KR101349295B1 - A producing method of biodiesel using a novel psychrophilic cyanobacterium oscillatoria sejongensis - Google Patents

Abstract

The present invention relates to a method for producing biodiesel using a new cyanobacteria oscillatoria sejong ensis KNUA009 KCTC 12034BP. According to the biodiesel manufacturing method according to the present invention, by using a new type of cold- cooled cyanobacteria Oscilltoria sejongensis KNUA009 KCTC 12034BP, it is possible to obtain sufficient biomass due to rapid growth in extreme environments. In addition, since the cell contains a large amount of fatty acids advantageous for the production of biodiesel, it is possible to effectively improve the biodiesel production yield.

Description

A method for producing biodiesel using a new cold-dried cyanobacteria oscillatoria sejong ensis {A PRODUCING METHOD OF BIODIESEL USING A NOVEL PSYCHROPHILIC CYANOBACTERIUM OSCILLATORIA SEJONGENSIS}

The present invention relates to a method for producing biodiesel using a psychrophilic new cyanobacterium oscillatoria Oscillatoria sejongesis isolated from Antarctica.

Cyanobacteria play an indispensable role in the global carbon and nitrogen cycle and are distributed in a wide variety of niches. Cyanobacteria are found in almost every possible environment, including polar and alpine areas due to their excellent resistance to a wide range of environmental conditions (Whitton and Potts 2000; Vincent 2007). Cyanobacteria play a large role in the composition of the entire ecosystem biomass in the cryosphere through photosynthesis, and accumulation of cyanobacteria in the polar regions is regularly observed (Broady 1996; Vincent 1988; Wynn-Williams 1996). Thus, algae in high and high hardness are of special interest, and research has also been carried out on the diversity of cyanobacteria in microbial mats of polar lakes and ponds, based on both morphological and molecular approaches. Tang et al. 1997; Nadeau et al. 2001; Taton et al. 2006a).

The Antarctic biota has been farther apart than any other world in the world for millions of years, so mat-forming algae dominating the Antarctic ecosystem are of great interest. Oscillatorian cyanobacteria, along with nostocales, are among the most predominant microbes in the Antarctic benthic microbial mat community (Brody 1996; Wynn-Williams 1996; Jungblut et al. 2005). ). This dominance is due to their high resistance to extreme conditions and due to the killing of competing species due to freeze-thaw cycles, extreme changes in ultraviolet light and nutrients.

Although recent culture-independent molecular techniques have been successfully applied to assess the diversity of Antarctic algae in Antarctic ecosystems (Taton et al. 2003; Wood et al. 2008), the isolation and characterization of Antarctic algae strains is still in many fields. It plays an important role. Furthermore, the number of Antarctic Antarctic strains held in culture collections and the molecular data in the public databases available are still very limited (Taton et al. 2006b).

 Thus, polyphasic studies of cultivable Antarctic algae may enable a better understanding of the diversity, physiology and biotechnological studies of Antarctic algae.

Meanwhile, the fossil fuel-based industry is facing a major crisis, due to the fact that fossil fuels will eventually be exhausted, resulting in supply instability and increased atmospheric CO2 concentrations. Therefore, carbon neutral, renewable alternative energy is needed, which should be an environmentally and economically sustainable alternative.

Among the renewable energy, biodiesel is receiving great attention as a transportation fuel. However, biodiesel, which is generally produced from oil and fat plants, currently replaces only a fraction of the transportation fuel, and biodiesel biomass from microalgae has various advantages to meet the demand for transportation fuel. Use as a raw material has been of interest to many people as an alternative to fossil fuels in recent years.

However, in order to put this into practical use, more efforts are needed to increase productivity, and more intensive research is still required.

The present invention, as part of the research on the South algae and the biodiesel as an alternative energy from the algae mat from the algae mat in King George Island in the western Antarctic region of King George Island (Osilatoria sejong ensis) Oscillatoria sejongensis ) KNUA009 KCTC 12034BP successfully completed the asenic culture, and aseptic cultures based on the results of morphological and molecular systems, physiological characteristics such as temperature, salinity and acidity tests, and chemical system analysis. Was identified as a new species, and from these results, to provide a method for producing biodiesel using the new strain.

In order to solve the above problems,

The present invention provides a method for producing biodiesel using microalgae in the method of producing biodiesel using microalgae, using microalgae oscillatoria sejongensis KNUA009 KCTC 12034BP. to provide.

In the production method, the microalgal culture temperature is preferably 5 ~ 20 ℃.

In the above production method, the pH range of the microalgae is preferably from 4.0 to 11.0.

In the above production method, the salt concentration of the microalgae is preferably 0.2M or less.

According to the method for producing biodiesel according to the present invention, by using Oscillatoria sejongensis KNUA009 KCTC 12034BP, which is a cold-cooled new cyanobacteria, sufficient biomass can be obtained due to rapid growth even in extreme environments. The fatty acid advantageously contained in the production of biodiesel can be contained in the biodiesel production yield can be effectively improved.

1 shows optical micrographs of Oscillatoria sejongensis KNUA009.
Figure 2 shows the lineage of the species closely related to Oscillatoria sejongensis KNUA009 interpreted from the sequencing data of Oscillatoria sejongensis KNUA009 and 16S rRNA. This includes Oscillatoria Five species of genus Oscillatoria sancta PCC7515 (Group 1), Oscillatoria nigroviridis PCC7112 (Group 2), Oscillatoria acuminate PCC6304 (Group 3), Oscillatoria formosa PCC6407 (Group 4) and Planktothrix agardhii CCAP1460 / 5 (Group 5) Included in the tree. Oscillatoria agardhii was renamed Planktothrix agardhii (Anagnostidis and Komarek 1988). Chloroflexus aurantiacus J-10-fl was used as outgroup. Bootstrap values are shown at each node. Ruler shows the difference of 1% of the nucleotide sequence.
3 is coming la thoria three kinds of N-Sys (Oscillatoria sejongensis) shows a relationship between the longitudinal lines, which are closely related to the three kinds of N-Sys come la thoria (Oscillatoria sejongensis) KNUA009 analysis from the ITS sequences of data KNUA009. Asterisk sequences are uncultivated cyanobacteria clones collected from Antarctica by Wood et al. (2008).
4 shows the growth curve of Oscillatoria sejongensis KNUA009 at various temperatures (a) and pH ranges (b).
5 shows an absorption spectrum of Oscillatoria sejongensis KNUA009.

Hereinafter, the present invention will be described in detail.

In the present invention, a method for producing biodiesel using microalgae, a method for producing biodiesel using microalgae, characterized in that Oscilltoria sejongensis KNUA009 KCTC 12034BP is used as microalgae. To provide.

The process of culturing the microalgae, extracting lipid components therefrom to produce biodiesel is well established, and the biodiesel manufacturing method of the present invention is well known except for using the new microalgae of the present invention. Algal biodiesel manufacturing process can be used as it is.

In addition, systems for microalgal mass culture can use open raceway ponds and closed photobioreactors.

Hereinafter, in the method for producing biodiesel using microalgae, a new cyanobacteria algae oscillatoria sejong ensis KNUA009 KCTC 12034BP, which is a feature of the present invention, will be described in detail.

Antarctica is one of the harshest ecosystems in the world. In particular, during the summer months, Antarctic algae are constantly exposed to daylight, which includes harmful ultraviolet rays, but southern algae are still commonly found throughout the Antarctic ecosystem.

Oscillatoria species of these algae are known as one of the major components of most freshwater ecosystems in Antarctica (Ellis-Evans 1996; Vincent and James 1996; Seckbach and Oren 2007).

In the present invention, the present inventors have isolated KNUA009 strain, a cold-cooled new alga, from a algae mat growing in meltwater of King George Island in western Antarctica, and at the morphological, molecular and physiological levels Identification was confirmed to be suitable strains for the production of biodiesel.

The KNUA009 strain of the present invention exhibited the morphological and molecular phylogenetic characteristics of Oscillatoria , which originated from sea ice water near Sejong Station, and was named Oscillatoria sejongensis KNUA009.

Accordingly, the present invention provides a cold- cooled new cyanobacteria oscillatoria sejong ensis KNU U009 KCTC 12034BP.

The new cyanobacteria of the present invention can be optimally grown at a salt concentration of 5-20 ° C., a pH of 4.0-11.0, and 0.2M or less.

Since cyanobacteria usually have more temperature flexibility than psychrophily (Tang et al. 1997; Chevalier et al. 2000), most polar rock algae cyanobacteria have temperatures above 15 ° C. Strict, cold-cooled strains with growth optimisation, which have optimal growth at temperatures below 15 ° C., are relatively uncommon (Tang and Vincent 1999; Nadeau and Castenholz 2000).

Nadeau and Castenholz (2000) propose an evolutionary scenario in which Antarctic cold-cooled algae strains may have lost their ability to live at higher temperatures as a reward for maintaining extensive resistance to other stressors during the evolution. It was. We estimate that this cold-cooled photoindependent nutrient may have lost resistance to higher temperatures in a similar manner.

Ribosome 16S-23S intergenic spacer region (ITS) sequencing analysis showed that KNUA009 strains were most similar to uncultivated Antarctic algae clones, with more than 97% homology. homology) (FIG. 3).

All similar clone sequences were samples analyzed by Wood et al. (2008) from southern algal mats or surface soils in eastern Antarctica. However, based on 16S rRNA sequencing data, the KNUA009 strain formed a cluster with 99% sequence homology with four environmental clones isolated at different geographical locations (Table 1, Figure 2).

These environmental clones include the Canadian Canadian Markham Ice Shelf (clone MIS92), the Western Onyx River (clone UOXA-b10) and the alpine Spanish Pyrenees. (Clone Llo_054) and China's Tianshan Mountain (clone KuyT-ice-32), respectively. Castenholz (1992) hypothesized that there would not be many species native to the polar region.

The present study also suggests that the genus Oscillatoria is distributed throughout the Antarctic region, but this cold-cooled algae may not be native to the Antarctic.

As shown in FIG. 1, the KNUA009 strain was also low in relation to the five reference strains constituting the genus Oscillatoria . Therefore, it can be concluded that the low level of DNA-DNA association obtained through genetic information analysis supports the novelty of the isolate.

Thus, this isolate represents a new taxa in the genus Oscillatoria , which we have found to be Oscillatoria sejongensis (se.jong.en'sis. NL fem. Adj.) Sp. nov. It was proposed as, Oscillatoria sejongensis KNUA009 dated October 14, 2011 was deposited with KCTC accession number KCTC 12034BP.

Cyanobacteria generally use several strategies to protect themselves from excessive ultraviolet radiation, which includes the production of matt or crust formation and photoprotective-quenching pigments.

In a similar manner, it is assumed that the KNUA009 strain can be protected from excessive light by forming a multi-layered mat.

As mates of Seckbach and Oren (2007) and Vincent (2007) have shown that lyophilized Antarctic algae mats resume photosynthesis and other physiological processes within minutes to hours after re-run, It is possible that the algae of the present invention have been endowed with the ability to maintain viability during the year-round freezing period.

Another common self-defense system observed in cyanobacteria is fatty acid desaturation, which increases the production of unsaturated fatty acids with reduced chain length to maintain fluidity at low temperatures. As expected, the cellular fatty acid composition of the KNUA009 strain was rich in C16: 1, C18: 1 C18: 2 and C18: 3 unsaturated fatty acids, effectively maintaining membrane fluidity at low temperatures.

Therefore, the new cyanobacteria of the present invention is characterized by containing a large amount of fatty acids in the cell and thus can be applied to biodiesel production.

Cyanobacteria are also known to have antioxidants such as carotenoids in their outer membrane and thylakoids to cope with harmful reactive oxygen species (ROS) (Pattanaik et al., 2007). Oren et al. (1995) noted that cyanobacteria communities exposed to high levels of light had a very high ratio of carotenoids to chlorophyll a .

From these results, it is assumed that the cyanobacteria of the present invention may have evolved a defense system by β-carotene synthesis to prevent oxidative damage by ROS.

Therefore, the new cyanobacteria of the present invention contain a large amount of β-carotene in the cell, and thus are suitable for separating β-carotene from it.

Hereinafter, the present invention will be described in more detail with reference to examples.

<Examples>

1. Isolation and Physiological Characteristics of New Microorganisms

Materials and methods

Sample Collection and Isolation of Cyanobacteria

Antarctic algae mat samples were collected in January 2010 from transient sea ice near King Sejong Station (62 ° 13'S, 58 ° 47'W) on the Barton Peninsula in King George Island, West Antarctica. Thereafter, 1 ml of cyanobacterial samples were treated with 100 ml BG-11 (+) medium (Rippka et al., 1979) containing 250 μg · ml ⁻¹ of cycloheximide (Sigma, St. Louis, MO, USA). Was inoculated. The flask was set at 160 rpm and 15 ° C. in an orbital shaker (Vision Scientific Co. Ltd., Bucheon, Korea) until approximately 70 μmole in day: night (16: 8 hours) cycles until blue algae growth became evident. M ^-2 -sec ^-1 were incubated at a light intensity.

Aseptic Culture Production

Centrifuge well grown algae culture (1.5 ml) at 3,000 g for 15 minutes and strayking dehydrated pellets on BG-11 agar medium containing cycloheximide (20 μg · ml ⁻¹ ) ). Thereafter, the plates were incubated under the same conditions as above and the growth of filaments was observed daily. When grown to the naked eye, the outgrowing cyanosa filament was aseptically transferred to a new BG-11 agar medium to isolate it from other contaminating bacteria. The cyanobacterial filaments were then inoculated on R2A and LB agar plates (Becton, Dickinson and Company, Sparks, MD, USA) and cultured in cancer conditions to check for aseptic conditions for cyanobacteria for 14 days. These procedures were repeated until a sterile cyanobacteria were obtained and named pure cyanobacteria strain KNUA009. This isolated strain represents a new taxa in the genus Oscillatoria , and the present inventors have identified it as Oscillatoria sejongensis (se.jong.en'sis. NL fem. Adj.) Sp. nov. It was proposed as, Oscillatoria sejongensis KNUA009 dated October 14, 2011 was deposited with KCTC accession number KCTC 12034BP.

Morphological identification

Strain KNUA009 was incubated in BG-11 (+) medium at 15 ° C. for 10 days. Cyanobacteria cells were harvested, suspended in a drop of immersion oil, and loaded at X 1,000 times on a Zeiss Axioskop 2 light microscope (Carl Zeiss, Standort Gottingen, Vertrieb, Germany) equipped with differential interference contrast (DIC) optics. Observed. See Bergey's Manual of Systematic Bacteriology (Castenholz 2001) for taxonomic description and morphotypic identification.

Molecular Identification

DNA was extracted using DNeasy Plant Mini kit (Qiagen, Hilden, Germany). PCR conditions and primer sets CYA106F (SEQ ID NO: 1) (5'-CGG ACG GGT GAG TAA CGC GTG A-3 '), CYA781R (a) (SEQ ID NO: 2) (5'-GAC TAC TGG GGT ATC TAA TCC CAT T -3 ') and CYA781R (b) (SEQ ID NO: 3) (5'-GAC TAC AGG GGT ATC TAA TCC CTT T-3') were used for 16S rRNA sequence analysis as described by Nubel et al. (1997). It was. Ribosome 16S-23S intergenic spacer region (ITS) was transferred to primers 16S1407F (SEQ ID NO: 4) (5'-TGT ACA CAC CGC CCG TC-3 ') and 23S30R (SEQ ID NO: 5) (5'-CTT CGC CTC TGT GTG CCT AGG T-3 ') (Taton et al. 2003). The RuBisCO ( Ribuloose -1,5-bisphosphate carboxylase / oxygenase) rbc LX was previously described (Rudi et al. 1998) primers CW (SEQ ID NO: 6) (5'-CGT AGC TTC CGG TGG TAT Amplification using CCA CGT-3 ') and CX (SEQ ID NO: 7) (5'-GGC GCA GGT AAG AAA GGG TTT CGT A-3'). Synthesis and DNA sequencing of the primers used in this study were performed in Macrogen (Macrogen, Seoul, Korea).

Phylogenetic analysis

The sequences analyzed in this study and the sequences closest to them genetically close were manually aligned in Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0 using ClustalW (Tamura et al. 2007). All sequences used for sorting were downloaded from the National Center for Biotechnology Information (NCBI) database. Evolutionary distances were calculated using the Maximum composite likelihood method, and phylogeny was generated from distance matrices using a neighbor-joining tree-building algorithm (Saitou and Nei 1987). The analyzed schematic was evaluated using the bootstrap test (1,000 bootstrap repetition) function in MEGA 4.0 (Felfenstein 1985) and only bootstrap values> bootstrap> 50% were displayed at the branch points. DNA sequences analyzed in the present invention were registered in the NCBI database as accession number HQ201392-HQ201394 (Table 1).

Growth experiment

KNUA009 (1 ml) strains cultured for 10 days were inoculated three times with BG-11 (+) medium and incubated for 20 days. To determine the optimal growth temperature of the isolate, the survival and growth of KNUA009 were examined at 5, 10, 15, 20, 25 and 30 ° C. Salinity and acid resistance tests were performed at 15 ° C. in the range of 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6M NaCl and pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 and 13.0 Each was performed. As described in Miura and Yokota (2006), carbon utilization tests were conducted using glucose, sucrose, fructose, ribose, sodium acetate, glycerol and glycolic acid. Cyanobacteria density was determined by measuring the optical density of the culture at 750 nm on an Optimizer 2120UV spectrophotometer (Mecasys Co. Ltd., Daejeon, Korea).

Pigment Identification

Pigment extraction was performed by the method of Azua-Bustos et al. (2010). Absorption spectra between 100 nm and 800 nm were measured on an Optimizer 2120UV spectrophotometer using Optizen View 3.02 software (Mecasys Co. Ltd., Daejeon, Korea).

GC content analysis

DNA G + C content was determined by HPLC method as described in Shin et al. (1996) by KCCM (Seoul, Korea).

Fatty acid analysis

Lipid extraction of microalgae was performed according to standard protocols (Sasser, 1990). Approximately 40 mg of cultured cyanobacterial cells were centrifuged to pellet and transferred into clean 13 × 100 mm culture test tubes. Put 1 ml of Reagent 1 solution containing NaOH and methanol into the test tube, briefly vortex, boil at 100 ℃ in a constant temperature water bath for 5 minutes, vortex again for 5-10 seconds, and saponify at 100 ℃ in a constant temperature water bath for 25 minutes. saponification) was performed. After the test tube was cooled, 2 ml of Reagent 2 solution containing hydrochloric acid and methanol was added to the test tube, briefly vortexed, and heated at 80 ° C. for 10 minutes, methylated, and cooled rapidly. 1.25 ml of Reagent 3 solution containing hexane and methyl tert-butyl ether were placed in a test tube, gently stirred for about 10 minutes, and the lower aqueous layer was pipetted away. 3 ml of Reagent 4 solution containing NaOH was added to the remaining organic solvent layer in the test tube, stirred for 5 minutes, then about 2/3 of the organic solvent layer was taken on Hewlett-Packard GC 6890 series gas chromatography at KBSI cod. Fatty acid composition was analyzed.

result

Morphological identification

Pure culture of Oscillatoria sejongensis KNUA009 was achieved by physical separation method. The microorganisms consisted of straight or slightly curved cell death (細胞 絲, trichome) consisting of disk-shaped vegetative cells forming filaments (Fig. 1). Cell deaths were about 3 μm wide and> 20 μm to hundreds of μm long. Its terminal cells were dome shaped. Heterocysts were not found, but the cross-walls were clearly visible in the KNUA009 strain. The color ranged from brown to olive, with the densely packed cell deaths forming a visible mat. Combining the morphological characteristics of strain KNUA009, it could be concluded that this isolate belongs to Oscillatoria .

Molecular Level Identification

Based on 16S rRNA sequencing data, strain KNUA009 showed the closest association with environmental clones derived from various ice cap regions, the closest cultivable strain of which was Phormidium pristleyi ANT with only 90% sequence homology. .LH66.1 (AY493581) (Table 1, FIG. 2). ITS sequencing also showed a 99% sequence homology to the uncultured cyanobacteria clone N184-8 (EU032395) isolated from the topsoil of the Meers Valley in eastern Antarctica, with the closest sequence match for the KNUA009 strain. (Table 1). Furthermore, the KNUA009 strain closely formed aggregates with Antarctic algae clones derived from the topsoil or algae mat of the Meyers valley (FIG. 3). Leptolyngbya sp. CCALA 094 (AM398976) was a culturable microorganism with the closest nucleotide sequence to KNUA009 strain, but its homology was only 86% (Table 1). Based on the rbc LX sequence comparison, the Prochlorothrix hollandica rbc L and rbc S genes (X57359) were the closest match for the KNUA009 strain, but the query coverage was only 29% (Table 1). This may be due to the lack of sequencing data in GenBank for the Antarctic algae rbc LX gene, and thus could not be confirmed by this result.

Physiological Characteristics of KNUA009

As shown in FIG. 4A, the optimum growth temperature of strain KNUA009 was 15 ° C., but growth was possible at 10 and 20 ° C. (FIG. 4A). However, this microorganism could not survive at 25 ° C. and above, indicating its quenching characteristics. However, markedly delayed growth was observed upon incubation at 5 ° C., which reached a stationary phase after 24 days of incubation. The KNUA009 strain began to disappear rapidly in most cases after reaching maximum growth, but this phenomenon did not occur when cultured in solid medium. Growth pH ranged from pH 4.0 to pH 11.0, while the optimum growth of the isolate was obtained at pH 7.0 (FIG. 4B). No growth was observed below pH 3 and above pH 12.0. Strain KNUA009 was able to withstand NaCl concentrations up to 0.2M. Since the KNUA009 strain did not utilize the other alternative carbon sources tested in this study, it was concluded that this cyanobacteria is an absolute photoautotroph. KNUA009 strain could not grow in BG-11 ₀ medium without nitrogen source. Physiological characteristics of KNUA009 strains are summarized in Table 2.

Chemical classification

The G + C content of DNA of strain KNUA009 is 47.5 mol%, which belongs to the average DNA sequence composition (40-50 mol% G + C) of the genus Oscillatoria deposited in Pasteur Culture Collection (Castenholz 2001). The fatty acid composition of strain KNUA009 was 16: 0 (34.6%), 16: 1 (24.9%), 16: 2 (0.8%), 18: 0 (1.2%), 18: 1 (5.2%), 18: 2 ( 15.5%) and 18: 3 (17.8%). Absorption spectra showed absorption peaks of chlorophyll a at 432 and 664 nm and β-carotene peaks at 485 nm (FIG. 5).

2. Culture of Microalgae and Extraction of Lipid Components

KNUA009 strains were inoculated into an 18 L photobioreactor made of transparent polycarbonate containing 16 L of BG-11 (+) medium and then cultured autotrophically. Cultivation conditions were incubated at a temperature of about 2 L · min ⁻¹ at 15 ° C. day and night (16: 8 hours) at a light intensity of about 70 μmo · m ⁻² · sec ⁻¹ . After about two weeks, the microalgal culture was centrifuged at 3,220 g for 10 minutes to harvest biomass and subjected to lyophilization to increase the extraction yield. The lyophilized biomass was mixed well with the chloroform: methanol (2: 1) mixed solution, placed in a separatory funnel, and allowed to stand for more than 8 hours to perform lipid extraction.

 3. Preparation of Biodiesel

 Chloroform extract isolated from 2. was dried on a rotary evaporator and treated with methanol and KOH to promote transesterfication reaction and hexane was added again to help biodiesel separation. The mixture was stirred and heated at 30 ° C. for 10 hours. After that, the mixture was allowed to stand and the methanol layer and the hexane layer were separated, and the biohexane was finally prepared by taking the hexane layer.

Korea Biotechnology Research Institute KCTC12034BP 20111014

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Claims (4)

Oscillatoria sejongensis KNUA009 ( Oscillatoria sejongensis KNUA009) strain (Accession No .: KCTC 12034BP), characterized in that the culture of biodiesel using microalgae, characterized in that at a temperature of 12 ℃ to 18 ℃, pH 6.5 to 7.5 Manufacturing method.
delete delete The method of claim 1,
Method for producing biodiesel using microalgae, characterized in that the culture at a salt concentration of 0.2M or less.

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