KR101398727B1 - A novel algal strain Chlorella vulgaris YSL001 for hydrogen production in both aerobic and anaerobic conditions - Google Patents

Abstract

The present invention relates to a novel strain, Chlorella vulgaris YSL001 KCTC 12144BP strain, more specifically, a hydrogen production example is extremely rare, and most studies are carried out under anaerobic hydrogen generation and organic matter abundant conditions, The present invention relates to a novel strain of Chlorella vulgaris YSL001 KCTC 12144BP which is capable of producing hydrogen even in an anaerobic condition, an organic condition, an exhalation condition, or an independent nutritional condition containing atmospheric carbon dioxide, by activating a hydrogen producing enzyme.

Description

A novel strain capable of producing hydrogen under aerobic / anaerobic conditions. Chlorella vulgaris YSL001 {A novel algal strain Chlorella vulgaris YSL001 for hydrogen production in both aerobic and anaerobic conditions}

The present invention relates to a novel strain Chlorella vulgaris YSL001 capable of producing hydrogen under aerobic / anaerobic conditions.

The increase in energy use on the earth continues to increase, causing a lot of environmental pollution, resulting in global warming [Non-Patent Document 1]. Biomass is a renewable energy source that does not affect the greenhouse effect. [Non-Patent Document 2] It is continuously being developed as energy for mass production (hydrogen, methane, ethanol). From another perspective, photosynthetic strains belonging to biomass release hydrogen during metabolic activity, and hydrogen is considered as the ultimate alternative energy source of the future as clean energy. Photosynthetic bacteria are classified into purple non-sulfur bacteria, purple sulfur bacteria, green non-sulfur bacteria and green sulfur bacteria, and aerobic bacteria Conditions, anaerobic dark conditions, and anaerobic light conditions. Rhodobacteria microorganisms, which are non-sulfur non-sulfur bacteria, have been used as a main object of development of alternative energy technology using microorganisms due to their high ability to convert solar energy into hydrogen, a clean chemical energy, during metabolism [Non-Patent Document 3]. Unlike photosynthesis of algae or plants, they do not produce oxygen during the process. On the other hand, chlorella genus has relatively unknown genes of components of hydrogen production nitrogenase and hydrogenase. Chlorella is the most widely known of birds, and its distribution is also easily found in various regions. Currently, the research direction of birds is mainly focused on the enhancement of hydrogen production through cultivation, unlike the research on genetic mutation of bacteria. This approach has led to a significant increase in hydrogen production, but this approach has limitations. Rhodobacter sphaeroides KCTC 12085 is a photosynthetic strain isolated from nature and has high hydrogen production ability and high resistance to salt and has similar or higher hydrogen production ability than other hydrogen producing photosynthetic strains, And it has been reported [Non-Patent Document 3]. The hydrogen production of the above photosynthetic strains is a result of proton (H ⁺ ) fixation mainly by nitrogen fixation enzyme, and the energy consumed at this time depends entirely on the light reaction. On the other hand, most photosynthetic bacteria and algae have high oxygen (O ₂ = 0.21 atm). However, hydrogenation enzymes that generate hydrogen are reported to be active mainly in anaerobic conditions, such as Ni-Fe-Hydrogenase (Non-Patent Document 4) and Fe-only Hydrogenase (Non-Patent Document 5) . In recent years, hydrogen-producing enzymes having oxygen resistance have been found in non-sulfur non-sulfur bacteria, and the possibility of hydrogen production in a natural environment (aerobic condition) is shown [Non-Patent Document 6].

Therefore, studies to clarify the culture conditions that can induce activation and stabilization of hydrogenation enzymes related to hydrogen production in more aerobic conditions are necessary to overcome these limitations.

Chisti, Y. 2007. Biotechnology Advances, 25: 294-306 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 Lee et al. 2002. Improvement of Photoheterotrophic Hydrogen Production of Rhodobacter sphaeroides by Removal of B800-850 Light-Harvesting Complex. Appl. Microbiol. Biotechnol. 60: 147-153 Appel et al. 2000. The bidirectional hydrogenase of Synechocystis sp. PCC 6803 works as an electron valve during photosynthesis.Arch Microbiol. 173: 333-338 Pan et al. 2003. Characterization of a basic [2Fe-2S] -cluster-containing protein from the Fe-only hydrogenase operon of Thermotoga maritime. J. Biol. Inorg. Chem. 8: 469-474 Liebgott et al. 2011. Original Design of an Oxygen-Tolerant [NiFe] Hydrogenase: Major Effect of a Valine-to-Cysteine Mutation near the Active Site. J. Am. Chem. Soc. 133, 986-997

The present inventors have studied to obtain a microalgae strain producing hydrogen, and as a result, it has been found that an independent nutrient microalga species in which hydrogen is generated in an aerobic state, which is a natural atmospheric state, Thereby confirming that hydrogen is useful in the production of hydrogen in a lake water environment, thereby completing the present invention.

Accordingly, the invention is a novel Chlorella vulgaris YSL001 (Chlorella vulgaris YSL001) KTCT12144BP strain.

The present invention also provides a method for solving the environmental problem of the greenhouse effect as well as the alternative energy by utilizing the high concentration of carbon dioxide as a carbon source in the course of energy production by growing the new strain in aerobic / There is a purpose.

As a means for solving the above problems, the present invention is to produce hydrogen Chlorella vulgaris YSL001 (Chlorella vulgaris YSL001) KTCT12144BP strain.

As another means for solving the above problems, the present invention provides a microorganism preparation for hydrogen production comprising a strain of Chlorella vulgaris YSL001 KTCT12144BP or a culture thereof.

As another means for solving the foregoing problems, the present invention is Chlorella vulgaris YSL001 (Chlorella The present invention provides a method for producing hydrogen, comprising culturing a KTCT12144BP strain.

As another means for solving the above problems, the present invention provides a hydrogenation enzyme isolated from a strain of Chlorella vulgaris YSL001 KTCT12144BP.

The invention Chlorella vulgaris YSL001 (Chlorella The present study was conducted to investigate the energy production and oxygen - tolerant hydrocortisone production by aerobic / anaerobic conditions and excess carbon dioxide injection using KTCT12144BP strain.

The hydrogenase of the new strain of the present invention has a strong oxygen tolerance and can be applied to carbon dioxide of the new strain, thus greatly improving the economical efficiency of the culture.

In addition, the grown chlorella bulgurris strain YSL001 KTCT12144BP shows hydrogen production not only in aerobic condition but also in anaerobic condition, and thus has an effect of contributing to the hydrogen production of microalgae in a lake water environment.

Figure 1 is a photograph of a strain of Chlorella vulgaris YSL001 KTCT12144BP.
Fig. 2 shows the nucleotide sequence of 23s r DNA of the strain Chlorella vulgaris YSL001 KTCT12144BP.
Fig. 3 shows the phylogeny of the strain Chlorella vulgaris YSL001 KTCT12144BP.
Fig. 4 shows hydrogen production in the anaerobic, aerobic and autotrophic (15% (v / v) / 15% (v / v) oxygen) condition of the strain of Chlorella vulgaris YSL001 KTCT12144BP.
Figure 5 shows the hydrogenase activity of the strain Chlorella vulgaris YSL001 KTCT12144BP.

Hereinafter, the present invention will be described in detail.

The novel strain of the present invention, chlorella bulgaris YSL01 ( Chlorella vulgaris YSL01) was first isolated and identified by the present inventors. The strains were isolated using BBM medium in a lake, and the isolated strains were analyzed by molecular genetic analysis by 28s rDNA sequencing search. As a result, It was confirmed that the strain is 98% similar to the virulence AB237642.1.

The present inventors named chlorella bulgaris YSL001 as the above-identified isolate, and deposited this on February 23, 2012 to the BRC of the Korea Biotechnology Research Institute and received the accession number KTCT12144BP.

Particularly, the chlorella bulgaric YSL001 ( Chlorella vulgaris YSL001) has a 28s r DNA sequence shown in SEQ ID NO: 1, and it can be confirmed that hydrogen production is possible under anaerobic, aerobic and autotrophic conditions. The anaerobic condition refers to a state in which oxygen is absent and the aerobic condition means atmospheric conditions in which oxygen exists, specifically, an oxygen concentration of 5% (v / v) or more (preferably 5 to 35% (v / v)) The autotrophic condition means an atmospheric state in which carbon dioxide is contained at a high concentration, specifically, an atmospheric state in which the concentration of carbon dioxide is 5 to 20% (v / v). Carbon Dioxide is closely related to the global greenhouse effect problem, and it takes a solution with environmental issue.

Hydrogen production can be achieved in all the natural environments of lake water because chlorine blebs YSL001 according to the present invention can produce hydrogen under all conditions such as aerobic and anaerobic high concentration carbon dioxide atmospheric conditions.

The present invention also relates to a method for the treatment of Chlorella vulgaris YSL001 YSL001 vulgaris) it is possible to provide a microbial agent for the hydrogen production, including KTCT12144BP strain or culture fluid thereof.

The present invention also relates to a method for the treatment of Chlorella vulgaris YSL001 YSL001 vulgaris) it is possible to provide a method for producing hydrogen, comprising the step of culturing the strain KTCT12144BP.

The chlorella bulgurris YSL001 is cultivated in BBM medium consisting of 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 at 20 to 30 ° C (Preferably 25 to 27 DEG C) at a pH of 7.0 to 9.0 (preferably at a pH of 7.5 to 8.5). The trace elements are ZnSO ₄ and 7H ₂ O, MnCl ₂ and _{4H 2 O, MoO 3, CuSO} 4 and _{5H 2 O, Co (NO 3} ) 2 and _{6H 2 O, Na 2 EDTA,} KOH, FeSO 4 and H ₂ SO _4, and the like. In particular, the medium is KH ₂ PO ₄ 17.3 to 17.7 g / L, CaCl ₂ .2H ₂ O 2.3 to 2.7 g / L, MgSO ₄ .7H ₂ O 7.3 to 7.7 g / L, NaNO ₃ 24.8 to 25.2 g / L, K ₂ HPO ₄ More preferably 7.3 to 7.7 g / L, 2.3 to 2.7 g / L of NaCl and 3 to 5 mL of trace elements.

The present invention also relates to a method for the treatment of Chlorella vulgaris YSL001 YSL001 vulgaris) it is possible to provide a hydrogenation enzyme having an oxygen resistant strain isolated from KTCT12144BP.

Hydrogenase activity of algae can be analyzed using methyl viologen, and the new strain of the present invention was also analyzed for hydrogenase activity by the conventional method as described above.

In addition, activity of the hydrolytic enzyme analyzed in this manner is observed not only in the anaerobic state but also in the aerobic condition, and hydrogen production can be performed by the hydrogenase activity, and the present invention can also include hydrogenase expression in the aerobic state.

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: New strain  detach

Samples were collected from Lake Magi, Yonsei University in Wonju, Gangwon Province. The collected samples were incubated in a 200 mL test tube for about 2 weeks in a BBM liquid medium as shown in Table 1 below. 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 the sample was taken from each test tube, inoculated into a Petri dish having a composition of BBM medium as shown in the following Table 2, And 50 [mu] mol / m < 2 > -sec of light intensity 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, the avian community and BBM medium were mixed at 1: 1 and cultured for 10-14 days (Table 1). In order to inhibit the growth of some microorganisms, only the algae were grown using the antibiotic Streptomycin in this Example. At this time, the amount of antibiotic used was 0.15 μm / mL per 1 L of medium. After a certain period of time, colonies cultured 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 (Table 2). After a certain period of time, the cultured algae colonies were predominantly cultured as one kind of colonies. The colonies were harvested and transplanted into a 250 mL Erlenmeyer flask containing 100 mL of BBM medium (Table 1).

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 Microelement Stock Solution ZnSO ₄ .7H ₂ O 8.82 g H ₃ BO ₃ 11.42 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 Microelement Stock Solution ZnSO ₄ .7H ₂ O 8.82 g H ₃ BO ₃ 11.42 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 Agar Agar powder 15 g

Example  2: New strain  culture

The culture medium of BBM [KH ₂ PO ₄ , CaCl ₂ .2H ₂ O, MgSO ₄ .7H ₂ O, NaNO ₃ , K ₂ HPO ₄ , NaCl and trace elements (H ₃ BO ₃ , ZnSO ₄ .7H ₂ O, MnCl ₂揃 4H ₂ O, MoO ₃ , CuSO ₄揃 5H ₂ O, Co (NO ₃ ) ₂揃 6H ₂ O, Na ₂ EDTA, KOH, FeSO ₄ and H ₂ SO ₄ ) The strain isolated in Example 1 was inoculated in a 250 mL Erlenmeyer flask at 25 to 27 DEG C under a pH of 7.5 to 8.5 with 10 mL of the above strain and cultured in a fluorescence agitating incubator for about 14 days. Stirring was maintained at 150 rpm and the illuminance was maintained at 50 μmol / m 2 -sec.

Example  3: New strain  Sympathy

The nucleotide sequences of the strains isolated in Example 1 were analyzed by 28s rDNA sequence sequencing.

The state of the sample was referred to as a plate culture medium with the colony of Example 1, and PCR products by Extraction-gDNA and PCR amplification were analyzed by sequencing in the request details. The sequences identified by the primers used in the analysis are as follows.

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

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

The results of the 28S rDNA sequence analysis of the isolated strain are shown in SEQ ID NO: 1 of the attached sequence listing [Fig. 2], and phylogenetic results are shown in the Neighbor-joining method using the Jukes-Cantor model, The results of the phylogenetic tree according to the method are shown in FIG.

The nucleotide sequence [SEQ ID NO: 1] of the isolate was classified as Chlorella vulgaris as shown in the following Table 3 as a result of homology to NCBI.

Isolated strain Sequence length (nt ^a ) Relative species %
Homology Chlorella vulgaris YSL001 883 Chlorella vulgaris AB237642.1   98

^a nucleotide

As described above, the microalgae isolated in the present invention were found to belong to chlorella bulgaricus in consideration of characteristics such as shape, optimum temperature and pH, except that they produced hydrogen even under aerobic conditions.

Thus, the Chlorella vulgaris strain according to the invention YSL001 (Chlorella vulgaris YSL001), and deposited on Feb. 23, 2012 with the Korea Research Institute of Bioscience and Biotechnology as the deposit number KTCT12144BP.

Example  4: Verification of hydrogen production

For the hydrogen production, chlorella bulguris strain YSL001 KTCT12144BP strain was used as a target strain. To grow the strain, the pH was adjusted to 7.5-8.5, the stirring speed was 150 rpm, the incubation temperature was 27 ° C using the minimum BBM medium shown in Table 1 At the same time, the culture was maintained at an intensity of 50 μmol / ㎡-sec for 5 weeks.

Here, in order to optimize the activity of the hydrogenation enzyme for the independent nutrition, Na ₂ EDTA contained in the BBM medium of Table 1 was not added and the experiment was carried out in the absence of a carbon component in the medium.

The cells were grown in an incubator in a bottle designed to prevent leaking of air in order to examine hydrogen production efficiency and hydrogen production of the bacteria. In addition, conditions in which the natural gas layer is grown as it is, a condition containing 15% (v / v) CO ₂ , 15% (v / v) O _{2 in} which carbon dioxide is increased about 150 times in the atmosphere, (Gas Chromatography, GC, Shimadzu). The gas phase was removed through a syringe designed to prevent air from leaking out of the gas phase.

4 and 5 are anaerobic (0% (v / v) O 2), aerobic (21% (v / v) O 2), autotrophic (15% (v / v) CO 2/15% (v / v) O ₂ ) conditions and the activities of chlorella bulgur hydrogen production and hydrogenation enzymes.

Until now, hydrogenation enzymes have been generally found in optical microorganisms or fermenting microorganisms, and their activity has been known in anaerobic conditions [Kessler E., 1974. Hydrogenase, photoreduction, and anaerobic growth. In Stewart WDP (ed.), Algal Physiology and Biochemistry. Blackwell, Oxford, pp. 456-473].

Hydrogenase was a major reaction mechanism in the production of hydrogen. In order to observe the production of hydrogen in the natural environment instead of the anaerobic condition, we observed hydrogen production and hydrogenase activity in anaerobic and aerobic conditions.

In addition, the chlorella bulgaris YSL001 KTCT12144BP strain is inhabited at pH 7.5 to 8.5, and the carbon dioxide in the atmosphere is dissolved in HCO ₃ ^- form in water, and microorganisms and the like take it as a carbon source. Therefore, we observed the activity of hydrogen production and hydrogenation enzyme by increasing the concentration of carbon dioxide to increase the byproducts generated by the strains when the activity of the strains increased. Hydrogenase activity of algae was determined by using methyl viologen.

In summary, in the present invention, the possibility of hydrogen production and the hydrogenation enzyme activity at the aerobic condition, which is the environment where the new strain, Chlorella vulgaris, is inhabited, have been investigated to try to produce hydrogen. To date, hydrogen production has been studied only in anaerobic conditions among photosynthetic strains [Rao, K.K. and Hall, D.O. 1996. Hydrogen production by cyanobacteria: potential, problems and prospects. J Mar Biotechnol. 4, 10-15] There is no report on new hydrogen production of algae under aerobic conditions.

Therefore, in the present invention, using the novel strain Chlorella vulgaris, the hydrogen production capacity is improved by securing a new strain in which hydrogen is produced even under aerobic conditions and in an autotrophic condition containing a large amount of carbon dioxide, as well as aerobic conditions.

Korea Biotechnology Research Institute KCTC12144BP 20120223

Attach an electronic file to a sequence list

Claims (14)

Chlorella Bulgarian YSL001 producing hydrogen ( Chlorella vulgaris YSL001) KTCT12144BP strain.
. The method according to claim 1,
Chlorella producing hydrogen in anaerobic condition vulgaris YSL001 (Chlorella vulgaris YSL001) KTCT12144BP strain.
The method according to claim 1,
Chlorella producing hydrogen under aerobic conditions vulgaris YSL001 (Chlorella vulgaris YSL001) KTCT12144BP strain.
The method according to claim 1,
Chlorella producing hydrogen under autotrophic conditions vulgaris YSL001 (Chlorella vulgaris YSL001) KTCT12144BP strain.
The method according to claim 1,
A strain Chlorella vulgaris YSL001 KTCT12144BP having the 28s r DNA sequence shown in SEQ ID NO: 1.
Chlorella vulgaris according to claim 1, wherein YSL001 (Chlorella vulgaris YSL001) microbial agent for hydrogen production, including KTCT12144BP strain or culture fluid thereof.
Chlorella vulgaris according to claim 1, wherein YSL001 (Chlorella vulgaris YSL001) method of hydrogen production which comprises culturing the strain KTCT12144BP.
8. The method of claim 7,
And culturing the strain under anaerobic conditions.
8. The method of claim 7,
And culturing the strain under aerobic conditions.
8. The method of claim 7,
And culturing the strain under autotrophic conditions.
8. The method of claim 7,
Culturing the strain in a medium consisting of 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. .
8. The method of claim 7,
Culturing the strain at a pH of 7.0 to 9.0 at 20 to 30 占 폚.
8. The method of claim 7,
And culturing the strain with carbon dioxide as a carbon source.
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