KR101232279B1 - Novel strain containing high conc. of lipid - Google Patents

Abstract

PURPOSE: A novel strain which contains a high lipid content is provided to produce bio-diesel and to ensure heavy metal resistance and excellent productivity. CONSTITUTION: A novel strain which contains a high lipid content is Nephroselmis sp.KGE8(deposit number: KCTC 12235BP). The strain is Nephroselmis pyriformis. The strain contains the DNA sequence of sequence number 1. The strain is cultured in a medium containing KH_2PO_4, CaCl_2, MgSO_4, NaNO_3, K_2HPO_4, NaCl, and H_3BO_3 at 25-30 deg.C. and pH 6-8. The medium further contains ZnSO_4, MnCl_2, MoO_3, CuSO_4, or Co(NO_3)_2.

Description

Novel strains containing a high content of lipids {NOVEL STRAIN CONTAINING HIGH CONC. OF LIPID}

The present invention relates to a novel strain comprising a high content of lipids.

The use of fossil fuels due to the high industrialization is a major cause of global warming by emitting a large amount of greenhouse gases. In addition, oil is being depleted due to excessive use of fossil fuels. Therefore, many researchers at home and abroad are developing alternative energy as an alternative means of fossil fuel. Alternative energy sources may include renewable energy, including solar, bio, wind, hydro, ocean, waste, geo, fuel cell and hydrogen. Among them, bioenergy can be used as a raw material for biodiesel, and the yield per unit area is much higher than that of other raw materials.

Conventional bioenergy sources are mainly beans, sugar cane and the like. However, the production efficiency of the biodiesel for the vast area required for cultivation thereof is low, and thus faces difficulties. Therefore, the study of microalgae as biomass is needed for stable and economic production of biodiesel.

Accordingly, the present inventors have made diligent efforts to solve the above problems, and have confirmed that the microalgae inhabiting acid mine drainage are novel strains containing lipids with high content and completed the present invention.

Republic of Korea Patent No. 10-0693865

It is an object of the present invention to provide novel strains containing high concentrations of lipids, especially fatty acids, especially palmitic acid.

In order to achieve the above object, the present invention provides a Nephroselmis sp. Strain, culture medium of the strain and a method for culturing the strain, comprising at least 35% by weight of lipids relative to the total weight of the dry strain. to provide.

The novel strains of the present invention contain high amounts of lipids, high amounts of fatty acids, in particular palmitic acid, and can be usefully used for biodiesel production. The novel strains of the present invention are also isolated from acid mines and have heavy metal resistance. In addition, the novel strains of the present invention are high in lipid high fatty acids, especially palmitic acid, palmitoleic acid, oleic acid, linoleic acid (18: 2n6t), or g-linoleic acid (18: 3n6). In addition to containing a high content, the growth rate is fast, the productivity is excellent.

1 is a neproselmis sp of the present invention. Strain ( Nephroselmis sp.KGE8, Accession No. KCTC 12235BP) is shown.
Figure 2 is neproselmis sp. Of the present invention in BBM (Bold's Basal Medium). Strain of Nephroselmis sp.KGE8, Accession No. KCTC 12235BP Growth and nitrogen, phosphorus removal effect is shown.
3 is neproselmis sp. It shows the lipid content of the strain ( Nephroselmis sp. KGE8, Accession No. KCTC 12235BP).
4 is neproselmis sp. Fatty acid distribution of strain ( Nephroselmis sp. KGE8, Accession No. KCTC 12235BP) is shown.

Neproselmis sp. Of the present invention. The strain was originally identified and identified by the inventors. The present inventors identified the isolated new strain of neproselmis sp. It was named KGE8 ( Nephroselmis sp.KGE8, Accession No. KCTC 12235BP), and was deposited on July 3, 2012 at the Korea Research Institute of Bioscience and Biotechnology.

The present invention, in one aspect, high lipid content, neproselmis sp. KGE8 (Nephroselmis sp.KGE8, Accession No. KCTC 12235BP) relates to a strain.

In another aspect, the invention relates to a Nephroselmis sp. Strain comprising at least 35% by weight of lipids relative to the total weight of the dry strain. KGE stands for KIST Guengnung Enviromental.

In general microalgae, the content of lipids is difficult to exceed mid 20%. However, the present invention contains more than 35% of the lipids, and a large amount of fatty acids can be utilized as a biomass. In view of the above, the nephroselmis sp. Strain of the present invention is at least 35.5 wt%, at least 36 wt%, at least 36.5 wt%, at least 37 wt%, at least 37.5 wt%, at least 38 wt% , At least 38.5 wt%, at least 39 wt%, at least 39.5 wt%, at least 40 wt%, at least 40.5 wt% or at least 41 wt% lipid. In view of the above, the nephroselmis sp. Strain of the present invention is 90% by weight, 88% by weight, 86% by weight, 84% by weight, 82% by weight, 80% by weight or less , 78% by weight, 76% by weight, 74% by weight, 72% by weight, 70% by weight, 68% by weight, 66% by weight, 64% by weight, 62% by weight, 60% by weight Or up to 58% by weight of lipids.

Neproselmis sp. In the strain, the neproselmis sp. Strain is neproselmis sp. KGE8 may be (Nephroselmis sp. KGE8).

Neproselmis sp. In the strain, the neproselmis sp. The strain was neproselmis sp. It may be Nephroselmis pyriformis .

Neproselmis sp. In the strain, the neproselmis sp. The strain was neproselmis sp. KGE8 (Nephroselmis sp.KGE8, accession number KCTC 12235BP) can be.

Neproselmis sp. In the strain, the neproselmis sp. The strain may comprise the DNA sequence of SEQ ID NO: 1.

Neproselmis sp. In the strain, the neproselmis sp. The strain may have an access number of KCTC 12235BP.

Neproselmis sp. In the strain, the neproselmis sp. Strain not only has a high content of fatty acids, but also palmitic acid, palmitoleic acid, oleic acid, linoleic acid (18: 2n6t) and g-linoleic acid (18: At least one fatty acid selected from the group consisting of 3n6).

Neproselmis sp. In the strain, the neproselmis sp. Strain is a high lipid content of neproselmis sp. KGE8 (Nephroselmis sp.KGE8, Accession No. KCTC 12235BP) relates to a strain.

In another aspect, the present invention, the neproselmis sp. It relates to a culture medium in which the strain is cultured. The culture solution was neproselmis sp. The product obtained by culturing a strain is contained, and it has a structure contained in a normal culture liquid.

In another aspect, the present invention, the neproselmis sp. It is a culturing method of strains, and relates to a culturing method of culturing the strains under conditions of 25 to 30 ℃, pH 6-8. Under these conditions, the growth rate of the new strain of the present invention is the highest. In view of the above, the strain may be incubated at a temperature of 25.5 to 29.5 ° C, 26 to 29 ° C or 26.5 to 28.5 ° C, and may be cultured at pH 6.2 to 7.8, pH 6.4 to 7.6 or pH 6.4 to 7.4. have.

In the culture method of one aspect of the invention, the culture method KH ₂ PO ₄ , CaCl ₂ , MgSO ₄ , NaNO ₃ , K ₂ HPO ₄ , NaCl and H ₃ BO _{3 In} a medium containing, the neprocell Miss sp. Strains can be cultured.

In the culture method of one aspect of the present invention, the medium may further include one or more elements selected from the group consisting of ZnSO ₄ , MnCl ₂ , MoO ₃ , CuSO ₄ and Co (NO ₃ ) ₂ .

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

New strain  Isolation and Cultivation

The samples were collected from the acid mine drainage area of Heungil Taebong, Mojeon-myeon, Gangneung-si, Gangwon-do. Sampling was carried out in the sterilized water sample pack (1L), the soil, water quality and cold storage using an ice box to the laboratory. The collected sample was partially separated and washed and centrifuged three times using sterilized DIW to obtain a new strain.

The new strain was placed in BBM liquid medium having the composition shown in Table 1 and cultured for 2 weeks. A 36 watt fluorescent lamp was used as the light source. Two weeks later, an independent colony was taken and first spread evenly spread using a platinum plate on an alar plate (15 g / L) containing BBM. After 8 days, the cultured colonies were second plated onto an alar plate containing BBM. After 12 days, the cultured colonies were harvested and the microalgae were dispersed in a Erlenmeyer flask containing a liquid medium containing BBM. And microalgae are incubated using a constant temperature shaker with a fluorescent lamp. At this time, the culture conditions were the temperature 25 ± 1 ℃, stirring speed 150rpm.

[Table 1]

Isolated strain  culture

Using the BBM medium of the composition shown in Table 1, 10 mL of the new strain of the present invention was put in a 250 mL Erlenmeyer flask at 25 ° C., pH 7 and incubated for 14 days in a constant temperature shaker with a fluorescent lamp. Stirring was maintained at 150 rpm and roughness 50 μmol / m 2 -sec.

Isolate  Sympathy and Homology  compare

New strain neproselmis sp. The sequencing of KGE 8 ( Nephroselmis sp. KGE8, Accession No. KCTC 12235BP) was analyzed by 28 s rRNA sequencing.

A portion of the microalgae was taken during the cultivation and centrifuged to use the sample for analysis. The microalgae were extracted using a DNA extraction kit (SolGent, Korea), and the extracted DNA was amplified by PCR. Primer 28S D1D2 was used.

Forward primer: 5'-AGCGGAGGAAAAGAAACTA-3 '

Reverse primer: 5'-TACTAGA-AGGTTCGATTAGTC-3 '

The resulting nucleotide sequence was described in SEQ ID NO: 1, using the nucleotide sequence was prepared a phylogenetic tree with reference to NCBI information (Fig. 2).

In addition, as a result of analyzing the homology by performing a Blast search in NCBI using the nucleotide sequence, Table 2 was obtained.

[Table 2]

As shown in Table 2, the new strain of the present invention was confirmed to have a homology of 89% with Nephroselmis pyriformis (HE561886).

In this regard, the present inventors have identified the strain of the present invention neproselmis sp. It was named KGE8 ( Nephroselmis sp.KGE8, Accession No. KCTC 12235BP), and was deposited on July 3, 2012 at the Korea Research Institute of Bioscience and Biotechnology.

Isolate  Growth rate measurement

New strain neproselmis sp. Of the present invention. In order to determine the rate at which KGE 8 removes nitrogen and phosphorus, the strain was incubated with BBM medium. After filling 300ml medium, a portion of the strain (OD 680nm 0.01) was inoculated and used. During the incubation, a fluorescent light source of 100umol / m ² s was used and incubated at 28 ° C.

The concentration of nitrogen and phosphorus with initial concentration of 50mg / L was measured using an analysis kit (C-Mac, Korea) with an initial concentration of 50mg / L of nitrogen and phosphorus, and absorbance at 680nm with UV meter (Hach, USA). (Optical Density) was measured to determine the growth rate.

The experiment was repeated three times in all.

As a result (FIG. 2), the absorbance was increased, nitrogen and phosphorus was consumed and the new strain was growing.

Lipid content analysis

New strain neproselmis sp. Of the present invention well cultured in BBM medium for 21 days. KGE 8 is recovered and centrifuged at 3500 rpm in a swing type centrifuge (Hanil, Korea). After freeze-drying the centrifuged microalgae using a freeze dryer for 3 days, 500mg of the sample was collected and placed in a container for lipid extraction (with a teflon liner inserted into a glass container and a lid capable of centrifugation), followed by solvent chloroforum 1.25. m and 2.5 ml of methanol were added using a glass pipette. Thereafter, the sample to which the solvent was added to the microalgae was mixed well with a high speed stirrer for 5 minutes and crushed using a sonicator for 30 minutes to start extraction. The crushed microalgae were extracted with stirring (30 ° C., 150 rpm) for one day with a constant temperature water shaker under conditions of 150 rpm and 30 ° C. 1.25ml of chloroforum was added to the extracted sample using a glass pipette, and further stirred in a constant temperature shaker for 2 hours. 1.25ml of distilled water was added to the extracted sample by using a micropipette and centrifuged to separate an organic layer and a water layer. The organic layer (primary extract) is transferred to a new container (with a Teflon liner inserted into a centrifuged glass container and lid) using a Paspan pipette and centrifuged once more by adding 1.25 ml of distilled water to the remaining material. The organic layer (secondary extract) and the water layer were separated, and the organic layer was separated using a Paspan pipette, and the primary and secondary extracts were combined to obtain a lipid extract. 5 ml of NaCl (5%) was added to the lipid extract thus obtained, followed by centrifugation to transfer only the organic layer. The organic layer was evaporated using a rotary vacuum evaporator to separate pure lipids. The weight of the separated lipid was measured to yield a weight of 207 mg of lipid.

[Equation 1]

Total lipid content (%) = dry weight of lipid extract (mg) / microalgae dry weight (mg) X 100

 [Table 3]

Neproselmis sp. As a result of measuring the growth rate of KGE8, the amount of microalgae produced per day was 560 mg, and the lipid productivity was 228 mg / L day.

&Quot; (2) "

Lipid productivity (mg / L day) = daily production of microalgae (mg / L day) X microalgal lipid content (%)

As a result (Table 3, Fig. 3), the new strain neproselmis sp. KGE 8 was confirmed to exhibit a lipid content of 41% or more with respect to the dry weight.

Fatty Acid Extraction and Analysis

Fatty acid content and composition were determined by Lepage and Roy [Lepage, G., C.C. Roy (1984) Improved recovery of fatty acid through direct transesterification without prior extraction or purification, Journal of Lipid Research, 25, 1391-1396.

A fatty acid methyl ester mixture, FAME Quantitative Standard Mix 37 comps. [AccuStandard, USA] was used as a standard. Place a mass of microalgal lipid sample in a glass tube [11 mL, DH.GL28020, Daihan Scientific, Korea] with a Teflon stopper, and inject 2 mL of chloroform-methanol (2: 1, vol / vol) at room temperature. It was mixed for a minute with a vortex mixer (Vorex Genius 3. Ika, Italy).

1 mL (500 μg / L) of chloroform, 1 mL of methanol, and 300 μl of sulfuric acid containing nonadecanoic acid (Sigma Co., USA), an internal standard, are sequentially added to the glass tube, followed by a mixer for 5 minutes. Mixed into. The tube was placed in a constant temperature water bath and reacted at 100 ° C for 10 minutes. After the tube was cooled to room temperature, 1 mL of distilled water was injected, mixed vigorously with a mixer for 5 minutes, and centrifuged at 4,000 rpm for 10 minutes to separate layers. The lower layer (organic phase) was extracted with a disposable PP syringe (Norm-ject, Germany) and filtered with a disposable 0.22 μm PVDF syringe filter (Millex-Gv, Millipore, USA), followed by gas chromatography with an automatic injector [Model 7890 , Agilent, USA].

As a result (FIG. 4), with respect to total fatty acid content, palmitic acid (16: 0) 27.3 wt%, palmitoleic acid (16: 1) 2.9 wt%, oleic acid (18: 1 n9) 13.4 wt%, linole Iksan (18: 2n6) 5.9 wt% and g-linoleic acid (18: 3n6) 43.7 wt% were detected. Therefore, it may be provided as an index for producing high quality materials in the production of biodiesel in the future.

As described above in detail the specific parts of the present invention, it is apparent to those skilled in the art that such specific description is merely a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Korea Biotechnology Research Institute KCTC12235BP 20120703

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> NOVEL STRAIN CONTAINING HIGH CONC. OF LIPID <130> 12P363IND_K06210 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 859 <212> DNA <213> Nephroselmis sp. KGE8 <400> 1 agcggaggaa aagaaactac tagcgtggag tgagtcggga cgagcgaacc gggaagagcc 60 caacttgaaa atctggcagc ttcgctgtcc gaattgtagt ctaaagaagc gtcctctgta 120 gcggaccggg cccaagttcc ctggaatggg acgtcagaga gggtgagagc cccgtcgacc 180 ccggaccctg ctactccacg aggcgctgtc gccgagtcgg gttgtttggg aatgcagccc 240 taaatgggtg gtaaattcca tctaaggcta aatactggcg agagaccgat agcgaacaag 300 taccgcgagg gaaagatgaa aagaactttg aaaagagagt taaaagtgct tgaaattgtt 360 gagggggaag cgaatggaag cagaggtgcg cctcggtttt atgtgggggt tcgcgccccc 420 gcctatcaac gcgaggcgct ggtcagcgtg ggttagcctg gcgggaaaaa agcaggggtt 480 gttaccctgt ccatatcgcc gggctgaccg aggtctgaag ggcgcgcttc ggcgagcttc 540 ggcatctgcg ccctcaggac gctggcttaa tgcttccatc cggcccgtct tgaaacacgg 600 accaaggagt ctaacatgta tgcgagccgg tgggtggcaa acccatgagg cgcaagtaac 660 ctgattggtg ggattccttt tggatgcacc atcgaccgac catgatcttc tgtgaaaggt 720 ttgagtagga gtatacctgt tgggacccga aagatggtga actatgcctg agcagggcga 780 agccagagga aactctggtg gaggctcgta gcgatactga cgtgcaaatc gttcgtcaga 840 ctaatcgaac cttctagta 859

Claims (10)

Neproselmis sp. KGE8 (Nephroselmis sp. KGE8, Accession No. KCTC 12235BP) strain.
delete delete The method of claim 1,
The neproselmis sp. The strain is Nephroselmis pyriformis , Neproselmis sp. Strain.
delete The method of claim 1,
The neproselmis sp. The strain comprises the DNA sequence of SEQ ID NO: 1, neproselmis sp. Strain. A culture medium in which the strain of any one of claims 1, 4 and 6 is cultured. The method of culturing the strain of claim 1,
The strain is incubated in a condition of 25 to 30 ℃, pH 6 to 8, the culture method. 9. The method of claim 8,
The culture method is a culture method of culturing the strain in a medium containing KH ₂ PO ₄ , CaCl ₂ , MgSO ₄ , NaNO ₃ , K ₂ HPO ₄ , NaCl and H ₃ BO ₃ . The method of claim 9, wherein the medium further comprises one or more elements selected from the group consisting of ZnSO ₄ , MnCl ₂ , MoO ₃ , CuSO _4, and Co (NO ₃ ) ₂ .

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