KR101496783B1 - Novel Microalgae Chlamydomonas reinhardtii KNUA021 and method for producing fatty alcohol and fatty acid from the same - Google Patents

Abstract

The present invention relates to a novel microalgae Clamidomonas reinhardtii KNUA021 strain (KCTC 12066BP) and to a process for the production of hexadecenol and C ₁₆ to C ₁₈ fatty acids therefrom. The Kramidomonas reinhardtii KNUA021 strain (KCTC 12066BP) of the present invention can produce hexadecenol (C ₂₀ H ₄₀ O) and C ₁₆ to C ₁₈ fatty acids which are high-value-added fatty alcohols. In addition, the biodiesel can be produced using the KNUA021 strain.

Description

[0001] The present invention relates to a novel microalgae Clamidomonas reinhardtii KNUA021 strain and a method for producing fatty alcohols and fatty acids from the microalgae strains KNUA021 and method for producing fatty alcohols and fatty acids from the same,

The present invention relates to a novel microalgae Clamidomonas reinhardtii KNUA021 strain and a process for producing hexadecenol and C ₁₆ to C ₁₈ fatty acids therefrom.

Humanity is faced with the dual challenges of global warming and depletion of energy sources through the use of indiscreet fossil fuels, and global demand for renewable and sustainable energy resources is increasing exponentially every year. In Korea, we are actively developing new and renewable energy, but it is still in the beginning stage. Unlike fossil energy, bio-energy is an environmentally friendly energy that does not generate carbon dioxide. It is a process of photosynthesis that captures carbon dioxide and solar energy, and produces organic carbon compounds that accumulate energy, thus extracting energy from the stored biomass.

Bioenergy is a non-toxic energy that is renewable and has almost no toxicity, is completely decomposed by microorganisms and is produced by fixing carbon dioxide, thus reducing the greenhouse effect. Currently, bio-energy research using land resources is underway, but the policy and research to utilize microalgae have been started all over the world as limitations of using land resources such as food resources, food cultivation area and competition are revealed.

Bio-energy is typically bioethanol / butanol and biodiesel. Research on the production of bioethanol / butanol around macroalgae is underway, and bio-diesel research is being conducted mainly on microalgae .

Biodiesel is produced in the form of methyl ester or ethyl ester by transesterification of fatty acids contained in biomass, and glycerol is produced as a by-product. Biodiesel, unlike petrodiesel, is an eco-friendly energy with significantly lower toxic emissions when burned.

The solar energy efficiency of microalgae is 5%, which is about 25 times higher than that of 0.2% of the land plants and the carbon dioxide immobilization rate is 15 times higher than that of pine trees. At present, microalgae are used not only as feeds and biological fertilizers but also as various health foods. Microalgae are microscopic in size and have similar photosynthetic mechanisms to land plants, but access to water, carbon dioxide and other nutrients is more effective in converting solar energy into biomass because it lives in an efficient underwater environment.

Recently, research on the production of biodiesel using microalgae has been actively conducted overseas. In December 2007, Shell, a world-renowned oil company, and HR Biopetroleum jointly installed a microalgae culture facility in Hawaii for the production of biodiesel. In January 2008, Solazyme of the United States launched a prototype called Soladiesel in Mercedes-Benz Diesel vehicles to test performance, and Chevron agreed with Solazyme to develop joint technology. In recent years, France has been actively researching the use of algae by allocating a budget of 2.8 million euros for three years to the Shamash program. The Shamash program is composed of seven research teams of French universities for the development of feasible production models by the National Research Agency. We are working on the production of fatty acids from algae and transforming them into bioenergy.

In Korea, we are conducting researches on biodiesel production process technology and biodiesel energy extraction, focusing on small and medium-sized companies. We are currently conducting research on biodiesel production using algae.

Microalgae containing cyanobacteria have been reported to produce various lipids, hydrocarbons, fatty alcohols and other complex oils (Metzger and Largeau 2005; Guschina and Harwood 2006; Schirmer et al 2010; Tan et al 2011). Although not all microalgae are suitable for biodiesel production, some microalgae strains have shown potential for use as biodiesel fuel (Chisti 2007). Chlamydomonas reinhardtii is one of the most widespread microalgae model systems, with desirable characteristics as a biofuel source. Chlamydomonas reinhardtii has been shown to accumulate lipids that can be used in biodiesel production in response to nitrogen deficiency (Riekhof et al . 2005; Wang et al . 2009; Siaut et al . 2011) lane hareuti (Sato 2000, etc.), which is also known bayida sikindaneun promote the total lipid content in the (Sato 2000, etc.). Metabolic engineering of the lipid production pathways has also been extensively studied using model systems strains of C. elegans (Li, Y. , 2010; Work , 2010; Siaut et al . , 2011). However, studies focusing on the potential for the biofuel candidates of the Clamidomonas Reinharti natural strain are relatively few compared to the model system Clamidomonas Reinhardt studies.

Therefore, the present inventors extracted asymmetrically the clamidomonas reinhardtii KNUA021 strain from the groundwater in the Chilgok Agricultural Technology Center. The KNUA021 strain was found to be not only hexadecenol (C ₂₀ H ₄₀ O) but also fine Showed that it is possible to produce algae biodiesel components autonomously, thus showing the possibility of biodiesel as raw material.

An object of the present invention aims to provide a novel micro-algae Chlamydomonas lane hareuti KNUA021 strain to produce fatty acids and C ₁₆ to C senol hexahydro to _18. The strain KNUA021 contains a mixture of fatty alcohols (hexadecenol (C ₂₀ H ₄₀ O)) and other common microalgae biodiesel components (palmitic acid (C _{16: 0} ), hexadecatetraenoic acid (C _{16: 4} ) , Linoleic acid (C _{18: 2} ) and linolenic acid (C _{18: 3} )) as major fatty acids.

It is another object of the present invention to provide a method for producing hexadecenol and a C ₁₆ to C ₁₈ fatty acid from the above-mentioned novel Clamidomonas reinhardtii KNUA021 strain.

Furthermore, the present invention relates to a method for producing biodiesel, which comprises transesterifying a C ₁₆ to C ₁₈ fatty acid produced in the above-mentioned Chlamydomonas reinhardtii strain KNUA021 (KCTC 12066BP) to produce a fatty acid ester And the like.

The present invention provides a microalgae, Chlamydomonas reinhardtii KNUA021 strain (KCTC 12066BP), which produces hexadecenol and C ₁₆ to C ₁₈ fatty acids.

In addition, the present invention is to hexa which comprises culturing the Chlamydomonas lane hareuti (Chlamydomonas reinhardtii) KNUA021 strain (KCTC 12066BP), and hexahydro to separate the senol and C ₁₆ to fatty acid of C ₁₈ from the culture medium senol and To provide a method for producing fatty acids of C ₁₆ to C ₁₈ .

The present invention also relates to a method for transesterifying a C ₁₆ to C ₁₈ fatty acid produced in the above-mentioned Chlamydomonas reinhardtii strain KNUA021 (KCTC 12066BP) to produce a fatty acid ester and glycerol, Of the present invention.

The present invention relates to a process for the production of high-value-added fatty alcohols hexadecenol (C ₂₀ H ₄₀ O) and C ₁₆ to C ₁₈ fatty acids using a novel microalgae Chlamydomonas reinhardtii strain KNUA021 (KCTC 12066BP) . In addition, the biodiesel can be produced using the KNUA021 strain.

Brief Description of the Drawings Fig. 1 is a diagram showing microscopic observation results of a strain of Chlamydomonas reinhardtii KNUA021 (KCTC 12066BP).
FIG. 2 is a graph showing the difference in growth of the Chlamydomonas reinhardtii strain KNUA021 (KCTC 12066BP) according to the temperature condition.
FIG. 3 is a graph showing the difference in growth according to the pH condition of the Chlamydomonas reinhardtii strain KNUA021 (KCTC 12066BP).
FIG. 4 is a graph showing the difference in growth according to the saltiness condition of the Chlamydomonas reinhardtii KNUA021 strain (KCTC 12066BP).
FIG. 5 is a graph showing the GC / MS analysis results of the Chlamydomonas reinhardtii strain KNUA021 (KCTC 12066BP).
6 is a diagram showing the electron impact (EI) mass spectrum of hexadecenol produced by the strain Chlamydomonas reinhardtii KNUA021 (KCTC 12066BP).

The present invention provides a microalgae, Chlamydomonas reinhardtii KNUA021 strain (KCTC 12066BP), which produces hexadecenol and C ₁₆ to C ₁₈ fatty acids.

The above-mentioned Chlamydomonas reinhardtii strain KNUA021 (KCTC 12066BP) was collected at groundwater (36 ° 02'N, 128 ° 22'E) at Chilgok Agricultural Technology Center in August 2011, and then fractionated on the agar streaking) were selected and identified through sequencing of 18S rRNA and 28s rRNA genes. The Chlamydomonas reinhardtii KNUA021 strain (KCTC 12066BP) was deposited with KCTC on Nov. 9, 2011 (deposit number: KCTC 12066BP).

The above Chlamydomonas reinhardtii strain KNUA021 (KCTC 12066BP) produces a high value-added fatty alcohol, preferably hexadecenol (C ₂₀ H ₄₀ O). In addition, the above-mentioned Chlamydomonas reinhardtii strain KNUA021 (KCTC 12066BP) is a major fatty acid having 16 carbon atoms and having a double bond number of 0 or 4 (16: 0, 16: 4 fatty acid) (18: 2, 18: 3 fatty acid) having a binding number of 2 or 3.

In addition,

1) culturing Clamidomonas reinhardtii KNUA021 strain (KCTC 12066BP);

It provides hexahydro to senol and C ₁₆ and C ₁₈ fatty acids in the production method of containing: 2) Hex to senol and C ₁₆ and the step of extracting the fatty acids of C ₁₈ from the culture broth obtained in the above 1) step.

As the growth conditions of the strain, the culture temperature is 15 ° C at 30 ° C and the culture pH is 4.0 to 12 ° C. The culture medium for the strain may be a medium commonly used in the art.

Any method known in the art can be used as a method for extracting hexadecenol and C ₁₆ and C ₁₈ fatty acids from the culture broth. The extraction solvent may be selected from the group consisting of pentane, nucleic acid, cyclohexane, toluene, methylene chloride, ethyl acetate, acetone, chloroform, methanol and mixed solvents thereof.

In addition, the present invention can produce biodiesel by transesterifying the triglyceride produced from Clamidomonas reinhardtii KNUA021 (KCTC 12066BP) to separate the fatty acid ester and glycerol. The fatty acid ester may preferably be a fatty acid methyl ester or a fatty acid ethyl ester, but is not limited thereto. The biodiesel can be produced in the form of a methyl ester or an ethyl ester by transesterifying the fatty acid contained in the biomass. The transesterification process refers to a method of transforming a large branched molecular structure of fat contained in biomass into a small, linear chain molecule required by a regular diesel engine. As the emulsifier in the transesterification process, Triton X-100 or Tween 60 may be added, but the present invention is not limited thereto. The emulsifier can be used to stabilize the interface of the bio-oil and mix well, thereby increasing the reaction yield and thus saving the biodiesel recovery cost. In addition, sodium hydroxide or potassium hydroxide may be used as a reaction catalyst in the transesterification process, but the present invention is not limited thereto.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

<Examples>

1. Sampling and separation of clamidomonas

Clamidomonas samples were collected from groundwater (36 ° 02'N, 128 ° 22'E) at Chilgok County Agricultural Technology Center in August 2011. Then, the collected samples were taken to a laboratory, and 1 ml of the sample was added to 100 ml of BG-11 containing meropenem (Meropenem, Yuhan Pharmaceuticals, Ochang-eup, Chungcheongbuk-do, South Korea) (+) Medium (containing nitrogen) (Rippka et al ., 1979). Flasks were incubated at 25 ° C with shaking at 160 rpm, until cultured biomass was observed.

Well-cultured medium (1.5 ml) was centrifuged at 3,000 g for 15 minutes (Centrifuge 5424, Eppendorf, Hamburg, Germany). The centrifuged pellets were fractionated on BG-11 agar and cultured at 25 캜 in a light-dark cycle (16: 8 hours). A single colony was then aseptically inoculated onto fresh BG-11 plates containing meropenem (20 ug / ml) to obtain an aseptic culture. The aseptic culture was named KNUA021.

2. Morphology and Molecular Identification

For the morphological analysis of KNUA021, the cells were cultured in BG-11 medium for 20 days. Live cells were harvested and suspended in sterile distilled water and observed at 400x magnification on a Zeiss Axioskop 2 light microscope equipped with a differential interference contrast (DIC) optical lens (Carl Zeiss, Standort Gottingen, Vertrieb, Germany).

The results are shown in Fig.

As shown in Figure 1, strain KNUA021 had the general characteristics of the genus Clamidomonas. The microorganism has a size of about 10 um and has a pair of flagella in the front part of the cell and contains a bowl-shaped chloroplast. Overall, morphological features of strain KNUA021 were suggested to belong to the genus Clamidomonas.

Sequence analysis of 18S rRNA and 28S rRNA was performed to confirm the taxonomic position of the strain through a molecular biological approach. The DNA of the isolate was extracted using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), and the PCR conditions used for 18S rRNA sequencing and primer set [NS1 (5'-GTA GTC ATA TGC TTG TCT C- ) And NS8 (5'-TCC GCA GGT TCA CCT ACG GA-3 ')] were referred to White et al. (1990). The D1-D2 region of the 28S rRNA of the strain was designated as NL1 (5'-GCA TAT CAA TAA GCG GAG GAA AAG-3 ') and NL4 (5'-GGT CCG TGT TTC AAG ACG G- Donnell et al., 1993). Synthesis and DNA sequencing of the primers used in this study were performed by Macrogen (Macrogen, Seoul, Korea). The DNA sequences obtained in this study are submitted to the NCBI database and their accession numbers are shown in Table 1.

As shown in Table 1, based on the molecular biological approach, the strain KNUA021 was found to be Chlamydomonas reinhardtii , and the deduced results from the sequence analysis of the D1-D2 region of 18S rRNA and 28s rRNA were consistent.

18S rRNA and 28s rRNA blast (BLAST) sequence comparison of KNUA021 Marker gene Access
number Length
(bp) Closest match
(GenBank access number) Overlap
(%) Sequence similarity (%) Taxonomic
relevance 18S rRNA JN863299 1,762 Chlamydomonas reinhardtii (M32703) 100 99 C. reinhardtii 28S rRNA JN863300 604 Chlamydomonas reinhardtii (EU410621) 100 99 C. reinhardtii

3. Physiological characteristics of strain KNUA021

 In order to confirm the physiological characteristics of strain KNUA021, experiments were carried out under different conditions of temperature, pH, salinity and salinity. The 20-day-old strain KNUA021 seed culture (1 ml) was inoculated into BG-11 medium (100 ml) three times and cultured for 20 days. Survival and growth of strain KNUA021 was observed at 5, 10, 15, 20, 25 and 30 ℃ in order to determine the optimal growth temperature of the culture. Salinity and acid tolerance tests were carried out at 25 ℃, the optimum growth temperature. Salinity was measured in the range of 0.1, 0.2, 0.3 and 0.4 M sodium chloride (NaCl), respectively, and the acidity was carried out in the range of pH 3.0 to pH 13.0. The optical density of the culture was measured at 680 nm in an Optimizer 2120 UV spectrophotometer (Mecasys Co. Ltd., Daejeon, Korea) to determine the density of strain KNUA021.

The results are shown in Figs. 2 to 4 and Table 2.

Physiological characteristics of strain KNUA 021 Physiological factor growth Temperature
pH
NaCl tolerance 15 ° C to 30 ° C
pH 4 to pH 12
Growth is inhibited by NaCl.

As shown in Figs. 2 to 4 and Table 3, the optimum growth temperature of strain KNUA021 was 25 占 폚, but growth and survival were possible even at a low temperature of 10 占 폚. Growth pH ranged from pH 4.0 to 12.0 and no growth was observed at pH 2.0 or pH 13.0. Strain KNUA021 could not grow in the presence of NaCl.

4. Confirm GC / MS analysis results

For GC / MS analysis, strain KNUA021 was inoculated into BG-11 (+) medium (pH 9.4) three times and cultured at 25 ° C for 20 days. Cells were harvested by centrifugation at 3,220 g (Centrifuge 5810R, Eppendorf, Hamburg, Germany) and microalgae lipids were extracted using the partially modified Bligh-Dyer method (Lee et al. 2010). Specifically, lipid components were extracted from lyophilized microalgae biomass using a chloroform: methanol (2: 1) solvent mixture. Biodiesel was obtained by transesterification between lipid and methanol, and potassium hydroxide (KOH) was used as the base catalyst. The fatty acid composition of the culture was measured by GC / MS (Jeol JMS700 mass spectrometer equipped with Agilent 6890N GC) at Daegu Center, Korea Basic Science Institute (KBSI).

The results are shown in FIG. 5 and Table 3.

Fatty acid and fatty alcohol composition of KNUA021 strain Peak No. Component Relative content (%) One Hexadecatrienoic acid (C _{16: 3} ω) 5.2 ± 0.3 2 Hexadecatetraenoic acid (C _{16: 4} ) 10.6 ± 1.1 3 Hexadecadienoic acid (C _{16: 2} 4) 4.0 ± 0.7 4 Hexadecatrienoic acid (C _{16: 3} ω 3) 2.3 ± 0.5 5 Palmitoleic acid (C _{16: 1?} 7) 1.7 ± 0.1 6 Palmitic acid (C16 _{: 0} ) 16.3 ± 0.4 7 δ-linolenic acid (C _{18: 3} ω) 5.7 ± 0.2 8 Unknown 1.4 ± 0.1 9 Linoleic acid (C _{18: 2} ω 6) 16.1 ± 2.3 10 α-linolenic acid (C _{18: 3} ω 3) 19.9 ± 1.9 11 Oleic acid (C _{18: 1?} 9) 3.4 ± 0.5 12 Hexadecenol (C ₂₀ H ₄₀ O) 11.3 ± 0.9 13 Stearic acid ( _{C18: 0} ) 2.1 ± 0.1

As shown in Figure 5 and Table 3, GC / MS results were included a total of 13 main peak, the strain KNUA021 the palmitic acid as the main fatty acid (C _{16: 0),} to hexadecafluorovanadyl tetracarboxylic acid (C _{16: 4} ), Linoleic acid (C _{18: 2} ) and linolenic acid (C _{18: 3} ).

In addition, it was confirmed that it is possible to synthesis a hexa senol (C ₂₀ H ₄₀ O, molecular weight = 296), having as one of the major cell components, as shown in Fig.

Korea Biotechnology Research Institute KCTC12066BP 20111109

Claims (2)

Hexahydro to senol and C ₁₆ to the microalgae, novel Chlamydomonas lane hareuti (Chlamydomonas reinhardtii) KNUA021 strain to produce fatty acids of C ₁₈ (KCTC 12066BP). The composition according to claim 1, wherein the C ₁₆ to C ₁₈ fatty acids are selected from the group consisting of palmitic acid (C16: 0), palmitoleic acid (C16: 1), linoleic acid (C18: 2) and linolenic acid A novel Chlamydomonas reinhardtii KNUA021 strain (KCTC 12066BP) which is at least one selected from the group consisting of:

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