal feed is called an animal feed, and the animal feed contains a feed additive which is added to the feed to supply nutrients that are not synthesized in the body as essential amino acids for animal feed addition. Biomass of microalgae according to the present application can be used as a feed additive.

In another aspect, the present disclosure also relates to a microbial agent, comprising the strains and / or cultures thereof disclosed herein, wherein the microbial agent is for the production of polyunsaturated fatty acids comprising omega-3 and / or omega-6, It can be effectively used for raw biomass production, lutein production, animal feed production, or removal of whole nitrogen and whole humans.

The culture medium of the strain according to the present invention is a medium cultured under appropriate conditions for the purpose of the microalgae according to the present application, the medium is a liquid composition containing the nutrients and / or a material for a special purpose necessary for the culture of the microalgae, Includes all that have been removed after the microalgae have been incubated, or include microalgae.

The strains according to the invention can be cultured in a variety of media used for the culture of microalgae. The medium used in the present invention is not limited thereto, but is preferably carried out in a medium containing a carbon source and a nitrogen source, and containing seawater and sodium chloride. The carbon source may be glucose, fructose, galactose, glucose, glycerol or a mixture thereof. It may be selected from the group consisting of, the nitrogen source may be selected from the group consisting of sodium glutamate, peptone, tryptone, yeast extract, corn steep liquor, sodium nitrate, ammonium sulfate, ammonium citrate or mixtures thereof. In one embodiment of the present application, R2A, which is a relatively inexpensive poor nutrient medium for the microalgal culture of the present application, is used. In addition, the medium used for culturing the microalgae according to the present application is not limited thereto but may not include sodium chloride or may be cultured at a sodium chloride concentration of up to 1.5 M. The microalgae of the present invention are resistant to sodium chloride due to the nature of the seawater source, so it is not limited to freshwater, and abundant seawater resources can be used as a culture medium.

Microalgae according to the present disclosure are cultivable over a wide temperature range, and in one embodiment of the present disclosure, may be cultured at a temperature of 5-35 ° C.

In another aspect, the present application also provides a method of culturing a strain according to the present disclosure at appropriate temperature and media conditions; And separating the lipid from the strain and extracting the polyunsaturated fatty acid from the lipid. Cultivation methods, including culture medium and temperature, may be referred to those disclosed herein, and separation or extraction may be performed using methods known in the art.

In another aspect the present disclosure also provides a method of culturing a strain according to the present disclosure at appropriate temperature and media conditions; And separating the pigment from the strain, and extracting lutein from the dye, and relates to a lutein production method using microalgae. Cultivation methods, including culture medium and temperature, may be referred to those disclosed herein, and separation or extraction may be performed using methods known in the art.

In another aspect the present application also relates to a method for removing nitrogen and phosphorus from wastewater, comprising culturing the strain according to the invention with wastewater. Methods of removing nitrogen and phosphorus through culturing can be carried out using those disclosed herein and methods known in the art.

In another aspect the present application also relates to a method for producing biofuel using biomass from a strain according to the present application. As mentioned above, the biomass of the strain according to the present application may be a raw material of the solid biofuel and the liquid biofuel, and the method of producing the bioraw material from the above-mentioned matters and methods known in the art using It may be carried out, the culture method may refer to that disclosed herein.

Hereinafter, examples are provided to help understand the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example

Example  1. Identification of MM0011 Strains

Sampling and Microalgae Separation

Seawater samples were collected in April 2016 from the tide pools around Turtle Rock near Ulleungdo, Seolleung-gun, Gyeongsangbuk-do, Korea. Samples were transferred to the laboratory and seeded in 100 ml BG-11 microalgae selection medium in sterile 250 ml flasks. In order to inhibit the growth of bacteria, chloramphenicol and imipenem, which are broad-spectrum antibiotics, were added to the medium at a concentration of 100 μg · ml ⁻¹ , respectively. The flask was incubated at 25 ° C using a fluorescent lamp to give a light quantity of about 40 μmole · m ⁻² · s ⁻¹ until the growth of microalgae was apparent at 16 hours: 8 hours (day: night). Was carried out. The microalgal biomass was secured by centrifuging 1.5 ml of the microalgal culture at 3,000 × g for 3 minutes. Centrifuged microalgal pellets were inoculated by fractional fractionation on R2A agar medium containing imipenem at a concentration of 20 μg · ml ⁻¹ and cultured in the same environment as described above. Single colonies were aseptically aliquoted on a new R2A agar medium and this process was repeated until aseptic cultures were obtained.

Sympathy

Pure microalgae culture was incubated for 8 days in R2A medium. Live cells were harvested by centrifugation at 3,000 × g for 3 minutes, washed three times with sterile distilled water, and observed at 1,000 × magnification using a Zeiss Axio Imager.A2 optical microscope. For scanning electron microscopy, 10 ml of the culture medium was prepared at a concentration of about 1,000 cells per ml, and fixed at a final concentration of 2% (v / v) osmium tetraoxide for 10 minutes. The fixed cells were collected in a polycarbonate membrane filter with a pore size of 3 μm, washed three times with distilled water to remove the components of the medium. The membrane filter was dehydrated by treating ethanol for 2 minutes each step (10, 30, 50, 70, 100% and 100% twice) .The supercritical dryer put the dried sample on an aluminum stub and a low vacuum coater. Gold-palladium coating was carried out using a field emission scanning electron microscope to observe the morphological characteristics of the cell surface.

10 ml of the culture was prepared for transmission electron microscopic observation and fixed for 2 hours at a final concentration of 2.5% (v / v) glutaraldehyde. The fixed cells were transferred to a 10 ml centrifuge tube and centrifuged at 1,610 × g for 10 minutes. Centrifuged microalgal pellets were transferred to a 1.5 ml microcentrifuge test tube, washed several times with a 0.2 M sodium cacodylate buffer solution at pH 7.4, and then with a final concentration of 1% (v / v) osmium tetraoxide. Second fixation was carried out for 30 minutes. The fixed pellets were embedded between agar and treated with ethanol in steps (50, 60, 70, 80, 90%, 100% and 100% twice) to proceed with dehydration. The dehydrated pellets were embedded in Spurr's resin, and then thin films were prepared using ultrathin slicers, and then stained with 3% (w / v) uranyl acetate and lead citrate. Thin film samples were observed at 100 kV with a H-7650 transmission electron microscope.

Molecular Biology Identification

For molecular biological analysis, genomic DNA was extracted using the DNeasy Plant Mini kit. NS1 / NS8 and ITS1 / ITS4 primers reported by White et al. Were used to amplify 18S rRNA and ITS fragments, respectively. Genetic analysis using the 18S rRNA sequence of MM0011 strain was MEGA ver. This was done using the 6.0 software package. The base sequences of oxenochlorella and chlorella related species of MM0011 were downloaded from the National Institute of Biological Information (NCBI) database and sorted using ClustalW. Kimura 2-parameter, the best-fit nucleotide substitution model, was selected based on the Bayesian information criteria. This model was used to generate the highest likelihood tree through 1,000 bootstrap iterations. Because of the high retention of rRNA sequences, the RuBisCO rbc L chloroplast gene was amplified using the rbc L 7F and rbc L 1391R primers proposed by Verbruggen et al. All genetic analyzes were repeated three times, and the sequence information obtained through this study was registered as MF040300, MF040301, and MF043910 in the US National Center for Biological Information (Table 1). Table 1 below shows the results of BLAST search using the sequences of the 18S rRNA, ITS, and rbc L genes of the MM0011 strain.

TABLE 1

The cells are solitary, non-motile, round in shape with a diameter of about 3-4 μm (FIGS. 1, 2). The cell wall consisted of three layers (FIG. 3B) with distinct cup-shaped chloroplasts (FIG. 1, 3). The isolates also lacked pyrenoids in chloroplasts (FIG. 3A). Overall, the MM0011 strain is A. It has typical morphological features of protothecoides species. Molecular biological characteristics of sequential analysis of 18S rRNA, ITS region, and rbc L genes were also isolated from A. appeared to belong to protothecoides ; All results were in agreement with this (Table 1, FIG. 4 above).

Thus, the marine microalgae of the present application have been identified as Auxenochlorella protothecoides MM0011 . The isolate was deposited with the Korean Collection for Type Cultures under accession number KCTC13291BP.

Example  2. Oxenochlorella Protothecoid  Culture condition of MM0011 strain

Temperature and Sodium Chloride Tolerance Tests: Pure cultures of oxenochlorella protothecoides were maintained by periodic passage on R2A agar slope medium. A single colony of MM0011 strains was fractionated in three replicates on R2A agar medium and incubated for 14 days. The optimal growth temperature was determined by investigating the survival and growth of MM0011 cells in the range of 5-35 degrees (5 degrees intervals). Sodium chloride resistance test was performed using a R2A agar medium at 20 degrees, and the experiment was performed by adding sodium chloride at concentrations of 0.0 M, 0.5 M, 1.0 M, 1.5 M and 2.0 M.

As a result of the experiment, maximum growth was observed at ambient temperature (Table 3). At low temperatures growth was observed to be inhibited. The isolate was also shown to be resistant to sodium chloride up to a 1.5 M NaCl concentration, although slow growth was observed in the presence of NaCl. However, it did not survive in 2.0 M NaCl. Table 2 below shows the growth of MM0011 strains at various temperatures and NaCl concentrations.

TABLE 2

Example  3. Oxenochlorella Protothecoid  Lipid Content Analysis of MM0011 Strains

Lipid content of the strain isolated herein was analyzed using gas chromatography as follows.

Specifically, the isolated strain was cultured heterotrophically for 8 days at 160 rpm using a shake incubator at 20 degrees in R2A medium. Biomass was harvested by centrifugation at 2,063 g for lipid analysis. Samples were lyophilized and homogenized to increase the efficiency of lipid extraction. Lipid extraction was performed using a method developed by Breuer et al. (Breuer, G. et al. 2013. J. Vis. Exp. 80, e50628). Fatty acid composition was analyzed using 7890A gas chromatograph (Agilent, USA) equipped with 5975C mass selective detector. DB-FFAP columns (30 m, 250 μm ID, 0.25 μm film thickness; Agilent) were used for lipid analysis. The original GC oven temperature started at 50 ° C. and held for 1 minute. The temperature was raised to 200 degrees for 30 minutes at 10 degrees per minute, and then increased to 10 degrees per minute to 240 degrees and maintained for 20 minutes. Samples were injected with 1 μl at a split ratio of 20: 1. Helium was used as the traveling gas at a flow rate of 1 ml per minute. The mass spectrometry parameters were as follows: injector temperature: 250 degrees / source temperature: 230 degrees, electron impact mode was used for ionization of the sample at an acceleration range of 70 eV acquisition range 50-550 m · z ^-1 . Fatty acid identification was found to be valid only when the match value was greater than 90% compared to the mass spectra of Wiley / NBS libraries.

As a result of the analysis, based on the mean ± standard deviation of the three measurements, A. FAME profiles of protothecoides are shown in Table 3 below. The major fatty acids of the strains isolated herein are C _{16: 0} (12.0% ± 0.5%), C _{18: 2} ω6 (27.6% ± 0.1%), and C _{18: 3} ω3 (39.6% ± 0.6%). In addition, unsaturated fatty acids, such as C _{16: 1} ω7 (3.2% ± 0.1%), C _{18: 1} ω9 (2.5% ± 0.3%), and C _{16: 2} ω6 (0.5% ± 0.0%), are also present in the photosynthetic microorganism. Detected. The content percentages in the table below are based on the extracted lipids.

TABLE 3

Intracellular fatty acid analysis of MM0011 strain showed C _{16: 0} saturated fatty acid (SFA), palmitic acid, and C _{18: 2} (27.6%) with omega 6 as unsaturated fatty acid and C _{18: 3} (39.6% with omega 3). A) rich in unsaturated fatty acids. In numerous studies, these essential unsaturated fatty acids have many health benefits, and various commercial products are widely used worldwide. Omega-3 unsaturated fatty acids are often extracted from fish oils, and omega-6 unsaturated fatty acids are preferentially obtained from plant sources such as sunflower, corn and soybean oils. Thus, the isolate may be used as a substitute for fish- and / or plant-derived resources. In addition, 16-carbon saturated palmitic acid may be usefully used for biofuel production.

Example  4. Oxenochlorella Protothecoid  Lutein of MM0011 Strain Productivity  analysis

Useful pigment components of the strains according to the present application were analyzed by high performance liquid chromatography. Pigment extraction was carried out using the method developed by Zapata et al. (Zapata, M. et al. 2000. Mar. Ecol . Prog . Ser. 195, 29-45). Specifically, photosynthetic pigments were extracted from the biomass of the 1 mg lyophilized strain using 90% HPLC grade acetone and filtered before injection with a Whatman PTFE syringe filter having a pore size of 0.2 μm. Samples were analyzed at 33 degrees using an Agilent 1260 Infinity HPLC system equipped with a Discovery C18 column (25 cm × 4.6 mm, 5 μm). The mobile phase gradient was maintained at a flow rate of 1 ml · min ⁻¹ using a method reported by Sanz et al., With methanol: 225 mM ammonium acetate (82:18, v: v) as solvent A and ethanol as solvent B ( Table 4) below. HPLC grade methanol and ethanol were used from JT Baker, and HPLC grade ammonium acetate was purchased from Fluka. Immediately before injection, 0.32 ml of HPLC grade water (Fisher) was added to 0.8 ml of sample to prevent distortion of the initial peak. Standard pigments (chlorophyll a , chlorophyll b and lutein) were purchased from Sigma-Aldrich. Lutein content was quantified by calculating the peak area from the calibration curve. Table 4 below shows concentration gradient profiles and mobile phase compositions of solvents A and B.

TABLE 4

A. The pigment profile of protothecoides MM0011 is shown in Table 5 below. As a result, the main pigments of the isolate according to the present application were chlorophyll a (Chl a , 38.4% ± 0.1%), chlorophyll b (Chl b , 14.4% ± 0.1%), and lutein (31.3% ± 0.1%). No other fine peaks were identified. The lutein content of strain MM0011 was found to be high as 3.5 ± 0.1 mg g ^-1 DW based on the microalgal biomass dry weight. % In Table 5 shows the content% of each pigment in the total pigment.

TABLE 5

This indicates that the isolated strain of the present application has a high concentration of lutein. Therefore, the strain according to the present application is a natural carotenoid, which is widely used in food, medicine, and cosmetics, and can be effectively used for the production of lutein, which has recently received more attention due to its function of protecting human eyes.

Example  5. Oxenochlorella Protothecoid  MM0011 strain Biomass  Ingredient analysis

The lyophilized biomass sample of the strain according to the present application was ground using a mortar and pestle and then ASTM No. Homogenization was performed using a 230 mesh (diameter size = 63 μm) precision standard mesh. Elemental analysis was performed in two iterations using a Flash 2000 elemental analyzer to determine the carbon, hydrogen, nitrogen and sulfur contents. Oxygen content calculation was calculated by subtracting the content of carbon, hydrogen, nitrogen, sulfur and ash from 100%. Total calorific value was estimated by the formula proposed by Friedl et al .: [Total calorific value = 3.55C ² -232C-2230H + 51.2C × H + 131N + 20,600 (MJkg ^-1 )]

Approximate analysis was performed using a DTG-60A thermal analyzer. 30 mg alpha-alumina powder was used as reference material to analyze about 10 mg of sample using a platinum pan. Nitrogen with a purity of 99.999% was used as the carrier gas at a flow rate of 25 ml · min ⁻¹ to prevent oxidation of the microalgal flour. The sample was heated at 50 ° C. to 900 ° C. with a 10 ° C. rise per minute. The thermogravimetric analysis data was obtained from ta60 Ver. 2.21 analyzed by software. The protein content was calculated by multiplying the nitrogen content from the elemental analysis by the conversion rate of 6.25.

In general component analysis conducted by thermogravimetric analysis, moisture content (MC) was determined by weight loss up to 110 ° C in N ₂ atmosphere, and organic matter (OM) was 110-900 in N ₂ atmosphere. It refers to the weight loss obtained as a result of thermal decomposition between ℃ and the rest weight refers to the inorganic matter (IM). The TGA profile is shown in FIG. 5 and the material composition of strain MM0011 is shown in Table 6. GCV (Gross Calorific Value) and protein content based on the final analysis were 20.3 MJ kg ^-1 and 51.4%, respectively (Table 6).

TABLE 6

The organic substance OM may be used as a solid fuel that emits gas when heated. In general, biomass of crops used as biomass is 63-80%, and trees are 72-78%. In comparison, the OM content of the present microalgae was 85.6%, which was significantly higher than that of crops and trees. GCV figures also suggest the potential to replace woody biofuel feedstocks, which are higher than the range of land-based energy crops (17.0–20.0 MJ · kg ⁻¹ ). In addition, the high protein content (51.4%) makes biomass itself a good animal feed.

Example  6. All nitrogen (total nitrogen, TN) and total phosphorus (TP) consumption

The total nitrogen and total phosphorus concentrations of R2A medium at day 0 and day 8 were analyzed by HS-TN (CA) -L and HS-TP-L water test kits (Humas, Daejeon, Korea) according to the manufacturer's instructions.

Microalgae were found to remove 40.5% TN and 27.9% TP in R2A, respectively (Table 7).

TABLE 7

These results indicate that the strain according to the present application can remove 40.5% total nitrogen and 27.9% total phosphorus from the culture, respectively. Therefore, the isolated strain of the present application can be effectively used for biofuel raw material production and wastewater treatment.

Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

<110> National Marine Biodiversity Institute of Korea <120> Auxenochlorella protothecoides MM0011 and use <130> DP201711005P <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1802 <212> DNA <213> Auxenochlorella protothecoides MM0011 <220> <221> misc_feature (222) (1) .. (1802) <223> rRNA <400> 1 gtcatatgct tgtctcaaag attaagccat gcatgtctga gtataacctg tttactactg 60 tgaaactgcg aatggctcat taaatcagtt atagtttatt tgatggtacc tgctactggg 120 atacccgtag taattctaga gctaatacct gcgcaaaccc cgactttggt ggaaggggtg 180 tatttattag atccaaggcc gaccgggctc gcccgactcg cggtgactca tgataacttt 240 acgaatcgca tggtctctgc accggcgatg tttcattcaa atttctgccc tatcaacttt 300 gtcggtagga tagaggccta ccgaggtgtt cacgggtgac ggaggatcag ggttcgattc 360 cggagaggga gcctgagaaa cggctaccac atccaaggaa ggcagcaggc gcgcaaatta 420 cccaatcctg acacagggag gtagtgacaa taaataacaa taccgggcct cacaggtctg 480 gtaattggaa tgagtacaat ctaaacccct taacgaggat caattggagg gcaagtctgg 540 tgccagcagc cgcggtaatt ccagctccaa tagcgtatat ttaagttgct gcagttaaaa 600 agctcgtagt cgaacttcgg ggggcccggg ccggtccttt ggcgcggtgg gttcacgccc 660 ccgccctctg cgcactggca ttccggcgcc cccctgttgc tggggacggc gctcctgggc 720 tttgctgtcc ggggcccgga gtcagcgagg ttactttgag taaattagag tgttcaaagc 780 aggcatccgc tctgaataca ttagcatgga ataacatgat aggactctgg cttatcacgt 840 tggtctgtaa gaccggagta atgattgaga gggacagtcg ggggcattcg tatttcattg 900 tcagaggtga aattcttgga tttatgaaag acgaactact gcgaaagcat ttgccaagga 960 tgttttcatt gatcaagaac gaaagttggg ggctcgaaga cgattagata ccgtcctagt 1020 ctcaaccgta aacgatgccg actcgggatc ggtggtcgtt cattcaatga caccaccggc 1080 acctcatgag aaatcaaagt ttttgggttc cggggggagt atggtcgcaa ggctgaaact 1140 taaaggaatt gacggaaggg caccaccagg cgtggagcct gcggcttaat ttgactcaac 1200 acgggaaaac ttaccaggtc cagacatagt gaggattgac agattgagag ctctttcttg 1260 attctatggg tggtggtgca tggccgttct tagttggtgg gttgccttgt caggttgatt 1320 ccggtaacga acgagacctc agcctgctaa atagttttgg ttgcggtttc accccagcct 1380 tgtaagactt cttagaggga ctctcggcga ctagccgagg gaagcttgag gcaataacag 1440 gtctgtgatg cccttagatg ttctgggccg cacgcgcgct acaatgatgc ggtcaacgag 1500 cccagccttg gccgacaggc ccgggtaatc tcgcaacccg catcgtgatg gggatagatc 1560 attgcaatta ttgatcttca acgaggaatg cctagtaagc gcaagtcatc agcttgcgtt 1620 gattacgtcc ctgccctttg tacacaccgc ccgtcgctcc taccgattga gtgtgctggt 1680 gaagtgttcg gattggcagc tcgatcgcgg ttctccgcga ccagcagccg agaagttcat 1740 taaaccctcc cacttagagg aaggagaagt cgtaacaagg tttccgtagg tgaacctgcg 1800 ga 1802 <210> 2 <211> 826 <212> DNA <213> Auxenochlorella protothecoides MM0011 <220> <221> misc_feature (222) (1) .. (826) <223> ITS <400> 2 tccgtaggtg aacctgcgga aggatcattg atttacattg atttttatac agtagtttga 60 ttcagaacac catcacgttg ttgcaacgtg tgacgagttt ttttattgtc aggtggacgc 120 gagtcccctg tcgctctcac acgctgcaca aacccaacaa ccttgaccca accaacccat 180 gcctgaagaa tcatgcgagc gtgcgtacta cccatgtaca cccctcgcca accagagaca 240 actctcaaca acggatatct tggctcccgt atcgacgaag aacgcagcga aatgcgatac 300 gtagtgtgaa ttgcagaatt ccgtgaacca tcgaatcttt gaacgcaact tgcgcccgag 360 gtcctagacc gagggcatgt ctgcctcagc gtcggcttta acccttccat ccagttgttg 420 gatgcattcc gcaactgcct gggacctggc tctcacgacc accaccccgg tggtcgtgtt 480 tgctgaagca cagaggcttg agcgaggacc ccgtttgcag ggcgctggct tggtaggtag 540 gcgaccccta caccacccgc cgacgcccga ggtgtccttg ctggaggccc agcaggaatt 600 gggaggtact ttttggtgag gttggtttgt tggggtgtgt gtgttgctgg atggatggat 660 ggattacaag ggttggtctt tgatcttccc tcttctcctc cctcatcatc atacaccccc 720 tactactact cctcaccacc caaaacaacc tcccaacccc caaacccttc gacctgagct 780 caggcaagac cacccgctga acttaagcat atcaataagc ggagga 826 <210> 3 <211> 340 <212> DNA <213> Auxenochlorella protothecoides MM0011 <220> <221> misc_structure <222> (1) .. (340) <223> rbcL <400> 3 ccacaaactg aaacaaaagc cagttcaggt tttaaagcag gtgttaaaga ttatcgttta 60 acttattata ctcctgatta ccaaacaaaa gatacggata ttttagcagc tttccgtatg 120 actcctcaac caggcgttcc accagaagaa tgtggtgcag ctgttgcagc agaatcttca 180 acaggtactt ggacgactgt ttggactgat ggtttaacta gtttagaccg ttataaagga 240 cgttgctatg atcttgaacc agtagcaggc gaagaaaatc aatatattgc atatgttgca 300 tatccaattg atctttttga agaaggctct gtaacaaact 340

Claims (14)

18S ribosomal ribonucleic acid (RSS) represented by SEQ ID NO: 1, an internal transcribed spacer (ITS) represented by SEQ ID NO: 2, and SEQ ID NO: 3 having a biooil-producing ability including high concentrations of polyunsaturated fatty acids and saturated fatty acids Having the base sequence of rbc L (the large subunit of the ribulose-bisphosphate carboxylase gene).
The polyunsaturated fatty acids are omega-3 and omega-6, the omega-3 and omega-6 are 60% to 70% of the total lipid weight,
The saturated fatty acid is palmitic acid, the palmitic acid is 10 to 15% of the total lipid weight, microalgae oxenochlorella protocoids ( Auxenochlorella protothecoides ) MM0011 strain deposited with accession number KCTC13291BP.
The method of claim 1,
The strain further has the ability to produce biomass, including organics and proteins, the ability to remove all nitrogen and whole humans, and lutein production, oxenochlorella protothecoide MM0011 strain.
delete delete The method of claim 2,
The lutein content is 3.5 ± 0.1 mg · g ⁻¹ based on the dry weight of the microalgae, oxenochlorella protothecoide MM0011 strain.
The method of claim 2,
The organic material contained in the biomass is more than 85% by weight, the protein is included in more than 50% by weight, oxenochlorella protothecoide MM0011 strain.
The method of claim 2,
The removal ability of the whole nitrogen and whole phosphorus is 40% or more and 27% or more, respectively, oxenochlorella protothecoid MM0011 strain.
The method of claim 2,
The microalgae will have a culture temperature of 5-35 ℃, culture conditions of NaCl concentration of 0 to 1.5 M, oxenochlorella protothecoide MM0011 strain.
A microbial preparation comprising the strain according to any one of claims 1, 2, or 5 to 8 or a culture thereof.
The microbial preparation is for biooil production, biomass production, lutein production, animal feed production, or removal of whole nitrogen and whole persons, microbial preparation.
Culturing the strain according to any one of claims 1, 2, or 5 to 8; And separating the lipid from the strain.
A method for removing nitrogen and phosphorus from wastewater, comprising culturing the strain according to any one of claims 1, 2, or 5 to 8 with wastewater.
Culturing the strain according to any one of claims 1, 2, or 5 to 8; And extracting a pigment from the strain; And separating lutein from the pigment.
A nucleic acid for detecting one or more oxenochlorella prototycoid MM0011 strains selected from the group consisting of 18S rRNA represented by SEQ ID NO: 1, ITS represented by SEQ ID NO: 2, and rbc L represented by SEQ ID NO: 3.
Oxenochlorella prototycoid MM0011 strain comprising a substance for detecting one or more genes selected from the group consisting of 18S rRNA represented by SEQ ID NO: 1, ITS represented by SEQ ID NO: 2, and rbc L represented by SEQ ID NO: 3 Detection composition.

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