KR101680048B1 - Novel strain of nephroselmis sp. kge2 and method for increasing fatty acid concentration in the strain - Google Patents

Abstract

The present invention relates to a novel neprose misantigen strain and a method for increasing the content of fatty acid in a strain using the same.

Description

[0002] NOVEL STRAIN OF NEPHROSELMIS SP. ≪ RTI ID = 0.0 > KGE2 < / RTI > KGE2 AND METHOD FOR INCREASING FATTY ACID CONCENTRATION IN THE STRAIN}

The present invention relates to a novel neprose misantigen strain and a method for increasing the content of fatty acid in a strain using the same.

The use of fossil fuels due to high industrialization has been pointed out as a main cause of global warming by emitting a large amount of greenhouse gas. In addition, the use of excessive fossil fuels is depleting oil. Many researchers at home and abroad are developing alternate energy as an alternative to fossil fuels. Alternative energy sources can include renewable energy, including solar, bio, wind, hydro, marine, waste, geothermal, fuel cells, and hydrogen. Among them, bioenergy can be used as a raw material for biodiesel, and the production per unit area is very superior to other raw materials.

Conventionally, soybeans, sugar cane, etc. have been used as energy sources for bioenergy. However, the production efficiency of biodiesel for the vast area required for cultivation thereof is low and it is facing difficulties. Therefore, it is necessary to study microalgae with biomass for stable and economical production of biodiesel.

However, to date, microalgae research for biofuel production has been limited to microalgae inhabiting the oceans, which requires the introduction of artificial minerals during culture and may cause problems with the transport of microalgae that are not dried from the oceans .

&Quot; Removal of metal from acid mine drainage using a hybrid system including a microalgae reactor ", Park Young-Tae et al., Bioresource Technology, Vol.150, Pages 242-248, 2013

It is an object of the present invention to provide novel strains and methods for increasing the fatty acid content in such strains.

To achieve the above object, one aspect of the present invention provides a strain of Nephroselmis sp. KGE2 (KGE2).

One aspect of the present invention also provides a method for increasing the fatty acid content in a strain, comprising culturing the strain and the wastewater together.

The novel strain of the present invention has a high growth rate and can contain lipids and fatty acids in a high content and can be usefully used for producing biodiesel. Furthermore, the novel strain of the present invention is isolated from acidic mines and has resistance to heavy metals, so that its activity can be maintained in wastewater. In addition, the novel strain of the present invention has an advantage that it can grow using minerals in wastewater by maintaining its activity in harsh environments such as wastewater.

In addition, the novel strain of the present invention can maximally increase the content of fatty acids when cultured together with mine drainage and wastewater, and thus can be used for producing biodiesel and can be used for producing alternative energy. In addition, the novel strains of the present invention comprise a high content of lipids and thus are associated with high levels of fatty acids such as palmitic acid, palmitoleic acid, oleic acid, linoleic acid (18: 2n6t), or g-linoleic acid (18: 3n6) in a high content, and thus can be usefully used.

1 is a microphotograph (1500 magnification) of Nefrocell missus KGE 2 (Nephroselmis sp. KGE2).
Fig. KGE 2 is a systematic diagram.
FIG. 3 is a graph showing the growth rate of KGE 2 in the necrosal sponge after treating mine drainage and livestock wastewater at various weight ratios.
FIG. 4 is a graph showing the results obtained by treating mine drainage and livestock wastewater at various weight ratios, The content of fatty acids in KGE 2 is shown.
FIG. 5 is a chart showing the content of fatty acid in the Nexrocellulosic KGE 2 after measuring mine drainage and livestock wastewater at various weight ratios.

In one aspect, the present invention relates to a strain of Nephroselmis sp. KGE2. The Nephroselmis sp. KGE2 strain, which is one aspect of the present invention, was isolated and identified by the present inventors for the first time. The present inventors named the new strain identified above as a strain of Nephroselmis sp. KGE2 (KGE2), and deposited it on the deposit number (KCTC 12456BP) on July 30, 2013 with the Korea Research Institute of Bioscience and Biotechnology, . KGE stands for KIST Gangneung Enviromental.

In one aspect of the present invention, the strain may comprise the nucleotide sequence of SEQ ID NO: 1.

A strain that is an aspect of the present invention may comprise at least 30 wt% lipid based on the total weight of the dry strain. The lipid content in general microalgae is difficult to exceed 20%. However, the present invention contains more than 30% of lipids and contains a large amount of fatty acid, and can be utilized as biomass. In view of the above, the strain of Nephroselmis sp. KGE2 according to the present invention contains 30.2 wt% or more, 30.4 wt% or more, 30.6 wt% or more, 30.8 wt% or more, 31 wt% or more, 31.2 wt% , 31.4 wt% or more, 31.6 wt% or more, 31.8 wt% or more, 32 wt%, 32.2 wt%, 32.4 wt%, 32.6 wt%, 32.8 wt% or 33 wt% or more.

In one aspect of the present invention, the strain may be the accession number KCTC 12456BP.

In one aspect of the present invention, the strain may include a high amount of lipid and fatty acid as described above. In addition to the palmitic acid that can be used as biomass, a strain such as palmitoleic acid, heptadichenoic acid Heptadecenoic acid, oleic acid, linolelaidic acid, and the like. Among them, palmitic acid is particularly suitable as a fuel for tropical climate regions because of its high oxidation stability and high cetane number (ease of ignition of fuel).

In another aspect, the present invention relates to a culture solution of a strain of Nexrocellulosus KGE2. The culture solution contains a product obtained by culturing a strain of Mycobacterium sp. KGE2, and has a constitution of being contained in a normal culture liquid.

In another aspect, the present invention relates to a culture method comprising the step of culturing a non-native Four Celec KGE2 strain. Specifically, the culturing method may include culturing the meprocellulosus KGE2 strain at 25 to 30 DEG C, pH 6 to 8, and the growth rate of the new strain of the present invention may be high under the above conditions . From the above viewpoint, the strain can be cultured at a temperature of 25.5 to 29.5 DEG C, 26 to 29 DEG C or 26.5 to 28.5 DEG C and can be cultured under the conditions of pH 6.2 to 7.8, pH 6.4 to 7.6 or pH 6.4 to 7.4 have. More specifically, in the method of culturing which is one aspect of the present invention, the culturing method comprises culturing in a medium containing KH ₂ PO ₄ , CaCl ₂ , MgSO ₄ , NaNO ₃ , K ₂ HPO ₄ , NaCl and H ₃ BO ₃ , And culturing the non-neprose missense KGE2 strain.

In one aspect of the present invention, the culture medium may further comprise at least one element selected from the group consisting of ZnSO ₄ , MnCl ₂ , MoO ₃ , CuSO ₄ and Co (NO ₃ ) ₂ .

The present invention, in another aspect, relates to a method for increasing the fatty acid content in a Neocrocellulosum KGE2 strain, comprising culturing a Nephroselmis sp. KGE2 strain with mine drainage and wastewater .

Specifically, the wastewater includes wastewater containing livestock manure, domestic wastewater, and the like. In addition, the mine drainage may include, but is not limited to, acid mine drainage. In this specification, the livestock manure may mean a livestock manure containing a large amount of N, P.

In one aspect of the present invention, there is provided a method for increasing fatty acid content in a strain, wherein the wastewater is at least one selected from the group consisting of wastewater containing livestock manure and domestic wastewater.

In one aspect of the present invention, a method for increasing fatty acid content in a strain, the weight ratio between the mine drainage and the wastewater may be 0.5 to 2.5: 1. The production of fatty acids is most excellent within the above range. In view of the above, the weight ratio between the mine drainage and the wastewater may be 0.6 to 2.4: 1, 0.7 to 2.3: 1, 0.8 to 2.2: 1, 0.9 to 2.1: 1 or 1 to 2:

In one aspect of the present invention, a method for increasing the fatty acid content in a strain, the strain may be the accession number KCTC 12456BP.

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

[ Experimental Example  One]

New strain  Isolation and Culture

Samples were taken from acidic mine drainage in Taebong abandoned mine area in Gangneung, Gangwon Province. Samples were taken from sterilized water quality sample packs (1 L). Soil and water quality were collected, stored at low temperature in an icebox, and transferred to the laboratory. A portion of the collected samples was separated, washed with sterilized DIW, and centrifuged three times to obtain a new strain. The isolated new strain samples were observed through an optical microscope at magnification of 1500 times.

The new strain was placed in a BBM liquid medium having the same composition as shown in Table 1 and cultured for 2 weeks. The BBM liquid medium can provide an environment in which microalgae can be optimally grown. The composition of the microelement stock solution and the solution 1 and the solution 2 constituting the composition of the BBM liquid medium are shown in Table 2. A 36-watt fluorescent light source was used as a light source. Two weeks after the culture, a single colony was collected and spread uniformly on platinum-coated alar plate (15 g / L) containing BBM. After 8 days, the cultured colonies were secondly plated on alar plates containing BBM. After 12 days, the cultured colonies were collected and the microalgae were dispersed in an Erlenmeyer flask containing a liquid medium containing BBM. And microalgae are cultured using a thermostatic horizontal shaker equipped with a fluorescent lamp. At this time, the culture conditions were temperature of 25 ± 1 ° C and stirring speed of 150 rpm.

Constituent content KH ₂ PO ₄ 175 mg / L CaCl ₂ * H ₂ O 25 mg / L MgSO ₄ * 7H ₂ O 75 mg / L NaNO ₃ 250 mg / L K ₂ HPO ₄ 75 mg / L NaCl 25 mg / L H ₃ BO ₃ 11.42 mg / L Microelement stock solution 1 ml Solution 1 1 ml Solution 2 1 ml

Microelement stock solution Solution 1 Solution 2 Constituent content Constituent content Constituent content ZnSO ₄ * 7H ₂ O 8.82 g / L Na ₂ EDTA 50 g / L FeSO ₄ 4.98 g / L MnCl ₂ * 4H ₂ O 1.44 g / L MoO ₃ 0.71 g / L KOH 3.1 g / L H ₂ SO ₄ 1 ml / L CuSO ₄ * 5H ₂ O 1.57 g / L _{Co (NO 3) 2 * 6H} 2 O 0.49 g / L

[ Experimental Example  2]

Isolate  culture

Using a BBM medium having the composition shown in Table 1, 10 mL of the new strain of the present invention was added to a 250 mL Erlenmeyer flask at 25 ° C and pH 7, and cultured for 14 days on a thermostatic shaker equipped with a fluorescent lamp. Stirring was maintained at 150 rpm and the illuminance was maintained at 50 μmol / m 2 -sec.

[ Experimental Example  3]

Isolate  Identification and Homology  compare

Sequence analysis of the new strain, Nephroselmis sp. KGE2, was performed by 28s rRNA sequencing.

A portion of the microalgae was collected and used as an analytical sample by centrifugation. The microalgae were extracted using a DNA extraction kit (SolGent, Korea), and the extracted DNA was amplified by PCR. The primer was 28S D1D2.

Forward primer: 5'-AGCGGAGGAAAAGAAACTA-3 '

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

The resulting nucleotide sequence is shown in SEQ ID NO: 1, and the nucleotide sequence was used to generate a gene flow diagram based on NCBI information (FIG. 2).

[SEQ ID NO: 1]

AGCGGAGGAAAAGAAACTAACAAGGATTCCCTCTAGTAACGGCGAGCGAACCGGGAAGAGCCCAACTTGAAAATCTGGCAGCTTCGCTGTCCGAATTGTAGTCTAAAGAAGCGTCCTCTGTAGCGGACCGGGCCCAAGTTCCCTGGAATGGGACGTCAGAGAGGGTGAGAGCCCCGTCGACCCCGGACCCTGCTACTCCACGAGGCGCTGTCGCCGAGTCGGGTTGTTTGGGAATGCAGCCCTAAATGGGTGGTAAATTCCATCTAAGGCTAAATACTGGCGAGAGACCGATAGCGAACAAGTACCGCGAGGGAAAGATGAAAAGAACTTTGAAAAGAGAGTTAAAAGTGCTTGAAATTGTTGAGGGGGAAGCGAATGGAAGCAGAGGTGCGCCTCGGTTTTATGTGGGGGTTCGCGCCCCCGCCTATCAACGCGAGGCGCTGGTCAGCGTGGGTTAGCCTGGCGGGAAAAAAGCAGGGGTTGTTACCCTGTCCATATCGCCGGGCTGACCGAGGTCTGAAGGGCGCGCTTCGGCGAGCTTCGGCATCTGCGCCCTCAGGACGCTGGCTCAATGCTTCCATCCGGCCCGTCTTGAAACACGGACCAAGGAGTCTAACATGTATGCGAGCCGGTGGGTGGCAAACCCATGAGGCGCAAGTAACCTGATTGGTGGGATTCCTTTTGGATGCACCATCGACCGACCATGATCTTCTGTGAAAGGTTTGAGTAGGAGTATACCTGTTGGGACCCGAAAGATGGTGAACTATGCCTGAGCAGGGCGAAGCCAGAGGAAACTCTGGTGGAGGCTCGTAGCGATACTGACGTGCAAATCGTTCGTCAGACTTGGGTATAGGGGCGAAAGACTAATCGAACCTTCTAGTA

In addition, Blast search was performed on NCBI with the above base sequence, and the homology was analyzed. As a result, Table 3 was obtained.

Microalgal strain Accession number Length (nt ^a ) Closest relative and
GenBank accession number Similarity (%) Nephroselmis sp. KGE2 HE861888 882 HE610144.1 89

Therefore, the present inventors have named the new strain identified above as Neisseria sp. KGE 2 ( Nephroselmis sp. KGE2) and deposited it on August 19, 2013 with the microorganism resource center of the Korea Research Institute of Bioscience & Biotechnology as deposit number KCTC 12456BP .

[ Experimental Example  4]

Identification of the growth of the strain by culture composition

Under the conditions of 25 ° C and pH 7, an Erlenmeyer flask was charged with a mixture of livestock wastewater and acidic mine drainage having the composition shown in Table 4 and 10 ml of Nephroselmus KGE 2 { Nephroselmis sp. KGE2} [KCTC 12456BP] were added to each well and cultured for 21 days on a thermostatic shaker equipped with a fluorescent lamp. Stirring was maintained at 150 rpm and the light intensity was maintained at 120 photos μmol / m 2 -sec. EDTA was added to prevent precipitation of iron.

Experimental group Sample composition BBM MBR Mine drainage Mine drainage + EDTA Distilled water Comparative Example 1 olny MBR 0 10 0 0 90 Comparative Example 2 BBM 10 0 0 0 0 Example 1 MBR + 5% AMD 0 10 5 0 85 Example 2 MBR + 10% AMD 0 10 10 0 80 Example 3 MBR + 5% AMD + EDTA 0 10 0 5 85 Example 4 MBR + 10% AMD + EDTA 0 10 0 5 80

MBR: livestock wastewater Primary raw water from which raw water was passed through the membrane to remove suspended matter

AMD: Acid Mine Drainage

EDTA: Eethylene Diamine Tetra Acetic acid

The KGE2 strains of the non-necropsy strains were cultured under the conditions as shown in Table 4, respectively, and then dried weight was measured. As can be seen from FIG. 3, when the strain was cultured using the wastewater and mine drainage together, it was confirmed that the growth of the strain was the best.

[ Experimental Example  5]

Lipid content analysis

The strain KGE2 was cultured for 21 days, and the strain was recovered and centrifuged at 3500 rpm in a swing type centrifuge (Hanil, Korea). The centrifuged microalgae were lyophilized for 3 days using a freeze dryer, 500 mg of which was collected and placed in a container for lipid extraction (a centrifugeable glass container and a lid with a teflon liner inserted therein) 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 the extraction was started by shredding using a sonicator for 30 minutes. The shredded microalgae were extracted with stirring (30 ° C, 150 rpm) for one day on a constant-temperature shaker at 150 rpm and 30 ° C. 1.25 ml of chloroprene was added to the extracted sample using a glass pipette, and further stirred for 2 hours on a thermostatic horizontal shaker. 1.25 ml of distilled water was added to the extracted sample using a micropipette, followed by centrifugation to separate the organic layer and the water layer. The organic layer (primary extract) was transferred to a new container (with a teflon liner inserted into a centrifugeable glass container and lid) using a Paschen pipette, and 1.25 ml of distilled water was added to the remaining material and centrifuged once more. The organic layer (secondary extract) and water layer were separated, and the organic layer was separated using a Paschen pipette to obtain a lipid extract by combining the first extract and the second extract. To the lipid extract thus obtained, 5 ml of NaCl (5%) was added and mixed. After centrifuging, only the organic layer was transferred, and the organic layer was evaporated using a rotary vacuum evaporator to separate the pure lipids. Thereafter, the dry weight of the pure lipid extract, the weight of the microalgae cultured, and the dry weight thereof were measured and substituted into the following equations (1) and (2) to obtain Table 5 below.

[Equation 1]

Lipid content (%) = dry weight of lipid extract (g) / dry weight of microalgae (g) X 100

 &Quot; (2) "

Lipid productivity (g / L) = microalgae production (g / L) X microalgae lipid content (%)

The results obtained by the above equations are shown in Table 5 below.

Experimental group
Production of microalgae (Biomass
, g / L) Lipid content
(Lipid contents,%) Lipid productivity
(Lipid productivity, g / L) KGE 2 Comparative Example 1 0.447 + 0.062 29.89 ± 3.48 0.133 + 0.018 Comparative Example 2 0.411 + 0.050 33.11 + -1.04 0.136 ± 0.001 Example 1 0.587 + 0.025 22.19 ± 1.03 0.0.130 ± 005 Example 2 0.580 ± 0.059 20.95 ± 1.49 0.121 + - 0.012 Example 3 0.513 + 0.034 20.78 ± 0.26 0.106 ± 0.007 Example 4 0.535 0.019 23.85 ± 2.71 0.084 ± 0.004

As shown in Table 5, when the strain was cultured using the wastewater and the mine drainage together, it was confirmed that the productivity of the strain was the most excellent.

[ Experimental Example  6]

Fatty acid extraction and content 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.

FAME Quantitative Standard Mix 37 comps. [AccuStandard, USA], a mixture of fatty acid methyl esters, was used as a standard. A microalgae lipid sample, which had been mass-measured, was added to a glass tube [11 mL, DH.GL28020, Daihan Scientific, Korea] with a teflon stopper and 2 mL of chloroform-methanol (2: 1, vol / vol) Minute vortex mixer [Vorex Genius 3. Ika, Italy].

1 mL (500 μg / L) of chloroform containing an internal standard substance nonadecanoic acid (Sigma Co., USA), 1 mL of methanol, and 300 μL of sulfuric acid were sequentially added to a glass tube, Lt; / RTI > The tube was placed in a constant-temperature water bath and reacted at 100 ° C for 10 minutes. After cooling the tube to room temperature, 1 mL of distilled water was poured, mixed vigorously for 5 minutes with a mixer, 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 analyzed by gas chromatography [Model 7890 (Millex-Gv, Millipore, USA) , Agilent, USA].

The necrosol KGE 2 was cultured for 21 days, and recovered and used. The distribution of fatty acids in each case was plotted in FIG. 4 for each type of fatty acid. As can be seen from Fig. 4, the contents of oleic acid (C18: 1 n9c) and palmitic acid (16: 0), which are the fatty acids which can be most stably used for the production of biodiesel of microalgae, Was found to be the highest.

The results are shown in Table 4. The results are shown in Table 4. The results are shown in Tables 4 and 5, and the results are shown in Table 5. [Table 4] < EMI ID = The results showed that the total amount of fatty acids increased rapidly when the strain was cultured using mine drainage. In addition, it was confirmed from Table 4 that the total amount of fatty acids increased sharply even under the condition that the lipid content and seismic productivity were deteriorated, and thus it was confirmed that the strain could be used as a biomass having excellent efficiency there was.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this 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 KCTC12456BP 20130730

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> NOVEL STRAIN OF NEPHROSELMIS SP. KGE2 AND METHOD FOR INCREASING          FATTY ACID CONCENTRATION IN THE STRAIN <130> 13P531IND_K06729 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 882 <212> DNA <213> NEPHROSELMIS SP. KGE2 <400> 1 agcggaggaa aagaaactaa caaggattcc ctctagtaac ggcgagcgaa ccgggaagag 60 cccaacttga aaatctggca gcttcgctgt ccgaattgta gtctaaagaa gcgtcctctg 120 tagcggaccg ggcccaagtt ccctggaatg ggacgtcaga gagggtgaga gccccgtcga 180 ccccggaccc tgctactcca cgaggcgctg tcgccgagtc gggttgtttg ggaatgcagc 240 cctaaatggg tggtaaattc catctaaggc taaatactgg cgagagaccg atagcgaaca 300 agtaccgcga gggaaagatg aaaagaactt tgaaaagaga gttaaaagtg cttgaaattg 360 ttgaggggga agcgaatgga agcagaggtg cgcctcggtt ttatgtgggg gttcgcgccc 420 ccgcctatca acgcgaggcg ctggtcagcg tgggttagcc tggcgggaaa aaagcagggg 480 ttgttaccct gtccatatcg ccgggctgac cgaggtctga agggcgcgct tcggcgagct 540 tcggcatctg cgccctcagg acgctggctc aatgcttcca tccggcccgt cttgaaacac 600 ggaccaagga gtctaacatg tatgcgagcc ggtgggtggc aaacccatga ggcgcaagta 660 acctgattgg tgggattcct tttggatgca ccatcgaccg accatgatct tctgtgaaag 720 gtttgagtag gagtatacct gttgggaccc gaaagatggt gaactatgcc tgagcagggc 780 gaagccagag gaaactctgg tggaggctcg tagcgatact gacgtgcaaa tcgttcgtca 840 gacttgggta taggggcgaa agactaatcg aaccttctag ta 882

Claims (8)

delete delete delete delete Comprising culturing a strain of Neisseria sp. KGE2 ( Nephroselmis sp. KGE2) with an acidic mine drainage and wastewater, wherein the strain comprises at least 20% by weight of lipase relative to the total weight of the dry strain, A method for increasing the fatty acid content in the genus KGE2.
6. The method of claim 5,
Wherein the wastewater comprises at least one selected from the group consisting of wastewater containing domestic animal manure and domestic wastewater.
The method according to claim 5 or 6,
Wherein the weight ratio between the acid mine drainage and the wastewater is 0.5 to 2: 1.
The method according to claim 5 or 6,
Wherein said strain is the accession number KCTC 12456BP.

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