KR101568566B1 - Novel strain Chlamydomonas mexicana YSL008 - Google Patents

Abstract

The present invention relates to a novel strain Clamidomonas Mexicana YSL008 [KCTC12343BP], more particularly, Clamidomonas mexicana or YSL008 has a high concentration of carbohydrate according to the optimal content culture of CO ₂ and an influent substance, And adds enzymatic hydrolysis to increase the glucose content in carbohydrates for ethanol production. In addition, since the wastewater resistance is strong, it is possible to remove nitrogen and phosphorus in the wastewater, and thus it can be used as a good energy source for producing bio energy simultaneously with wastewater treatment.

Description

Novel strain Chlamydomonas mexicana YSL008}

The present invention relates to a strain of Chlamydomonas mexicana YSL008 [KCTC12343BP] having a high carbohydrate content.

Bioethanol is in the spotlight as a representative renewable resource energy in addition to biodiesel. It can be mixed with gasoline or used as a fuel alone, and it is also possible to extract it from grains / woody / vegetable materials. In each country, alternative energy development is emerging as a global issue based on exhaustion of fossil fuels and strengthening of greenhouse gas regulations. In addition to reducing petroleum consumption by 20% by 2017, the United States will expand use of alternative energy such as bioethanol, The country is also pursuing bioethanol production policy. However, the grain system that mainly produces bioethanol is at a time when it is difficult to further develop due to the viewpoint of food supply, and the woody material contains a lot of lignin components, and thus, it has many problems in the pretreatment to solve this problem. Recently, as a source of energy for bioethanol, domestic and foreign research groups are studying the technology for obtaining bioethanol from microalgae rich in carbohydrates and saccharides. Most microalgae consume about 35% or more carbohydrates, and the constituents vary depending on the growth environment. In addition, most of carbohydrate substances in algae are composed of glucose, so it is easy to produce energy by sugar fermentation process. In order to change the composition of such carbohydrates, microbial growth proceeds according to changes in the surrounding environment.

Microalgae are composed of carbohydrates, proteins, lipids, etc., and when a carbohydrate is used efficiently, a large amount of ethanol can be easily obtained. In order to cultivate algae for bioethanol production, the composition of temperature, carbon source, and mineral element is important, and algae resistant to wastewater are likely to be converted to ethanol upon fermentation because of their rapid growth and high carbohydrate content.

However, the algae have a rigid cell wall, requiring pretreatment to destroy the cell wall, and hydrolysis of the extracted carbohydrate should be carried out in order to use the fermenting bacteria. In this process, mannose, galactose, and glucose, which are carbohydrate constituents, can be obtained, and the glucose can be increased to about 60% (Non-Patent Document 1).

R. Harun, M.K. Danquaha, Chemical Engineering Journal 168 (2011) 1079-1084

Accordingly, the inventors of the present invention have developed a new microalgae culture study for increasing the production efficiency of bioethanol, and have been studying resistance to wastewater and rapidly ingesting carbon dioxide, and by selecting microalgae for bioethanol production by high carbohydrate content Thereby completing the invention.

Accordingly, the present invention relates to a novel strain Clamidomonas mexican or YSL008 {Novel} which can be used for the treatment of wastewater to solve the environmental pollution problem and to apply the microalgae to the production of bioethanol by applying culture conditions suitable for carbohydrate production in the process. strain Chlamydomonas mexicana I would like to provide YSL008.

As a means for solving the above problems, the present invention is Chlamydomonas Mexicana YSL008 (Chlamydomonas mexicana YSL008) [KCTC12343BP].

As another means for solving the above problems, the present invention provides a microorganism preparation for treating wastewater comprising Clamidomonas mexican or YSL008 [KCTC12343BP] strain or a culture thereof.

As another means for solving the above problems, the present invention provides a method for culturing the new strain in wastewater.

As another means for solving the above problems, the present invention provides a method for producing a microorganism which comprises hydrolyzing a strain of Clamidomonas mexican or YSL008 [KCTC12343BP], which has been pretreated with an ultrasonic wave, with an enzyme and then fermenting it with anaerobes to obtain bioethanol Wherein the bio-ethanol is produced by a method comprising the steps of:

The present invention has investigated the wastewater resistance and the content of glucose in carbohydrates and carbohydrates, which are the producers of bioethanol, by using Clamidomonas mexican strain YSL008 [KCTC12343BP], and secured the possibility as a new strain for bioethanol production.

The new strain of the present invention has a strong resistance to wastewater, and it is possible to directly apply N, P and carbon dioxide contained in wastewater, thereby greatly improving the economical efficiency of culture.

In addition, the grown Clamidomonas mexican strain YSL008 [KCTC12343BP] has 50% by weight of carbohydrate in the strain and contains glucose (more than 60%) in the carbohydrate, thereby contributing to the production of bioethanol.

1 is a photograph of a strain of Clamidomonas mexicanus YSL008 [KCTC12343BP].
Fig. 2 shows the phylogenetic tree of the Clemimonas mexicana YSL008 [KCTC12343BP] strain.
Figure 3 shows the removal of nitrogen and phosphorus in the wastewater with Clamidomonas mexicana strain YSL008 [KCTC12343BP].
Figure 4 shows the carbohydrate, lipid and protein distribution of the strain per weight of Clamidomonas mexicana YSL008 [KCTC12343BP].
Figure 5 shows the carbohydrate concentration according to the culture conditions of Clamidomonas mexicana YSL008 [KCTC12343BP] strain.
Figure 6 shows the composition of carbohydrate constituents contained in Clamidomonas mexicana YSL008 [KCTC12343BP] strain [[E]: enzyme; [S]: Clamidomonas Mexicana YSL008 Biomass].
FIG. 7 shows changes in glucose concentration by enzymatic hydrolysis of carbohydrates generated from Clamidomonas mexicana YSL008 [KCTC12343BP] strain. This is the system tree.
FIG. 8 shows the production of ethanol by anaerobic fermentation of carbohydrates generated from Clamidomonas mexicana YSL008 [KCTC12343BP] strain [FB: anaerobic microorganism; DW: distilled water; Glu: glucose, MB: Clamidomonas Mexicana YSL008 Biomass, SB: Ultrasonically pretreated Clamidomonas mexican YSL008 Biomass, En: Enzyme pretreatment].

The inventors of the present invention identified the first strain of Chlamydomonas mexicana YSL008 (Chlamydomonas mexicana YSL008) [KCTC12343BP] according to the present invention. The strains were isolated using a BBM medium under carbon dioxide conditions at the wastewater treatment plant, Molecular genetic analysis of the strain by 28s rDNA sequencing showed that it was 98% similar to the standard strain, Clamidomonas mexicana AF395501.1.

The present inventors have named the new strain identified above as Clamidomonas Mexicana or YSL008, and deposited it on December 28, 2012 at the BRC of Korea Research Institute of Bioscience and received the accession number KCTC12343BP.

The Clamidomonas mexicana YSL008 is a new species having a carbohydrate content of 50% by weight or more (preferably 50 to 60% by weight) and containing a large amount of carbohydrate components 1.5 to 2 times as much as other algae, and the content of glucose in carbohydrates Is 60% by weight or more (preferably 60 to 70% by weight) based on the total carbohydrate. Specifically, Clamidomonas mexicana YSL008 may contain a carbohydrate of 0.5 g / L or more (preferably 0.5 to 1.0 g / L) per 1 g of the cell dry weight.

The abovementioned Clamidomonas mexicana YSL008 can be cultivated in wastewater and is cultivated at a pH of from 7.0 to 8.0 (more preferably from pH 7.2 to 7.6) at 25 to 35 캜 (more preferably 27 to 32 캜) . In addition, culturing in a medium containing 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 trace elements) More preferable. 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.

The climidomonas mexican or YSL008 according to the present invention can be grown under the conditions of nitrogen, phosphorus (20 mg / L, 4 mg / L), so that culture can be performed without greatly diluting the dilution ratio of wastewater.

Also, since it shows the effect of removing nitrogen and phosphorus, which are pollutants in the wastewater, it can be used for wastewater treatment.

As described above, the present invention can provide a microorganism preparation for treating wastewater comprising Clamidomonas mexican strain YSL008 [KCTC12343BP] or a culture thereof.

The cultivar Clamidomonas mexican YSL008 [KCTC12343BP] according to the present invention cultivated by using wastewater has a high growth rate and a high carbohydrate-rich species More than 70% carbohydrate content), which can be used as a component for producing bioethanol from carbohydrate extraction.

The present invention also relates to a method for producing bioethanol comprising hydrolyzing microalgae having cell walls broken by pretreatment with ultrasonic waves with an enzyme, followed by acid fermentation with anaerobic bacteria to obtain bioethanol.

The method for producing bioethanol according to the present invention will be described in more detail as follows.

First, the cell wall is disrupted by pretreating Clamidomonas mexican or YSL008 [KCTC12343BP] and glycosylated.

Clomidomonas Mexicana YSL008 [KCTC12343BP] is attempted to destroy cell walls by sonication so that the monosaccharide of carbohydrate or saccharide in the algae is easily dissolved. Dissolved carbohydrates undergo hydrolysis with enzymes to increase the glucose content.

The enzyme is applicable to any condition capable of breaking the link of the cellulose series, and in the embodiment of the present invention, Trichoderma reesei ) was used.

In particular, by treating the ultrasonic waves for 5 to 30 minutes, it is possible to extract more than 50% of sugar components contained in the microalgae.

The ultrasonic waves are preferably processed at an intensity of 20 to 60 kHz and 1.0 to 5.0 Kw.

In the above, the cell wall is disrupted and hydrolyzed Clamidomonas mexican or YSL008 [KCTC12343BP] is used as a substrate and anaerobically fermented by anaerobic bacteria to obtain bioethanol.

That is, pretreated monosaccharide (crushed microalgae) is converted into ethanol in anaerobic acid fermentation process, and the selection of acid fermentation process is because sugar is rapidly converted and ethanol production is possible.

The acid fermentation is preferably performed under the conditions of 30 to 40 캜 (more preferably 33 to 37 캜) and a pH of 5.0 to 7.0 (more preferably, a pH of 5.5 to 6.5).

Since the biomass (crushed microalgae) generated above contains a large amount of carbohydrate, it can be utilized as an energy source for the next generation fermentation process, and it is very likely to be commercialized because of its utilization value of pulmonary biomass.

In addition, Clamidomonas mexican YSL008 [KCTC12343BP], which is used as a substrate for anaerobic acid fermentation, can be extracted as a carbohydrate and thus can be used as a component for producing ethanol.

As a result of analyzing the glucose in the separated carbohydrate, it was confirmed that glucose was contained in an amount of 60% by weight or more based on the whole carbohydrate, and bioethanol can be produced using the glucose.

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 1: Isolation and culture of new strain

Samples were collected from the wastewater treatment plant in Wonju, Gangwon Province. The collected samples were incubated in a 200 mL test tube with BBM liquid medium and 15% CO ₂ as shown in Table 1 for about 2 weeks. The concentration of wastewater was 20 mg / L and 4 mg / L of nitrogen and phosphorus, respectively, and the irradiance was adjusted to about 24 watts on the surface of the culture tube using a fluorescent lamp as a light source. 1 mL of the pre-incubated sample was added to a 30 mL test tube containing 10 mL of sterilized distilled water and mixed well to disperse the microalgae cells. From this, 0.1 mL of sample was taken using a micropipette and placed in a test tube containing sterile distilled water. After repeating this operation 5 times, about 0.1 mL of each sample was taken from each test tube, and the sample was inoculated into a Petri dish having a composition of BBM medium as shown in the following Table 2, And cultured in a 40 [mu] mol / m < 2 > -sec incubator for 2 to 3 weeks. When colonies of microalgae were observed, each group was transferred to a well cell culture plate using platinum under microscope and separation was started. In each hole, 5% CO ₂ was added every 10 minutes for 20 minutes at a ratio of 1: 1 to the avian community and BBM medium, followed by incubation for 10 to 14 days. In order to suppress the growth of some microorganisms, only antibiotic streptomycin was used to grow algae in this example. At this time, the amount of antibiotic used was 0.15 μm / mL per 1 L of medium. After a period of time, the cultures in each hole were observed under a microscope. At this time, only the characteristic microalgae grew in most holes, and the same shaped seeds were cultured in petri dishes prepared with BBM medium. After a certain period of time, the cultivated algae community was predominantly cultivated with one kind of community, and this community was collected and transplanted into a 250 mL Erlenmeyer flask containing 100 m of BBM medium.

Composition of liquid medium (BBM) for microalgae culture ingredient Content (per liter) KH ₂ PO ₄ 17.5 g CaCl ₂ .2H ₂ O 2.5 g MgSO ₄ .7H ₂ O 7.5 g NaNO ₃ 25 g K ₂ HPO ₄ 7.5 g NaCl 2.5 g H ₃ BO ₃ 11.42 g Trace elementary mother liquor 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 Solution (1) Na ₂ EDTA 50 g KOH 3.1 g Solution (2) FeSO ₄ 4.98 g H ₂ SO ₄ 1 mL

Composition of microbial culture medium (BBM) agar ingredient Content (per liter) KH ₂ PO ₄ 17.5 g CaCl ₂ .2H ₂ O 2.5 g MgSO ₄ .7H ₂ O 7.5 g NaNO ₃ 25 g K ₂ HPO ₄ 7.5 g NaCl 2.5 g H ₃ BO ₃ 11.42 g Trace elementary mother liquor 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 Solution (1) Na ₂ EDTA 50 g KOH 3.1 g Solution (2) FeSO ₄ 4.98 g H ₂ SO ₄ 1 mL Baby Agar powder 15 g

Example  2: Culture of isolated strains

10 ml of the above strain was added to a 250 ml Erlenmeyer flask at 30 ° C and pH 7.2 to 7.6 at 30 ° C using 15% CO ₂ in the BBM medium shown in Table 1 for about 14 days in a fluorescent stirring incubator Respectively. Stirring was maintained at 150 rpm and the illuminance was maintained at 40 μmol / m 2 -sec. Figure 1 shows the isolated strain, Clamidomonas mexicus or YSL008 strain. After adapting to wastewater, it was shown to maintain its original shape without being hindered by growth. The climidomonas Mexicana strain YSL008 contained a large amount of carbohydrates and showed that the productivity of microalgae was faster than other algae.

Example  3: Identification of isolated strains

The nucleotide sequence analysis of the isolated strain of Example 1 was analyzed by 28s rDNA sequence sequencing analysis. The state of the sample was requested in the state of a plate culture with the colonies of CML or YSL008, and PCR products by extraction-gDNA and PCR amplification were analyzed through sequencing. The primers used in the analysis are as follows.

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

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

28s rDNA sequence analysis results are shown in the attached SEQ ID NOs and phylogenetic results are shown in the phylogenetic tree results according to the Neighbor-joining method using the Jukes-Cantor model Respectively.

The nucleotide sequence of the isolated strain [SEQ ID NO: 1] was classified as CLAMIDOMONUS MICKERA AF395501.1 as shown in the following Table 3 as a result of homology in NCBI.

Birds Relative species Homology Chlamydomonas Mexicana YSL008 Chlamydomonas Mexicana
( Accession Number : AF395501 .One) 98%

As described above, the microalgae isolated in the present invention were found to belong to Clamidomonas mexicanus when the characteristics such as shape, optimum temperature and pH were taken into consideration.

Accordingly, the new strain according to the present invention was named as Clamidomonas mexicella or YSL008, and was deposited with the KRC Biotechnology Center on Dec. 28, 2012 and received the accession number KCTC12343BP.

Example  4: Wastewater resistance and waste water My inn , Nitrogen removal confirmation

Nitrogen in MF (Micro Filtration) the rough livestock wastewater [N, P (20 mg / L, 4 mg / L) contained] The embodiment of birds Chlamydomonas Mexicana YSL008 (Chlamydomonas Mexicana YSL008) obtained by Example 1 and Algae were seeded to examine removal of phosphorus. Each of the reactors was kept at a temperature of 20 to 22 ° C, pH was maintained at 7.7 to 7.9 in a natural state, and a 500 mL bottle was subjected to an ash experiment with an initial algae concentration of 0.5 g / L. During the reaction period, air aeration was carried out, and a 24 W white bulb was installed to constantly irradiate the light. The time it takes to adapt to the first post-breeding birds was about 5 days, and then the removal of nitrogen and phosphorus occurred regardless of the initial concentration of wastewater and BBM.

In FIG. 3, the reduction of nitrogen and phosphorus rapidly proceeded within 7 to 10 days after the adaptation of the algae, and it is considered that rapid phosphorus and nitrogen removal will occur when a steady-state algae is applied to the reactor.

Example  5: Identification of cell components

The isolated strain of Example 1, Clamidomonas Mexicana or YSL008 ( Chlamydomonas Mexicana YSL008) was composed of carbohydrates in more than 50% of the dry cell weight. Analysis of each part was carried out by phenol-sulfuric acid method for carbohydrate, bradford assay for protein and Lowry Assay for lipid. Composition analysis of 1 g algae revealed that the carbohydrate was more than 50 wt% of the total weight (Fig. 4). However, in the case of not pretreating, only 1% (0.01 g by weight) of dissolved carbohydrate is present in water and only trace amounts of carbohydrate are present.

In order to investigate the distribution of carbohydrates contained in Clamidomonas mexicana YSL008, a pelleted sample of 1 g algae was pretreated to examine the effect of carbohydrate distribution on the pretreatment. The sample was analyzed using Bio-LC (ICS-5000) and the column used was CarboPac PA1 (Dionex). As a result, about 65% of the carbohydrates appeared as glucose components, and the amount of dissolved carbohydrates that could be easily used for energy conversion increased with pretreatment [Table 4].

Example  6: Identification of carbohydrate changes according to microbial growth conditions

FIG. 5 shows changes in carbohydrate concentration of Clamidomonas mexicana and YSL008 according to culture conditions.

Algae growth depends on light and carbon source concentrations, and the composition of the carbohydrates, proteins, and fats that make up the algae is also affected by the external conditions supplied. The distribution of carbohydrates in the algae showed 5 ~ 10% difference between the BBM medium made of the organic elements required for the growth of algae, the wastewater containing abundant organic elements and the carbon source from carbon dioxide. The wastewater used was the same as in Example 4, and the BBM medium was as shown in Table 1.

Example  7: Identification of carbohydrate changes by enzymatic hydrolysis

As confirmed in Example 6 above, in the case of Clamidomonas mexicana or YSL008, a high carbohydrate content of 50 wt% based on the dry weight of the cells and at least 60 wt% of the carbohydrate constituents formed glucose, and Trichoderma Hydrolysis of the microalgae was carried out for about 18 hours at pH 6 with reesei enzyme, and the changes in carbohydrate composition were confirmed.

FIG. 6 shows hydrolysis results according to the amount of enzyme injected. [E] is the enzyme Trichoderma reesei , and [S] is the rate according to the injection rate with Clamidomonas mexicana or YSL008 biomass.

 As a result of enzyme hydrolysis, as shown in Table 4, in order to examine the change of glucose content in the components of carbohydrate and the maximum amount of enzyme to be injected as shown in FIG. 6, the maximum carbohydrate conversion rate by enzyme injection amount was examined and a bio- . The optimal conditions for hydrolysis of the enzyme by pH were examined and shown in FIG. The pH range was 3-9 in acid and base conditions. The optimal pH condition for the hydrolysis of the enzyme is 5.5, which does not greatly affect the anaerobic fermentation process (pH) immediately after the enzyme treatment.

Therefore, it can be applied as an energy source without additional process because it is a pH condition that can ferment the fermentation process without increasing the glucose content by enzymatic hydrolysis of the bio-ethanol compatible microalgae Clamidomonas mexicana or YSL008 have.

Example  8: Manufacture of bioethanol

1) Carbohydrate extraction (preprocessing)

1% of the cultured Clamidomonas mexicana YSL008 was recovered, diluted with distilled water, and treated with ultrasonic waves of 40 kHz and 2.2 Kw for 15 minutes to break the cell wall by 60% or more. The enzyme, Trichoderma Reesei ) to increase the glucose content in the carbohydrate to 60% by weight or more.

It was confirmed that the dissolved carbohydrate was eluted at about 0.6 g / L per 1 g of the dry weight of the cells after hydrolysis of hydrolyzed Clamidomonas mexicana YSL008 (hereinafter referred to as crushed algae). This increased the dissolved carbohydrate by 19-21% over the total weight compared to the dissolved carbohydrate measured before shredding.

Comparison of dissolved carbohydrate content before and after pretreatment of Clavidomonas Mexicana YSL008 division Concentration of dissolved carbohydrate before crushing
(mg / g) Dissolved carbohydrate concentration after crushing + hydrolysis (mg / g) Dissolved carbohydrate growth rate (%) Clamidomonas Mexicana YSL008 10-14 188-204 19-21

2) Anaerobic acid fermentation

The crushed algae of 1) above is mixed with the anaerobic bacteria Clostridium sp.) was added to the fermenter. Anaerobic bacteria were glucose 10,000 mg / L; NH ₄ HCO ₃ , 4,700 mg / L; K ₂ HPO ₄ , 125 mg / L; MgCl ₂ .6H ₂ O, 100 mg / L; MnSO ₄ .6H ₂ O, 15 mg / L; FeSO ₄ .7H ₂ O, 25 mg / L; CuSO ₄ .5H ₂ O, 5 mg / L; And CoCl _{2 5H 2 O, 0.1 mg / L} ; NaHCO ₃ , 2,000 mg / L, and the retention time (HRT) was adjusted to 12 hours. The reaction tank stabilized with Clostridium was fermented using Clamidomonas Mexicana or YSL008 as a substrate and recovered high quality bioethanol. At this time, the shredded algae and anaerobic bacteria were treated at 35 ° C and pH 5.5 Lt; RTI ID = 0.0 > to 6.0 < / RTI >

The efficiency of 34 ~ 43% more than the conventional materials such as sugar cane and corn was better.

The bioethanol generated in the acid fermentation tank is ethanol produced per biomass, and pure bioethanol can be obtained by the subsequent distillation.

FIG. 8 shows ethanol production according to Clamidomonas mexicana and YSL008 biomass. Ethanol production was about 0.3 g / L in the case of pretreatment using ultrasound in the production of ethanol and about 0.33 g / L in the case of adding enzyme pretreatment (18 hours). This is the conversion of 0.5 g / L of carbohydrate to about 67% of ethanol in 1 g / L of total biomass. After pretreatment of the enzyme, the amount of dissolved carbohydrate that the anaerobic bacteria could ingest was increased (Fig. 6), and the production lag time of ethanol was about 4 days faster than the other pretreatment conditions. The ethanol production rate was found to be faster as the concentration of dissolved carbohydrate was higher in pretreatment of microalgae. The level of ethanol production was about 7 times higher than that of untreated Clamidomonas Mexica or YSL008.

Korea Biotechnology Research Institute KCTC12343BP 20121228

<110> Yonsei University Wonju Industry-Academic Cooperation Foundation <120> Novel strain Chlamydomonas mexicana YSL008 <150> 1020130002096 <151> 2013-01-08 <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 870 <212> DNA <213> Chlamydomonas mexicana YSL008 <400> 1 agcggaggaa aagaaactaa ccaggawagc cctagtaacg gcgagtgaag cggctatagc 60 ccaacttgaa aatctccctc tggagaattg tagtctatag aagtgccctc tgtatcgggc 120 ggagctcaag tccactggaa agtggcgtca gagagggtga gaaccccgtc ggctcctgct 180 tcgatgctcc acgaggtact ttccacgagt cgggttgttt gggaatgcag ccctaatttg 240 gtggtaaatc ccatctaagg ctaaatactg gcgagagacc gatagcgaac aagtaccgtg 300 agggaaagat gaaaagaact ttgaaaagag agtcaaaaag tgcttgaaat tgttgagggg 360 gaagcgattg gatgccgtgt gtgcgcccag gctcaagcgg ttctaacgag ccgttgaatg 420 tgctgggtgc tggtcaactc gggttgcttt ggcgggataa cagtcgggaa cgtaggtagc 480 ctggccgatg ccgccgaagc gaccaaggcg tgatgggcgc tccgtccttc gggaactgcg 540 tccaaaagga gttggcagaa ggctttcaat cggcccgtct tgaaacacgg accaaggagt 600 ctaacatgta tgcgagttgg cgggtggcaa acccgtaagc gcaagtaacc tgactggtgg 660 gatggcgtaa gcctgcacca tcgaccgacc atgagcttct gcgaaaggtt tgagtgcgag 720 catacctgtt gggacccgaa agatggtgaa ctatgcctga gcagggtgaa gccagaggaa 780 actctggtgg aggctcgtag atgtgctgac gtgcaaatcg cttwtcggac ttgggtgata 840 ggggcgaaag actaatcgaa ccttctagta 870 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 agcggaggaa aagaaacta 19 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 tactagaagg ttcgattagt c 21

Claims (12)

A Chlamydomonas Mexicana YSL008 strain with a carbohydrate content of at least 50% by weight based on the dry weight of the composition and having a deposit number of KCTC 12343BP.
The method according to claim 1,
A clamidomonas mexican strain YSL008 having the nucleotide sequence of SEQ ID NO: 1.
delete A microorganism preparation for treating wastewater comprising the Clamidomonas mexicana strain YSL008 of claim 1 or a culture thereof.
A method for culturing Clamidomonas mexican or YSL008 strain of claim 1 in wastewater.
6. The method of claim 5,
Wherein the strain is cultured at 25 to 35 占 폚 and at a pH of 7.0 to 8.0.
6. The method of claim 5,
Characterized in that the strain is cultured in 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 trace elements Way.
8. The method of claim 7,
Wherein the trace element is 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 .
Hydrolyzing a strain of Chlamydomonas Mexicana YSL008 with KCTC 12343BP, which has been pretreated with an ultrasonic wave and having a cell wall disrupted, with an enzyme, followed by fermentation with anaerobic bacteria to obtain bioethanol &Lt; / RTI &gt;
10. The method of claim 9,
Wherein the ultrasonic waves are treated at 20 to 60 kHz, 1.0 to 5.0 Kw, and 5 to 30 minutes.
10. The method of claim 9,
Wherein the enzyme is Trichoderma reesei.
10. The method of claim 9,
Wherein the acid fermentation is carried out at 30 to 40 DEG C and at a pH of 5.0 to 7.0.

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