KR20080012083A - Culture method for high concentration of thiocapsa reseopersicina - Google Patents

Culture method for high concentration of thiocapsa reseopersicina Download PDF

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KR20080012083A
KR20080012083A KR1020060073105A KR20060073105A KR20080012083A KR 20080012083 A KR20080012083 A KR 20080012083A KR 1020060073105 A KR1020060073105 A KR 1020060073105A KR 20060073105 A KR20060073105 A KR 20060073105A KR 20080012083 A KR20080012083 A KR 20080012083A
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thiocapsa
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김미선
오유관
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한국에너지기술연구원
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/0004—Oxidoreductases (1.)
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Abstract

A culture method for high concentration of Thiocapsa roseopersicina is provided to maximize the growth of cells under photo-autotrophic conditions, so that a large quantity of hydrogenase is produced. A high concentration of Thiocapsa roseopersicina NCIB 8347 is photo-autotrophically cultured in a medium containing acetate as a carbon source under the carbon dioxide-containing nitrogen condition at 26 deg. C for 3-5 days, wherein the medium further contains ammonium chloride and a trace amount of elements including Na2S.9H2O, Na2.EDTA, FeSO4.7H2O, CoCl2.6H2O, MnCl2.2H2O, ZnCl2, NiCl2.6H2O, Na2MoO4.2H2O, H3BO3 and CuCl2.2H2O.

Description

티오캅사 로세오페르시키나의 고농도 균체 배양 방법{Culture Method for high concentration of Thiocapsa reseopersicina}Culture method for high concentration of Thiocapsa reseopersicina

도 1은 광합성 종속영양 조건에서의 티오캅사 로세오페르시키나의 성장 곡선 및 수소생산을 측정하여 나타낸 그래프이다. Figure 1 is a graph showing the growth curve and hydrogen production of thiocapsa roseeo Pershina in photosynthetic heterotrophic conditions.

도 2는 티오캅사 로세오페르시키나에 대한 아세테이트 첨가배지에서의 생육곡선 및 하이드로게나아제 생산을 측정하여 나타낸 그래프이다. (○, cell concentration; ●, hydrogenase activity)FIG. 2 is a graph showing the growth curve and the hydrogenase production in the acetate addition medium to thiocapsa roseeopercyna. (○, cell concentration; ●, hydrogenase activity)

도 3은 티오캅사 로세오페르시키나 배양 시, 연속적으로 아세테이트를 첨가하는 배양조건에서의 생육곡선 및 하이드로게나아제 활성을 측정하여 나타낸 그래프이다. (↓ 아세테이트 첨가 0.05%)Figure 3 is a graph showing the growth curve and the hydrogenase activity in the thiocapsa Roseeo Pershina culture, culture conditions in which the addition of acetate continuously in the culture. (↓ 0.05% acetate added)

도 4는 티오캅사 로세오페르시키 NCIB 8347을 연속적으로 영양물질을 공급하면서 배양할 때 균체 성장 및 하이드로게나아제 생산을 측정하여 나타낸 그래프이다. (▽, cell concentration; ○, hydrogenase specific activity; ●, hydrogenase total activity)4 is thiocapsa Roseeo Persiki It is a graph showing the measurement of cell growth and hydrogenase production when NCIB 8347 is cultured continuously with feeding nutrients. (▽, cell concentration; ○, hydrogenase specific activity; ●, hydrogenase total activity)

본 발명은 티오캅사 로세오페르시키나 NCIB 8347의 고농도 배양방법에 관한 것으로, 더욱 상세하게는 홍색 유황 광합성세균인 티오캅사 로세오페르시키나 NCIB 8347을 탄소원으로 아세테이트를 포함하는 배지에 광합성 독립영양 조건으로 배양하여 고농도 균체를 배양하는 방법에 관한 것이다. The present invention is thiocapsa Roseeo Pershina The present invention relates to a high-concentration culture method of NCIB 8347, and more particularly, to a method of culturing high-concentration cells by culturing red sulfur sulfur photosynthetic bacterium thiocapsaceopercyna or NCIB 8347 in a medium containing acetate as a carbon source under photosynthetic autotrophic conditions. It is about.

수소를 생산하는 광합성 세균은 홍색 비유황세균 (purple non-sulfur bacteria), 홍색 유황세균 (purple sulfur bacteria), 녹색 유황세균 (green sulfur bacteria)으로 나누어진다. Hydrogen-producing photosynthetic bacteria are divided into purple non-sulfur bacteria, purple sulfur bacteria, and green sulfur bacteria.

홍색세균은 세포막에서 광합성 메카니즘이 일어나며, 이때 존재하는 박테리오클로로필에 의해 빛을 흡수하고 광합성 작용Ⅰ을 한다. 흡수파장 중 일부는 800㎚ 이상에서 높은 흡광도를 내기 때문에 붉은색을 띄는데 이러한 점이 광합성 작용Ⅰ과Ⅱ를 동시에 하는 녹조류나 고등식물과 다르다. Photosynthetic mechanism occurs in the cell membrane, at this time absorbs light by the existing bacteriochlorophyll and performs the photosynthetic action I. Some of the absorption wavelengths are red because of their high absorbance at 800 nm and above, which is different from green algae and higher plants that simultaneously perform photosynthesis action I and II.

홍색세균은 비교적 작은 분류군으로 30~40여 개의 균종으로 구성되어 있지만 유전정보 중 GC 함량이 46~73%의 광범위한 분포를 나타내는 것으로 보아서 작은 분류군이지만 유전적으로는 다양한 유연성을 갖는 균종으로 구성되어있다. The red bacterium is a relatively small taxon and is composed of 30-40 species, but it is a small taxon because the GC content is 46-73% of the genetic information.

황화수소와 같은 환원 유황 화합물을 산화하여 환원력을 공급할 수 있는 여부에 따라 홍색 유황세균과 홍색 비유황세균으로 구분되며, 이들은 유황화합물 산 화력 외에도 생리학적 및 생태학적인 차이점이 뚜렷하게 다르다.Red sulfur bacteria and red non-sulphur bacteria are classified according to whether or not reducing sulfur compounds such as hydrogen sulfide can be oxidized to supply reducing power, and physiological and ecological differences in addition to sulfur compound oxidation power are distinctly different.

홍색 유황세균에 속하는 대표적인 세균은 Thiocapsa roseopersicina, Chromatium rinosum, C. okenii, Thiospirillum jenense 등이 있고, 홍색 비유황세균에 속하는 세균은 Rhodobacter spaeroides, Rhodospirillum rubrum, Rhodopeudomonas palustris 등이 있다. Representative bacteria belonging to the red sulfur bacteria include Thiocapsa roseopersicina , Chromatium rinosum , C. okenii , Thiospirillum jenense , and bacteria belonging to the red non-sulfur bacteria include Rhodobacter spaeroides , Rhodospirillum rubrum and Rhodopeudomonas palustris .

홍색 유황세균은 광합성 독립 영양(photo-autorophic)을 하므로 공기 중의 이산화탄소를 고정하여 탄수화물 등 고분자 물질을 합성하고, 광합성 인산화 과정에서 ATP를 생산하며, 동시에 H2S가 산화하는 과정 중이나 분자 상의 수소가 산화할 때 발생하는 전자가 환원력을 공급한다. Since red sulfur bacteria are photosynthetic photo-autorophic, they fix carbon dioxide in the air to synthesize high-molecular substances such as carbohydrates, and produce ATP during photosynthetic phosphorylation, while H 2 S oxidizes and The electrons generated when oxidizing provide reducing power.

Figure 112006055730538-PAT00001
Figure 112006055730538-PAT00001

홍색 비유황세균은 반면 광합성 종속영양(photo-heterotropic)을 하기 때문에 외부에서 공급되는 유기물질을 탄소원으로 사용하며, 이로부터 전자가 공급된다.On the other hand, because the red non-Sulfur bacteria are photosynthetic heterotrophic (photo-heterotropic) using an organic material supplied from the outside as a carbon source, the electron is supplied from this.

티오캅사 로세오페르시키나(Thiocapsa roseopercisina)는 홍색 유황세균이지만 광합성 종속영양과 독립영양을 할 수 있는 대표적인 균주인데, 광합성 독립영양으로 배양할 때만 내열성이 좋으며, 산소에도 안정한 하이드로게나아제(Hydrogenase: 수소생산효소)를 생산한다. 그러나 광합성 독립영양 배양할 때 균체는 성장이 매우 느려서 원하는 하이드로게나아제를 얻기 위해서는 상당한 시일이 필요하다. Thiocapsa roseopercisina is a red sulfur bacterium, but it is a representative strain capable of independent nutrition with photosynthetic heterotrophs. It is heat resistant only when cultured with photosynthetic autotrophs and is stable to oxygen. Hydrogenase (Hydrogenase: Hydrogen) Production enzymes). However, when photosynthetic autotrophs are cultured, the cells grow very slowly, which requires a considerable amount of time to obtain the desired hydrogenase.

본 발명은 상기 종래기술이 가지는 문제를 해결하기 위하여 제시되는 것으로, 본 발명의 목적은 수소를 생산하는 홍색 유황 광합성세균인 티오캅사 로세오페르시키나NCIB 8347을 최적 배지에 배양하여 고농도 균체를 배양하는 방법을 제공하는데 있다.The present invention is presented to solve the problems of the prior art, an object of the present invention is to cultivate high concentration cells by culturing thiocapsa roseeopercyna or NCIB 8347, a red sulfur photosynthetic bacterium that produces hydrogen in an optimal medium To provide a method.

상기 목적을 달성하기 위한 수단으로, 본 발명은 티오캅사 로세오페르시키나(Thiocapsa roseopercisina) NCIB 8347을 탄소원으로 아세테이트를 포함하는 배지에 배양하여 고농도 균체를 배양하는 방법을 제공한다. As a means for achieving the above object, the present invention is Thiocapsa roseopercisina Provided is a method for culturing high concentration cells by culturing NCIB 8347 in a medium containing acetate as a carbon source.

본 발명에 의하면 상기 균체를 배양하는 배지는 암모늄 클로라이드, 미량원소 Na2S·9H2O, Na2·EDTA, FeSO4·7H2O, CoCl2·6H2O, MnCl2·2H2O, ZnCl2, NiCl2·6H2O, Na2MoO4·2H2O, H3BO3, CuCl2·2H2O를 추가로 포함하는 것을 특징으로 하는 균체 배양 방법을 제공한다. According to the present invention, the medium for culturing the cells is ammonium chloride, trace elements Na 2 S · 9H 2 O, Na 2 · EDTA, FeSO 4 · 7H 2 O, CoCl 2 · 6H 2 O, MnCl 2 · 2H 2 O, It provides a cell culture method characterized in that it further comprises ZnCl 2 , NiCl 2 · 6H 2 O, Na 2 MoO 4 · 2H 2 O, H 3 BO 3 , CuCl 2 · 2H 2 O.

본 발명에 의하면 상기 균체의 배양은 광합성 독립영양 배양하는 것을 특징으로 하는 균체 배양 방법을 제공한다. According to the invention the culture of the cells provides a cell culture method characterized in that the photosynthetic autotrophic culture.

이하, 본 발명의 내용을 더욱 상세히 설명하면 다음과 같다.Hereinafter, the content of the present invention in more detail as follows.

본 발명에서 사용한 티오캅사 로세오페르시키나(Thiocapsa roseopercisina) NCIB 8347은 수소를 생산하는 광합성 세균으로 홍색 유황세균이다. 티오캅사 로세오페르시키나 NCIB 8347은 탄소원으로 아세테이트를 포함하는 배지를 사용한 광합성 독립영양 조건에서 최적 배양조건을 나타낸다. Thio kapsa Rosedale Ofer sikina used in the present invention (Thiocapsa roseopercisina) NCIB 8347 is a hydrogen-producing photosynthetic bacterium. Thiocap Corporation roseopercyna NCIB 8347 shows optimal culture conditions under photosynthetic autotrophic conditions using a medium containing acetate as the carbon source.

본 발명에 따르면 티오캅사 로세오페르시키나 NCIB 8347 균체의 생장을 촉진하기 위해 페니그 배지에 배양하며, 2±0.5%의 이산화탄소를 함유한 질소를 공급하며 26±0.5℃에서 광합성 독립영양 배양하는 것이 바람직하다. According to the present invention, cultivation in Phenig medium to promote the growth of thiocapsaceopercyna or NCIB 8347 cells, and supplying nitrogen containing 2 ± 0.5% of carbon dioxide to photosynthetic autotrophic culture at 26 ± 0.5 ° C desirable.

또한, 배양 시작 후 20~24시간마다 0.05±0.01% 아세테이트를 탄소원으로 추가하며 3~5일 동안 배양하는 것이 바람직하다.  In addition, it is preferable to incubate for 3 to 5 days while adding 0.05 ± 0.01% acetate as a carbon source every 20 to 24 hours after the start of the culture.

본 발명의 실시예에 의하면 탄소원으로 아세테이트의 첨가는 균체의 최대 생장속도를 7일에서 1일로 7배 증가시키며, 이로부터 얻어진 하이드로게나아제의 비활성역가 (specific activity)는 유사한 것으로 미루어 7배 많은 하이드로게나아제 양을 확보할 수 있다. According to an embodiment of the present invention, the addition of acetate to the carbon source increases the maximum growth rate of the cells 7 times from 7 days to 1 day, and the specific activity of the hydrogenase obtained therefrom is similar, indicating that 7 times more hydro The amount of genease can be obtained.

또한, 균체의 생장속도를 더욱 높이기 위해 아세테이트 외에 암모늄 클로라이드와 미량원소 Na2S·9H2O, Na2·EDTA, FeSO4·7H2O, CoCl2·6H2O, MnCl2·2H2O, ZnCl2, NiCl2·6H2O, Na2MoO4·2H2O, H3BO3, CuCl2·2H2O를 약 24시간 간격으로 추가하는 것이 바람직하다. Further, in addition to acetate to further increase the growth rate of the cells of ammonium chloride and trace elements Na2S · 9H2O, Na 2 · EDTA , FeSO 4 · 7H 2 O, CoCl 2 · 6H 2 O, MnCl 2 · 2H 2 O, ZnCl 2, Preferably, NiCl 2 · 6H 2 O, Na 2 MoO 4 · 2H 2 O, H 3 BO 3 , CuCl 2 · 2H 2 O are added at about 24 hour intervals.

상기에서 암모늄 클로라이드는 1ℓ당 0.24~0.44g 첨가하는 것이 바람직하다. 또한, 미량원소는 1ℓ당 Na2S·9H2O 1.0~2.0g, Na2·EDTA 0.001~0.005g, FeSO4·7H2O 0.001~0.0015g, CoCl2·6H2O 0.00015~0.0002g, MnCl2·2H2O 0.00003~0.00005g, ZnCl2 0.00004~0.00005g, NiCl2·6H2O 0.00002~0.00003g, Na2MoO4·2H2O 0.00001~0.00002g, H3BO3 0.0002~0.0004g, CuCl2·2H2O 0.000001~0.000003g 첨가하는 것이 바람직하다.In the above ammonium chloride is preferably added 0.24 ~ 0.44g per 1L. In addition, the trace elements are 1ℓ Na 2 S · 9H 2 O 1.0 ~ 2.0g, Na 2 · EDTA 0.001 ~ 0.005g, FeSO 4 · 7H 2 O 0.001 ~ 0.0015g, CoCl 2 · 6H 2 O 0.00015 ~ 0.0002g per MnCl 2 2H 2 O 0.00003 to 0.00005 g, ZnCl 2 0.00004 to 0.00005 g, NiCl 2 6H 2 O 0.00002 to 0.00003 g, Na 2 MoO 4 2H 2 O 0.00001 to 0.00002 g, H 3 BO 3 0.0002 to 0.0004 g It is preferable to add 0.000001 to 0.000003g of CuCl 2 · 2H 2 O.

이하 본 발명의 내용을 실시예에 의해 더욱 상세하게 설명하기로 한다. 다만, 이들 실시예는 본 발명의 내용을 이해하기 위해 제시되는 것일 뿐 본 발명의 권리범위가 이들 실시예에 한정되는 것으로 해석되어서는 안 된다. Hereinafter, the content of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, and the scope of the present invention should not be construed as being limited to these embodiments.

[실시예 1] 광합성 종속영양조건에서의 균체 배양 및 수소생산Example 1 Cell Culture and Hydrogen Production in Photosynthetic Heterotrophic Conditions

페니그 배지는 1ℓ당 CaCl2·2H2O 0.25g, NH4Cl 0.34g, KCl 0.34g, MgSO4·7H2O 1.025g, KH2PO4 0.34g, Vitamin B12 0.00002g, Na2·EDTA 0.003g, FeSO4·7H2O 0.0011g, CoCl2·6H2O 0.00019g, MnCl2·2H2O 0.00005g, ZnCl2 0.000042g, NiCl2·6H2O 0.000024g, Na2MoO4·2H2O 0.000018g, H3BO3 0.0003g, CuCl2·2H2O 0.000002g, NaHCO3 1.5g, Na2S·9H2O 0.4g, Mg-acetate 0.125g, Ammonium-acetate 0.125g으로 조성되었다.Phenig medium contains 0.25 g CaCl 2 · 2H 2 O, 0.34 g NH 4 Cl, 0.34 g KCl, 0.34 g MgSO 4 · 7H 2 O 1.025 g, KH 2 PO 4 0.34 g, Vitamin B 12 0.00002 g, Na 2 EDTA 0.003 g, FeSO 4 · 7H 2 O 0.0011 g, CoCl 2 · 6H 2 O 0.00019 g, MnCl 2 · 2H 2 O 0.00005 g, ZnCl 2 0.000042 g, NiCl 2 · 6H 2 O 0.000024 g, Na 2 MoO 4 2H 2 O 0.000018g, H 3 BO 3 0.0003g, CuCl 2 · 2H 2 O 0.000002g, NaHCO 3 1.5g, Na 2 S.9H 2 O 0.4g, Mg-acetate 0.125g, Ammonium-acetate 0.125g It became.

T. roseopersicina NCIB 8347 균주를 페니그 배지에서 30℃ 배양온도에서 할로겐램프로 8-9 Klux를 조사하면서 종속 영양조건으로 배양하였다. 그 결과 T. roseopersicina NCIB 8347은 도 1과 같은 성장과 수소생산을 보였다. 첨가한 숙신 산(succinate)은 배양 초기부터 분해되기 시작하여 약 48-50시간에 모두 소비되었다. 초기 pH 7.0은 배양 15-18시간까지 pH 9로 증가하다가 점점 저하하여 32시간까지 pH 8.1-8.2로 내려갔고 이후는 계속 유지되었다. 균체는 초기부터 성장하여 배양 22-24시간에 최대로 성장하였고, 수소는 성장대수기 중간인 18시간부터 발생하여 숙신산이 소비되는 45시간까지 발생하였고, 그 이후는 더 이상 생산되지 않았다. 배양 55시간까지는 생산된 수소가 감소되는 현상은 보이지 않았다. T. roseopersicina NCIB 8347 strains were cultured under heterotrophic conditions, irradiating 8-9 Klux with halogen lamps at 30 ° C. in Phenig medium. As a result, T. roseopersicina NCIB 8347 showed the growth and hydrogen production as shown in FIG. The added succinate started to degrade from the beginning of the culture and was consumed in about 48-50 hours. Initial pH 7.0 increased to pH 9 until 15-18 hours of incubation, then gradually decreased to pH 8.1-8.2 until 32 hours, and then continued. Cells grew from the beginning to the maximum growth in culture 22-24 hours, hydrogen was generated from 18 hours in the middle of the logarithmic growth stage until 45 hours of succinic acid consumption, after which it is no longer produced. There was no decrease in hydrogen produced up to 55 hours of incubation.

[실시예 2] 광합성 독립 영양 조건에서의 균체 배양 및 수소생산Example 2 Cell Culture and Hydrogen Production in Photosynthetic Independent Nutrition Conditions

Thiocapsa roseopersicina NCIB 8347을 최소배지인 페니그 배지(ℓ당 CaCl2·2H2O 0.25g, NH4Cl 0.34g, KCl 0.34g, MgSO4·7H2O 1.025g, KH2PO4 0.34g, Vitamin B12 0.00002g, Na2·EDTA 0.003g, FeSO4·7H2O 0.0011g, CoCl2·6H2O 0.00019g, MnCl2·2H2O 0.00005g, ZnCl2 0.000042g, NiCl2·6H2O 0.000024g, Na2MoO4·2H2O 0.000018g, H3BO3 0.0003g, CuCl2·2H2O 0.000002g, NaHCO3 1.5g, Na2S·9H2O 0.4g, Mg-acetate 0.125g, Ammonium-acetate 0.125g)에서 2%의 이산화탄소를 함유한 질소를 공급하며 26℃에서 광합성 독립영양 배양하였다. 그러나 상기의 조건에서는 균체의 생장속도가 매우 느려 일주일 배양 시 0.3g 건조균체량/L-배양액이 생성된다. (도 2). Thiogcapsa roseopersicina NCIB 8347 is the minimum medium for penigue medium (CaCl 2 · 2H 2 O 0.25g / l, NH 4 Cl 0.34g, KCl 0.34g, MgSO 4 · 7H 2 O 1.025g, KH 2 PO 4 0.34g, Vitamin B 12 0.00002g, Na 2 · EDTA 0.003g, FeSO 4 · 7H 2 O 0.0011g, CoCl 2 · 6H 2 O 0.00019g, MnCl 2 · 2H 2 O 0.00005g, ZnCl 2 0.000042g, NiCl 2 · 6H 2 O 0.000024g, Na 2 MoO 4 2H 2 O 0.000018g, H 3 BO 3 0.0003g, CuCl 2 · 2H 2 O 0.000002g, NaHCO 3 1.5g, Na 2 S · 9H 2 O 0.4g, Mg-acetate 0.125g , Ammonium-acetate 0.125g) was supplied with nitrogen containing 2% of carbon dioxide and photosynthetic autotrophic culture at 26 ℃. However, under the above conditions, the growth rate of the cells is very slow, and thus 0.3g dry cell weight / L-culture solution is produced during the week culture. (FIG. 2).

[실시예 3] T. roseopersicina NCIB 8347 고농도 균체배양 방법 Example 3 T. roseopersicina NCIB 8347 high concentration cell culture method

T. roseopersicina NCIB 8347 고농도 균체 성장 조건을 찾기 위해, 본 발명에서는 균체의 생장을 촉진하기 위해 페니그 배지에 배양 시작 후 24시간마다 0.05% 아세테이트를 탄소원으로 추가하였다. 2%의 이산화탄소를 함유한 질소를 공급하며 26℃에서 광합성 독립영양 배양하였으며, 배양시간의 경과에 따른 균체 성장과 이때 균체에서 분리한 하이드로게나아제의 활성 변화를 측정하였다(도 3). 그 결과, 배양 20시간까지 지수성장기가 계속되었으며, 0.33g(건조균체량/L-배양액)의 균체 성장을 보였다. 이와 같은 현상은 T. roseopersicina NCIB 8347이 젖산과 글루타믹산을 각각 탄소원 전자 공여체 및 질소원으로 공급하는 광합성 종속 영양조건으로 배양(도 1)할 때와 상이하였다. 광합성 종속 영양 배양에서는 수소가 균체의 지수성장기 후반부터 생성되어 균체 성장이 끝난 후에도 지속되었다. 이와 같은 현상은 수소를 발생하는데 주로 나이트로게나아제(nitrogenase)가 관여하기 때문이며, 균체가 최대로 성장한 후 질소원이 고갈될 때, 나이트로게나아제가 양성자를 수소로 환원하여 발생한다. T. roseopersicina NCIB 8347 In order to find high concentration of cell growth conditions, in the present invention, 0.05% acetate was added as a carbon source every 24 hours after the start of the culture in Penny medium to promote the growth of the cells. Photosynthetic autotrophic culture was carried out at 26 ° C. while supplying nitrogen containing 2% carbon dioxide, and cell growth over time and the activity change of hydrogenase isolated from the cells were measured (FIG. 3). As a result, the exponential growth period was continued until 20 hours of culture, and showed cell growth of 0.33 g (dry cell weight / L-culture solution). This phenomenon was different from when T. roseopersicina NCIB 8347 was cultured under photosynthetic heterotrophic conditions in which lactic acid and glutamic acid were supplied to a carbon source electron donor and a nitrogen source, respectively (FIG. 1). In photosynthetic heterotrophic culture, hydrogen was produced from the late stage of exponential growth of cells and continued even after growth of cells. This phenomenon is mainly due to the nitrogenase (nitrogenase) involved in generating hydrogen, when the nitrogen source is depleted after the maximal growth of the cells, the nitrogenase is generated by reducing protons to hydrogen.

탄소원으로 아세테이트의 첨가는 균체의 최대 생장속도를 7일에서 1일로 7배 증가시켰고, 이로부터 얻어진 하이드로게나아제의 비활성역가 (specific activity)는 유사한 것으로 미루어 7배 많은 하이드로게나아제 양을 확보하였다. The addition of acetate to the carbon source increased the maximum growth rate of the cells 7 times from 7 days to 1 day, and the specific activity of the obtained hydrogenase was similar, thus securing 7 times as much hydrogenase amount.

이로부터 균체의 생장속도를 더욱 높이기 위해 필요한 3가지 필수 영양소인 아세테이트(0.5g/ℓ), 암모늄 클로라이드(0.34g/ℓ), 미량원소(ℓ당 Na2S·9H2O 1.5g, Na2·EDTA 0.003g, FeSO4·7H2O 0.0011g, CoCl2·6H2O 0.00019g, MnCl2·2H2O 0.00005g, ZnCl2 0.000042g, NiCl2·6H2O 0.000024g, Na2MoO4·2H2O 0.000018g, H3BO3 0.0003g, CuCl2·2H2O 0.000002g)를 24시간 간격으로 첨가하면서 3주 동안 균체의 생장과 하이드로게나아제의 활성을 측정하였다(도 4). 균체의 성장은 16일간 지속적으로 증가하여 4.5g(건조균체량/ℓ 배양액)의 균체 농도를 보였고, 이후 정체기에 이르렀다. 이에 반해 하이드로게나아제의 활성은 배양 3-5일(72-120 시간)일 때 가장 높은 활성을 보이고, 이 후 감소하여 7 일째부터는 최대 활성의 60% 정도를 계속 유지하였다. 이상의 결과로부터 균체는 3-4 일간의 배양 후 수확하여 이용하였다.From this, three essential nutrients necessary to further increase the growth rate of the cells: acetate (0.5 g / l), ammonium chloride (0.34 g / l), trace elements (1.5 g of Na 2 S · 9H 2 O per l, Na 2 · EDTA 0.003g, FeSO 4 · 7H 2 O 0.0011g, CoCl 2 · 6H 2 O 0.00019g, MnCl 2 · 2H 2 O 0.00005g, ZnCl 2 0.000042g, NiCl 2 · 6H 2 O 0.000024g, Na 2 MoO 4 2H 2 O 0.000018g, H 3 BO 3 0.0003g, CuCl 2 · 2H 2 O 0.000002g) was added for 24 hours to measure the growth of the cells and the activity of the hydrogenase for 3 weeks (Fig. 4). The growth of the cells was continuously increased for 16 days, showing a cell concentration of 4.5 g (dry cell weight / l culture solution), and then reaching a plateau. In contrast, the activity of hydrogenase showed the highest activity at 3-5 days (72-120 hours) of cultivation, and then decreased to maintain 60% of the maximum activity from day 7. From the above results, the cells were harvested and used after 3-4 days of culture.

본 발명에 의하면 티오캅사 로세오페르시키나 NCIB 8347은 탄소원으로 아세테이트를 포함하는 배지를 사용한 광합성 독립영양 조건에서 배양하여 균체 성장을 최대로 할 수 있으며, 이를 이용하여 많은 양의 하이드로게나아제를 생산할 수 있다. According to the present invention, thiocaps roseopercyna or NCIB 8347 can be cultured under photosynthetic autotrophic conditions using a medium containing acetate as a carbon source to maximize cell growth, and can produce a large amount of hydrogenase. have.

Claims (3)

티오캅사 로세오페르시키나(Thiocapsa roseopercisina) NCIB 8347을 탄소원으로 아세테이트를 포함하는 배지에 배양하여 고농도 균체를 배양하는 방법 Thiocapsa ( Thiocapsa) roseopercisina ) Method of culturing high concentration cells by incubating NCIB 8347 in a medium containing acetate as a carbon source 제1항에 있어서, 배지는 암모늄 클로라이드, 미량원소 Na2S·9H2O, Na2·EDTA, FeSO4·7H2O, CoCl2·6H2O, MnCl2·2H2O, ZnCl2, NiCl2·6H2O , Na2MoO4·2H2O, H3BO3, CuCl2·2H2O를 추가로 포함하는 것을 특징으로 하는 균체 배양 방법 The medium according to claim 1, wherein the medium is ammonium chloride, trace elements Na 2 S.9H 2 O, Na 2 EDTA, FeSO 4 7H 2 O, CoCl 2 6H 2 O, MnCl 2 2H 2 O, ZnCl 2 , Cell culture method characterized in that it further comprises NiCl 2 · 6H 2 O, Na 2 MoO 4 · 2H 2 O, H 3 BO 3 , CuCl 2 · 2H 2 O 제1항에 있어서, 배양은 광합성 독립영양 배양하는 것을 특징으로 하는 균체 배양 방법 The cell culture method of claim 1, wherein the culture is photosynthetic autotrophic culture.
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