WO2014021508A1 - Novel strain containing high lipid content - Google Patents

Abstract

The present invention relates to a high strain containing high lipid content. The novel strain of the present invention contains high lipid content, high fatty acid content, and in particular contains palmitic acid, and therefore, can be used effectively in the production of bio diesel. In addition, the novel strain of the present invention is separated from an acid mine and has heavy metal resistance. Furthermore, the novel strain of the present invention not only contains high lipid content, high fatty acid content, and in particular contains high palmitic acid content but also has high growth speed, and therefore, has excellent productivity.

Description

Novel strains containing high amounts of lipids

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.

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 (Nephroselmis sp.KGE8, accession number 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 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 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.

Example 1

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 5 and allowed to stand for 2 weeks. The composition of the solution constituting the BBM liquid medium is shown in Tables 1 to 4. 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 ℃, stirring speed 150rpm.

Table 1

Solution 1 (mg / L)
CaCl 2 * H 2 O	175 mg
MgSO 4 * 7H 2 O	25mg
NaNO 3	75mg
K 2 HPO 4	250 mg
NaCl	75mg
H 3 BO 3	25mg

TABLE 2

Solution 2 (mg / L)
Na 2 EDTA	50 g
KOH	31 g

TABLE 3

Solution 3 (mg / L)
FeSO 4	4.98 g
H 2 SO 4	1ml

Table 4

Microelement stock solution (g / L)
ZnSO 4 * 7H 2 O	8.82 g
MnCl 2 * 4H 2 O	1.44 g
MoO 3	0.71 g
CuSO 4 * 5H 2 O	1.57 g
Co (NO 3 ) 2 * 6H 2 O	0.49 g

Table 5

BBM constellation
Solution
1	1L
Solution
2	1ml
Solution 3	1ml
Microelement stock solution	1ml

Example 2

Isolated strain culture

Using the BBM medium of the composition shown in Table 5 10 mL of the new strain of the present invention in a 250 mL Erlenmeyer flask at 25 ℃, pH 7 and incubated for 14 days in a constant temperature shaker attached to a fluorescent lamp. Stirring was maintained at 150 rpm and roughness 50 μmol / m 2 · sec.

Example 3

Identification and homology comparison of isolates

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 is described in SEQ ID NO: 1, using the base sequence was prepared by referring to the NCBI information (Fig. 1).

In addition, as a result of analyzing the homology by performing a Blast search in NCBI using the nucleotide sequence, Table 6 was obtained.

Table 6

Microalgal strain	Accession number	Length (nt a )	Closest relative and Genebank accession number	Similarity (%)
Nephroselmissp. KGE8	HE561886	859	HE610144.1	89

As shown in Table 6, the new strain of the present invention was identified 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.

Example 4

Growth rate measurement of isolates

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.

Example 5

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). Centrifuged microalgae was lyophilized for 3 days using a lyophilizer to obtain 500 mg of dried microalgae. The dried microalgae were placed in a lipid extraction container (with a teflon liner inserted into a glass container and a lid capable of centrifugation), and then 1.25 m of solvent chloroforum 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 thus obtained lipid extract, followed by centrifugation to transfer only the organic layer. The organic layer was evaporated using a rotary vacuum evaporator to separate 207 mg of the dried lipid extract.

Dry lipid (207 mg) and microalgae dry weight (500 mg) of the lipid extract obtained above were substituted in Equation 1 below to obtain total lipid content (%) as shown in Table 7 and FIG. 3.

[Equation 1]

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

In addition, to measure the lipid productivity, the growth rate of the microalgae was measured during the period from 7 days to 17 days after the start of microalgae culture. During the above period, the culture solution containing the microalgae was taken 1 L daily, centrifuged and lyophilized, and the difference in weight from the previous day's dry weight was calculated. As a result, the growth rate of the microalgae of the present invention was 560 mg / L day. The lipid productivity of the microalgae of the present invention was calculated by substituting the growth rate and the total lipid content obtained in Equation 1 into Equation 2, and the results shown in Table 7 were obtained.

[Equation 2]

Lipid productivity (mg / L day) = Growth rate (mg / L day) × total lipid content (%)

TABLE 7

Microalgal strain	Growth rate (mg / L day)	Lipid Productivity (mg / L day)	Total lipid content (% lipid / biomass)
Nephroselmissp. KGE8	560	231.8	41.4

Example 6

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. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

[Accession number]

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC 12235BP

Deposited Date: July 9, 2012

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.

[Microbial deposit]

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY

<120> NOVEL STRAIN CONTAINING HIGH CONC. OF LIPID

<130> OF12P245PCT

<150> KR 10-2012-0084922

<151> 2012-08-02

<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)

1. High in lipid, neproselmis sp. KGE8 ( Nephroselmis sp. KGE8, Accession No. KCTC 12235BP) strain.
2. Neproselmis sp. Comprising at least 35% by weight of lipids relative to the total weight of the dry strain. ( Nephroselmis sp.) Strain.
3. The method of claim 2, wherein the neproselmis sp. Four strains Pro cell Miss KGE8 (Nephroselmis sp. KGE8) of, four Pro cell Miss sp. Strain.
4. The method of claim 2, wherein the neproselmis sp. The strain is Nephroselmis pyriformis , Neproselmis sp. Strain.
5. The method of claim 2, wherein the neproselmis sp. Four strains Pro cell Miss KGE8 (Nephroselmis sp.KGE8, Accession No. KCTC 12235BP) of, four Pro cell Miss sp. Strain.
6. The method of claim 2, wherein the neproselmis sp. The strain comprises the DNA sequence of SEQ ID NO: 1, neproselmis sp. Strain.
7. A culture medium in which the strain of any one of claims 1 to 6 was cultured.
8. The method of culturing the strain of any one of claims 1 to 6,

   The strain is incubated in a condition of 25 to 30 ℃, pH 6 to 8, the culture method.
9. The method of claim 8, wherein the culturing method comprises any one of claims 1 to 6 in a medium containing KH ₂ PO ₄ , CaCl ₂ , MgSO ₄ , NaNO ₃ , K ₂ HPO ₄ , NaCl, and H ₃ BO ₃ . Culture method of culturing one strain.
10. 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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