KR101114426B1 - Novel strain Chlamydomonas pitschmannii YSL03 - Google Patents

Abstract

The present invention is Mycobacterium Klimidomonas Fitschmanny YSL03 [KCTC 11715BP] More specifically, the new strain Chlamydomonas pitschmanny YSL03 [KCTC 11715BP] has a high lipid content, which is very effective for biodiesel production and strong wastewater resistance, which can remove nitrogen and phosphorus in the wastewater.

Description

Novel strain Chlamydomonas pitschmannii YSL03}

The present invention, Chlamydomonas pitschmanny high in wastewater resistance and lipid content YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain.

Increasing use of energy on the planet continues to increase, resulting in many environmental pollutions, resulting in global warming [Chisti, Y. 2007. Biodiesel from microalgae. Biotechnology Advances, 25: 294-306.]. Biomass is a renewable energy source that does not affect the greenhouse effect [Widjaja, A., Chao-Chang Chien, Yi-Hsu Ju. 2009. Study of increasing lipid production from fresh water microalgae Chlorella vulgaris. J. Taiwan Inst. Chem. Eng.40, 13-20.] It is continuously developed as it is used as energy in mass production. Biodiesel is obtained from triglyceride oils and monohydric alcohols. It is an energy source belonging to biomass and has been mainly produced from soybeans, sugar cane and animal fats [Lang, X., Dalai, AK. , Bakhshi, NN, Reaney, MJ, Hertz, PB, 2002. Preparation and characterization of biodiesels from various Bio-Oils. Biores. Technol. 80, 53-62.]. However, it is difficult to develop due to the food shortage theory.

Recently, algae, which is an energy source capable of producing biodiesel in a stable and economical manner, are being developed due to the following characteristics of algae. 1) Algae are independent, heterotrophic photosynthetic bacteria that grow in the presence of solar energy, water, and carbon dioxide (sea, lake, river, etc.). 2) Better productivity than other materials (eg soybeans, corn, sugar cane, etc.) [Becker, 1994. Measurement of algal growth. In: Microalgae biotechnology and microbiology. Cambridge University Press, P 56-62.], 3) has been reported to have a high lipid content, which is a biodiesel product [Sheehan, J., Dunahay, T., Benemann, J., Roessler, P., 1998.A look back at the US Department of Energy ’s Aquatic Species Program: Biodiesel from Algae. Close-Out report. National Renewable Energy Lab, Department of Energy, Golden, Colorado, U.S.A. Report number NREL / TP-580-24190.]. However, most of the algae studied until recently are marine algae inhabiting the ocean, and have the disadvantage of artificially supplying elements such as nitrogen and phosphorus during cultivation. Algae that live in rivers and lakes grow by using trace elements in wastewater or by nitrogen and phosphorus in rivers and rivers. Therefore, if the microalgae strains having wastewater resistance are separated, the nutrient is ingested from the wastewater supplied to the microalgae incubator, and thus the economic efficiency of the process may be improved without considering the artificial injection of nitrogen-phosphorus. However, most of the common microalgal strains currently deposited in the strain banks are very poorly reported in these wastewaters and lack of data on lipid content and growth rate, so the isolation of new microalgae is urgently needed.

Thus, the present inventors conducted a study to secure microalgae strains resistant to wastewater. As a result, microalgae species having resistance to wastewater, growing rapidly in wastewater, and having a lipid content of 50% or more of biodiesel production material are separated from the natural environment, and thus, are useful for biodiesel production. .

Accordingly, the present invention is to solve the environmental pollution problem by using a novel microalgae in wastewater treatment, and to provide a method for solving the alternative energy by using the microalgae generated in the process as a raw material for biodiesel production.

In order to achieve the above object, the present invention

The present invention Chlamydomonas Fitschmann YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strains and cultures thereof are characterized.

In addition, the present invention Chlamydomonas Fitschmann Chlamydomonas pitschmannii YSL03 [KCTC 11715BP] strains or cultures thereof, and the method for producing biodiesel using the separated lipids are another feature.

In addition, the present invention Chlamydomonas Fitschmann Another feature is a microbial agent for wastewater treatment comprising a strain YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] or a culture thereof.

The present invention also includes a method for culturing the new strain in wastewater.

The present invention Chlamydomonas Fitschmann YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain was used to examine wastewater resistance and lipid content as a biodiesel production source, thus securing the potential as a new strain for biodiesel production.

The new strain of the present invention has a strong wastewater resistance, and can directly apply N and P contained in the wastewater, thereby greatly improving the economics of the culture.

Also, grown Chlamydomonas Fitschmann. YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] contains high content (more than 50%) lipids, which contributes to biodiesel production.

1 is Chlamydomonas Fitschmann YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP].
Fig. 2 Chlamydomonas Fitschmann The phylogenetic tree of YSL03 [KCTC 11715BP] is shown.
Figure 3 Chlamydomonas Fitschmann in wastewater and BBM YSL03 [KCTC 11715BP] shows the effect of nitrogen and phosphorus removal.
Figure 4 Chlamydomonas Fitschmann Fatty acid distribution of YSL03 [KCTC 11715BP] [linolenic acid (C18: 3n6), linolenic acid (C18: 3n 3), oleic acid (C18: 1n9c), stearic acid (C18: 0), heptadecanoic acid (C17: 0 ), Palmitoleic acid (C16: 1), palmitic acid (C16: 0), mystic acid (C14: 0), lauryl acid (C12: 0)].

Hereinafter, the present invention will be described in detail.

Chlamydomonas pitschmanny, a new strain of the present invention YSL03 was first identified and identified by the present inventors, and strains were separated using BBM medium in sewage and wastewater treatment plants. As a result of analyzing the isolated strains in a molecular genetic manner by 28s rDNA sequencing sequence search, a standard strain was identified. Inn Klamidomonas Fitschmann It was confirmed that the strain was 99% similar to AF183462.

The present inventors named the isolated and identified mycobacterium Klamidomonas Fitschmanny YSL03, and deposited on June 18, 2010 to the Korea Institute of Bioscience and Biotechnology Biological Resource Center was given accession number KCTC 11715BP.

The Chlamydomonas pitschmanny YSL03 can be cultured in the waste water, in particular, it is preferable to incubate at 25 to 27 ℃, pH 7.2 to 7.6. In addition, culturing in BBM medium (KH ₂ PO ₄ , CaCl ₂ · 2H ₂ O, MgSO ₄ · 7H ₂ O, NaNO ₃ , K ₂ HPO ₄ , NaCl, H ₃ BO ₃ and a trace element containing trace elements) More preferred. The trace element may be at least one selected from the group consisting of ZnSO ₄ .7H ₂ O, MnCl ₂ .4H ₂ O, MoO ₃ , CuSO ₄ .5H ₂ O, and Co (NO ₃ ) ₂ .6H ₂ O.

Chlamydomonas pitschmanny YSL03 according to the present invention can be grown under high concentrations of nitrogen and phosphorus (800 mg / L, 10 mg / L) conditions can be cultured without significantly reducing the dilution ratio of the waste water.

In addition, since it shows the effect of removing nitrogen, phosphorus, which is a pollution source in wastewater, it can be used for wastewater treatment.

Domestic livestock farms have more than 110,000 households, and large amounts of nitrogen and phosphorus flow into the wastewater treatment plant. Existing nitrogen and phosphorus removal are all physical, chemical, and biological methods, and there are many disadvantages in the economic part. However, the light needed for photosynthesis during algae cultivation is taken from the natural sun and wastewater is added to the additional growth factors nitrogen and phosphorus. Ingestion naturally, so the process is simple. Therefore, the investment cost and operation cost required for the process facility is very low, so the possibility of commercialization is very high.

As described above, the present invention is Chlamydomonas Fitschmann Chlamydomonas pitschmannii YSL03 [KCTC 11715BP] strain or culture medium thereof may be provided.

On the other hand, Chlamydomonas pitschmanny according to the invention cultured using wastewater YSL03 provides fast growth rates and lipid-rich species ( Lipid content of 50% or more), which can be used as a component providing element capable of producing biodiesel from lipid extraction.

Therefore, the present invention, Chlamydomonas Fitzmann Chlamydomonas pitschmannii YSL03 (KCTC 11715BP) strain or a culture medium thereof, and a method for producing biodiesel using the lipids separated therefrom are also included.

The lipid analysis of algae can be carried out using the Sulfophosphovanillin method, Kunkel method, and Brugdon method.

The new strain of the present invention was also analyzed for lipids by the conventional lipid analysis method as described above.

In addition, as a result of analyzing the fatty acids in the lipids thus separated, it was confirmed that palmitic acid and oleic acid contained more than 30% by weight relative to the total fatty acid content, using the separated lipid (fatty acid) The biodiesel can be produced, and the present invention may also include a biodiesel production method using the lipid.

Hereinafter, the present invention will be described in more detail by way of examples. It should be noted, however, that the following examples are illustrative of the invention and are not intended to limit the scope of the invention.

Example  One: Neomycology  Isolation and Cultivation

Samples were collected from the sewage and wastewater treatment plant in Wonju, Gangwon-do. The collected sample was placed in a BBM liquid medium as shown in Table 1 in a 200 mL test tube and left to incubate for about 2 weeks. Nitrogen-phosphorus was fed at 800 mg / L and 10 mg / L, respectively, and the concentration of wastewater was adjusted to about 24 watts on the surface of the culture tube using a fluorescent light source, followed by direct incubation. 1 mL of the pre-cultured sample was placed in a 30 mL test tube containing 10 mL of sterile distilled water and mixed well to disperse the microalgal cells. From this, a 0.1 mL sample was taken using a micro pipette, and then mixed into a test tube containing sterile distilled water. After this procedure was repeated five times, about 0.1 mL of the sample was taken from each test tube, which was inoculated in a Petri dish containing BBM medium having a composition as shown in Table 2 below, followed by a temperature of 25 to 27 ° C. And stationary culture for 2-3 weeks in an incubator of roughness 50 μmol / m 2 -sec. When colonies of microalgae appeared, micron was used to check each microscope and each colony was transferred to a well cell culture plate using platinum teeth to start separation. Each hole was incubated for 10 to 14 days after administration of avian colonies and BBM medium 1: 1. In order to inhibit the growth of some microorganisms, we used antibiotic streptomycin to grow only algae. The amount of antibiotic used was used 0.15 μm / mL per 1L of medium. After a period of time, the colonies cultured in each hole are examined under a microscope. At this time, most of the holes grow only the characteristic microalgae, the same shape was incubated in Petri dishes prepared with BBM medium. After a period of time, the cultivated algae colonies were predominantly cultured as one kind of colonies. The colonies were harvested and transplanted into 250 mL Erlenmeyer flasks containing 100 m of BBM medium.

Example  2: New strain  culture

Chlamydomonas pitschmanny using the BBM medium of Table 1 above YSL03 ( Chlamydomonas pitschmannii YSL03) strain was placed in a 250 mL Erlenmeyer flask at 25 to 27 ° C., pH 7.2 to 7.6, and 10 mL of the strain was incubated in a fluorescent stirred incubator for about 14 days. Stirring was maintained at 150 rpm and roughness 50 μmol / m 2 -sec.

Example  3: Neomycology  Sympathy

Chlamydomonas Fitzmanny The sequencing of the strain YSL03 was analyzed by 28s rDNA sequencing. The state of the sample is Chlamydomonas Fitschmann. YSL03 colonies were plated, and as a request, PCR products by Extraction-gDNA and PCR Amplication were analyzed by sequencing reaction. Primers used for analysis are as follows.

Forward primer: 5'-AGCGGAGGAAAAGAAACTA-'3 (SEQ ID NO: 2)

Reverse primer: 5'-TACTAGA-AGGTTCGATTAGTC-'3 (SEQ ID NO: 3)

The 28s rDNA sequencing results are described in the attached SEQ ID NO., And the phylogenetic results are shown in the phylogenetic tree results according to the neighbor-joining method using the Jukes-Cantor model. 2 is shown.

Chlamydomonas Fitzmanny The base sequence [SEQ ID NO: 1] of the strain YSL03 was classified as Chlamydomonas pitschmanny as shown in Table 3 below when the homology was examined in NCBI.

As described above, the microalgal bacteria isolated from the present invention, when considering the characteristics of morphology, optimal temperature, pH, etc., except that it contains wastewater resistance and high content of lipids, Chlamydomonas Fitsch Manni Turned out to belong.

Thus, the new strain according to the present invention Chlamydomonas Fitschmann YSL03 ( Chlamydomonas pitschmannii YSL03), and it was deposited on June 18, 2010 at the Korea Institute of Bioscience and Biotechnology, and was assigned accession number KCTC 11715BP.

Example  4: Check wastewater resistance and nitrogen and phosphorus removal

Alga Chlamydomonas pitschmanny secured by Example 2 in livestock wastewater after MF (Micro Filtration) YSL03 ( Chlamydomonas pitschmannii In YSL03, algae were seeded according to different sources for consideration of nitrogen and phosphorus removal. Each reactor maintained a room temperature of 20-22 ° C., pH was maintained at 7.7-7.9 in nature and proceeded in the form of batch experiments. After the first planting, algae took about 10 days to acclimate, after which the removal of nitrogen and phosphorus occurred regardless of the initial concentrations of wastewater and BBM.

In FIG. 3, the reduction of nitrogen and phosphorus proceeded rapidly within 10 to 12 days after the algae adaptation proceeded, and it is determined that rapid removal of phosphorus and nitrogen occurs when the steady-state algae is applied to the reactor.

Example  6: lipid production and fatty acid extraction

Algae are heterotrophic / independent nutrients composed of carbohydrates, proteins, and lipids that grow through photosynthesis.

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 mixture of fatty acid methyl esters Mix RM3, Mix RM5, GLC50, GLC70 [Supelco, USA], heptadecanoic acid and gamma-linolenic acid [Supelco, 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].

Chlamydomonas pitschmanny according to the present invention The strain YSL03 ( Chlamydomonas pitschmannii YSL03) was found to contain about 50% by weight of dry weight lipids in the cells [Table 4].

Among the fatty acids, palmitic acid (16: 0), stearic acid (C18: 0), oleic acid (C 18: 1n9c), and linolenic acid (C18: 3n3) are known as fatty acids for biodiesel production.

Figure 4 Chlamydomonas Fitschmann Palmitic acid (16: 0) is the most widely distributed fatty acid distribution of YSL03 strain. In particular, the most stable fatty acids for biodiesel production of all the microalgae reported to date are known as oleic acid (C 18: 1n9c) and palmitic acid (16: 0).

Klimidomonas pitschmanny, a new strain according to the present invention YSL03 is made up of 40% by weight of all fatty acids composed of oleic acid and palmitic acid for biodiesel production.

Korea Research Institute of Bioscience and Biotechnology KCTC11715BP 20100618

Attach an electronic file to a sequence list

Claims (12)

Chlamydomonas pitschmanny with high wastewater resistance and lipid content YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain.
According to claim 1, Chlamydomonas Fitschmanny having a nucleotide sequence of SEQ ID NO: YSL03 ( Chlamydomonas pitschmannii YSL03 [KCTC 11715BP] strain.
The method of claim 1, wherein the lipid content is more than 50% by weight of the dry cell weight Chlamydomonas Fitschmanny YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] Strain
The method of claim 1, wherein Chlamydomonas Fitschmanny containing at least 30% by weight of oleic acid and palmitic acid in the total fatty acids YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain.
Chlamydomonas Fitzmanny YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain culture.
In the method for separating lipids from algae or its culture,
The bird is Chlamydomonas Fitschmann YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain, characterized in that the lipid separation method.
In the method of producing biodiesel using lipids,
The lipid is Chlamydomonas Fitschmann YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] Biodiesel production method characterized in that the lipid isolated from the strain.
Chlamydomonas Fitschmanny of claim 1 YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain or microbial agent for wastewater treatment comprising the culture solution of claim 4.
Chlamydomonas Fitschmanny of claim 1 YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain is cultured in wastewater.
The method of claim 9, wherein the strain is incubated at 25 to 27 ℃, pH 7.2 to 7.6.
The method of claim 9, wherein the strain is added to a medium containing KH ₂ PO ₄ , CaCl ₂ .2H ₂ O, MgSO ₄ .7H ₂ O, NaNO ₃ , K ₂ HPO ₄ , NaCl, H ₃ BO _3, and a trace element. Culturing.
12. The method of claim 11, wherein the trace element is one selected from the group consisting of ZnSO ₄ .7H ₂ O, MnCl ₂ .4H ₂ O, MoO ₃ , CuSO ₄ .5H ₂ O and Co (NO ₃ ) ₂ .6H ₂ O. The method characterized by the above.

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