KR100755507B1 - Method for Producing Hydrogen Gas Using Novel Strain of Rhodobacter sphaeroides Having High productivity of Hydrogen and Capable of Photosynthesis in High Concentration of Salts - Google Patents

Abstract

The present invention provides a method for producing hydrogen from the cultivation of a new strain belonging to Rhodobacter spheroides capable of producing hydrogen by photosynthesis and capable of photosynthesis at a salt concentration of 3% NaCl in a medium. According to the present invention, the photosynthetic bacterium may contain pRK415, which is a shuttle vector, and may be genetically manipulated. The photosynthetic bacterium is 4 times higher than that of the Rhodobacter spheroides 2.4.1 strain, which is known in the art. It finds a fermentation condition suitable for hydrogen production rather than conditions and provides a manufacturing process for generating hydrogen with high efficiency.

Description

Method for Producing Hydrogen Gas Using Novel Strain of Rhodobacter sphaeroides Having High productivity of Hydrogen and Capable of Photosynthesis in High Concentration of Salts}

The present invention relates to a method for producing hydrogen using a novel photosynthetic bacterium, and more particularly, to produce hydrogen by photosynthesis, and belongs to a novel Rhodobacter spheroides species capable of photosynthesis at a salt concentration of 3% NaCl. It relates to a method for producing hydrogen using photosynthetic bacteria.

The nature of microorganisms generating or absorbing hydrogen gas has been observed by some photosynthetic microorganisms and anaerobes, and their hydrogen production mechanisms differ in the path of hydrogen production depending on the presence or absence of a light source, as well as substrate types and growth conditions. It is therefore affected.

Hydrogen-producing microorganisms are largely divided into photosynthetic bacteria, non-photosynthetic anaerobic bacteria, algae, and the like, and their hydrogen production mechanisms, usable substrates, and hydrogen generation amount vary considerably. .

Photosynthetic bacteria are separated into purple non-sulfur bacteria, purple sulfur bacteria, and green sulfur bacteria. Among them, photosynthetic bacteria obtain electrons from organic matter and photosynthesis Hydrogen is produced by the reaction. Generally, they inhabit freshwater lakes or ponds that contain organic compounds but do not contain or contain trace amounts of H ₂ S. Double Rhodobactor genus has its own red pigment called bacteriochlorophyll, which receives long wavelengths of light and performs nitrogen immobilization by electron transfer mechanism. The final enzyme involved in nitrogen fixation is nitrogenase, and when no nitrogen source such as nitrogen, ammonia, ammonium ion (NH ₄ ⁺ ) is present in the vicinity, protons (H ⁺ ) are converted to hydrogen (H ₂ ). Reduction produces hydrogen gas.

In addition, Rhodobacter spp. Microorganisms are known to be able to grow both under aerobic and anaerobic dark conditions due to various metabolism, as well as photosynthesis. Among photosynthetic microorganisms, red non-sulfur bacteria have better hydrogen productivity than other groups, and because of this diversity, monosaccharides, disaccharides, and various organic acids can all be used as a culture substrate.

Theoretically, hydrogen can generate 12 molecules of hydrogen gas from one molecule of glucose and 6 molecules of water, and 4, 6 and 7 molecules of hydrogen are produced from one molecule each of the organic acids acetate, lactate and butyrate. . But Rhodobacter, a photosynthetic bacterium, There is a difference in the efficiency of converting organic matter to hydrogen depending on the genus. It is known that the maximum substrate conversion rate is 30 to 40% for glucose and 85 to 90% for lactate. There are also organic acids or sugars that do not degrade depending on the substrate. The conversion efficiency from algae or photosynthetic bacteria to biological hydrogen is known to produce more hydrogen as the maximum quantum efficiency that can use light energy is increased, which is due to the number of photoreaction centers and antenna size, which are photochemical variables in the strain. Proportional.

Various methods for maximizing hydrogen production by photosynthesis from organic material using microorganisms in Rhodobacter have been studied.

First, research on manipulating related genes such as modification of nitrogenase gene, an enzyme involved in hydrogen generation, and removal of uptake hydrogenase (Hup) gene, which reuses the generated hydrogen. Genetic engineering studies are underway, including studies on the genes involved in photoreaction. In addition, metabolic-related molecular biology studies are conducted to maximize hydrogen evolution by eliminating the production pathways of macromolecules such as polyhydroxybutyrate (PHB) and aminolevulonic acid, which microorganisms accumulate as metabolites simultaneously with hydrogen. have.

Second, research on the type and optimal hydrogen production conditions of photosynthetic hydrogen producing bioreactors has been actively conducted, which aims at efficient light supply and dispersion with a focus on practical application of large scale reactors. Each type of photosynthetic bioincubator has advantages and disadvantages related to cell mass production and hydrogen generation, but most of them have been developed for the purpose of securing a large number of cells, and have not been properly studied for gas production.

Third, photosynthetic microbial cultivation technology is an important part of biohydrogen production as well as securing excellent microorganisms or developing appropriate reactors, and various kinds of microorganism immobilization technology and organic wastewater or waste resources to secure the maximum microorganisms in culture medium. Pretreatment, temperature and light control to remove salt and ammonia are mainly studied.

Despite these research efforts, the hydrogen production capacity of the microorganisms in Rhodobacter is known until now, and furthermore, when a substrate having a high salt concentration such as organic wastewater is applied, its activity is lost or killed and thus hydrogen is produced. The problem arises that it cannot be used industrially.

The present invention is presented to solve the problems of the prior art,

An object of the present invention is to provide a method for producing hydrogen using a new photosynthetic bacterium capable of photosynthesis even in a high salt environment, and having the ability to produce four times or more hydrogen with respect to a conventionally known strain by photosynthesis from an organic material.

As a means for achieving the above object, the present invention provides a method for producing hydrogen by culturing Rhodobacter spheroides capable of producing hydrogen by photosynthesis, photosynthetic capable of photosynthesis at a salt concentration of 3% NaCl. .

According to the present invention, the photosynthetic bacterium may contain pRK415, a shuttle vector, and provides a method for producing hydrogen, characterized by Rhodobacter spheroides, which is genetically engineered.

According to the present invention, the photosynthetic bacterium provides a method for producing hydrogen, characterized in that Rhodobacter spheroides KD131 KCTC 12085.

According to the present invention, the medium provides a method for producing hydrogen, wherein the medium contains D, L, or DL organic acids as carbon sources.

According to the present invention, the organic acid provides a method for producing hydrogen, characterized in that the maleate, acetate, lactate, or butyrate.

According to the present invention, the medium provides a method for producing hydrogen, characterized in that the medium medium or cystrome medium.

According to the present invention, it provides a method for producing hydrogen, characterized in that the culture is carried out in a vertical plate incubator.

According to the present invention, the vertical flat medium provides a method for producing hydrogen, characterized in that the front surface is made of a transparent material.

According to the present invention, it provides a method for producing hydrogen, characterized in that the light is irradiated at 7 to 10 klux in culture.

Hereinafter, the content of the present invention in more detail as follows.

Photosynthetic bacteria used in the present invention belong to the genus Rhodobacter as red non-sulfur bacteria. In particular, the photosynthetic bacterium according to the present invention is capable of growing even at high concentrations of salinity, and thus has properties suitable for treating organic wastewater having high salt concentrations.

The photosynthetic bacterium according to the present invention belongs to the Rhodobacter speroides species, which are preferably capable of growing under a salt concentration of 3% NaCl, and preferably contains the plasmid when mobilizing pRK415, known as a shuttle vector. It can be genetically manipulated and has colony forming ability.

As mentioned above, the strain having colony-forming ability was named KD131, and the strain was deposited with KCTC 12085 (May 6, 2002) at the Korea Biotechnology Research Institute Gene Bank. In addition, the strain is capable of photosynthesis in any of aerobic or anaerobic conditions.

The Rhodobacter spheroides KD131 strain is 99.5% similarity to the Rhodobacter spheroids IFO2203, Rhodobacter spheroids IL106 as a known strain in the sequence analysis of 16S rRNA. see.

In addition, even when the salt concentration is 3% NaCl, it shows a doubling time of about 3 hours, more than twice as much as Rhodobacter spheroides 2.4.1 in the amount of light fixtures, and four times the hydrogen productivity. It has the above difference. From this, it can be seen that the photosynthetic bacterium according to the present invention can achieve the dual purpose of treating wastewater and generating hydrogen gas.

The Rhodobacter spheroides KD131 strain according to the present invention can efficiently produce hydrogen using organic acids or sugars, preferably organic acids. The organic acid is not particularly limited, but acetate, lactate, butyrate, maleate, and the like may be preferably used.

In the case of culturing the Rhodobacter spheroides KD131 strain according to the present invention, GL medium or sistrom medium may be used, and in the case of using minerals and minerals of GL medium, hydrogen production efficiency surface. Good at In the case of the use of the cystrom medium, the maleate is superior to other organic acids as the carbon source, but when the Ziel medium is used, butyrate is superior to other organic acids.

In addition, since the strain produces hydrogen by nitrogenase, when the concentration of nitrogen source is high, nitrogenase undergoes nitrogen fixation, which is the main reaction, and cell growth becomes active, and protons (H ⁺ ) are hydrogen in an insufficient nitrogen environment. The reaction to reduce to (H ₂ ) may be carried out to increase the hydrogen production amount.

In addition, the incubator for the cultivation of the Rhodobacter spheroides KD131 strain according to the present invention may be a cylindrical, coiled, vertical flat type incubator, but the vertical flat type incubator is recommended in terms of hydrogen production, preferably It is preferable to use an incubator made of a transparent material (for example, acrylic) in order to have excellent light transmittance. In this case, cell growth and hydrogen productivity are highest when irradiated with light of about 8 to 10 klux / m 2.

In addition, the photosynthetic bacterium according to the present invention may be used for the purpose of removing heavy metals because it accumulates heavy metals in its body during photosynthesis.

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.

Example 1 Isolation and Identification of Bacteria

(1) Securing domestic indigenous photosynthetic bacteria

In order to secure strains with high hydrogen production and resistance to salinity, aquatic soil collection was conducted in Daebudo, Hwaseong-gun, Gyeonggi-do Province. Strain selection resistant to salinity is intended to prevent the availability of the strain to be inhibited by salinity after strain improvement. Samples collected from five of the nine samples were able to grow in a medium containing 3% NaCl, and showed relatively high hydrogen generating ability among the isolates. As a result of measuring the absorption wavelength by the photosynthetic apparatus of these isolates, the photosynthetic apparatus of the typical non-sulfur purple photosynthetic bacteria was shown (FIG. 1).

(2) Identification of selected isolates

PRK415 (Keen NT, Tamaki S, Kobayashi D, Trollinger D (1988) Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria, known as a shuttle vector of photosynthetic bacteria and E. coli to all the isolates shown in Figure 1). Gene 70: 191-197) was mobilized. As a result, only KD131 formed colonies containing plasmid. For the identification of KD131, a portion of the gene encoding 16S rRNA was degenerated primer of upstream primer 5'-ACA CAT GYA AGT CGA RCG-3 'and downstream primer 5'-TCC CAT GGT GTG ACG GGC-3'. PCR was performed using degenerated primers. As a result, a 1.28 kb DNA fragment was obtained from the 5 'end to the 60th nucleotide to the 1339th nucleotide. Sequencing of the parts showed 99.5% homology with Rhodobacter spheroides 2.4.1, IFO2203, IL106, and thus the strain was named Rhodobacter spheroides KD131 (KCTC 12085).

(3) Investigation of similarity between isolates of uptake hydrogenase gene, PHB forming gene and laboratory strain

For this purpose, the hydrogenase gene (Dischert W. et al. 1999) and PHB forming gene (Kim JH. Lee JK 1997) of Rhodobacter spheroids 2.4.1 were described as "Kim JH, Lee JK (1997) Cloning, nucleotide sequence." and expression of gene coding for polyhydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1.J Microbiol Biotechnol 7: 229-236) using a probe as a Southern blot analysis (hydrogenase gene probe: Pst I- Hind 5,023bp, PHB gene probe III hupSL: Xho I- Xho 1,869bp of I phbC). Southern blot analysis of the isolates showed that KD131 strain had high similarity with genomic DNA of Rhodobacter spheroides 2.4.1 (FIG. 2). Therefore, KD131 was used in subsequent experiments, which showed excellent hydrogen production performance compared to other isolates, and showed similar DNA sequence similarity to that of conventional strains.

In hydrogen productivity, KD131 showed about 4 times higher hydrogen production rate than the conventional strain (Table 1).

Table 1 Comparison of hydrogen production rates between Rhodobacter spheroides KD131 and Rhodobacter spheroids 2.4.1.

Strain Hydrogen production rate Rhodobacter spheroides 2.4.1. 48 uL / hr KU * Rhodobacter spheroides KD131 228 uL / hr KU

* KU = 1 × 10 ⁷ cells

Example 2 Physiological Characteristics of Bacteria

Basic physiological characteristics of Rhodobacter spheroides KD131 strain according to the present invention were investigated. As a result of measuring the growth of salts (0.05%, 3%, 5% NaCl) and the absorption wavelength by photosynthetic apparatus, KD131 showed 0.05%, 3%, 5% NaCl containing Sistrom's minimal media) (Sistrom WR (1962) The kinetics of the synthesis of photopigments in Rhodopseudomonas sphaeroides.J Gen Microbiol 28: 607-616) showed doubling times of 1.6, 3.1 and 9.5 hours (Figure 3), In terms of the amount of light fixtures, the amount of B875 complex and B800-850 complex was up to 2 times higher than that of Rhodobacter spheroides 2.4.1 (FIG. 4).

Photosynthetic bacteria according to the present invention can produce hydrogen even at high concentrations (3%) of NaCl. Therefore, since hydrogen can be generated from the organic waste containing high salt concentration, the dual effects of wastewater treatment and hydrogen gas generation can be achieved. In addition, photosynthetic bacteria can accumulate heavy metals in their bodies during photosynthesis, and thus may be applied for the purpose of removing heavy metals during wastewater treatment. In particular, if the formation of light fixtures is enhanced, the concentration of physiologically active substances such as pigments and electron transporters may be increased, thereby increasing the value-added value of the feed of more than 100 million dollars annually in the world.

Example 3 Optimization of Culture Conditions

end. Use strain

(1) Use strain

In the present invention, Rhodobacter sphaerodies KD131 (KCTC 12085) strain, which is an anaerobic photosynthetic strain, was used. The Rhodobacter spheroides RV used as a comparative strain was obtained from Dr. National University of Bioscience and Health. Used by J. Miyake.

(2) Medium composition

Rhodobacter spheroides KD131 used a modified cystrome medium for cell growth (Table 2) for seed culture, and a modified cystrom for hydrogen production (Table 3) for this culture. Organic acids (acetate, propionate, butyrate, lactate, maleate) and glucose added as carbon sources were added separately after sterilization of 30 mM. Rhodobacter spheroides RV strains were observed for cell growth and hydrogen production using GL and aSY medium (Table 4).

(3) Culture conditions

All cultures of Rhodobacter spheroides KD131 were inoculated to have an initial cell concentration of 660 nm at an absorbance of 0.5, and were replaced with argon in the incubator to make anaerobic conditions, and then made at 30 ° C. Halogen lamps were used as the light source. As a culture medium, a culture medium for fermentation of cystrom cells containing succinate as a carbon source was used, and the culture medium for fermentation of cyst hydrogen produced with maleate as a carbon source was used for culture. In order to compare the hydrogen productivity of Rhodobacter spheroides KD131 according to the type of organic acid, GL medium containing malate, acetate, or lactate was adjusted to pH 6.8 using 2N KOH and sterilized. In outdoor culture using sunlight, a non-sterile medium was used. In the case of serum bottle culture, the cells were shaken by shaking 2-3 times a day, and the 3.6L incubator was stirred at 100 rpm using a magnetic stirrer, and the 50 L incubator was stirred by using an impeller. The Rhodobacter spheroides RV strain was inoculated with 5% of the strain stored in the liquid state in aSy medium (Table 4) in a new aSY medium, maintained at 30 ° C, incubated for 24 hours while irradiating 8 klux, and then again aSY medium. After 25% inoculation to incubate under the same conditions, hydrogen production was observed using a gL medium. At this time, the cell inoculation concentration was 25% and Rhodobacter spheroides. It was higher than KD131.

<Table 2> Cystrom medium (for cell growth)

KH ₂ PO ₄ 2.72g (NH ₄ ) ₂ SO ₄ 0.5g Succinate 4.0g L-aspartate 0.04g NaCl 0.5g Nitrilotriacetate 0.2 g MgCl ₂ 6H ₂ O 0.244 g 5% CaCl ₂ ㆍ 2H ₂ O (5g / 100ml) 0.668ml 2% FeSO ₄ 7H ₂ O (2g / 100ml) 0.1ml Trace Element Solution / 100ml EDTA 1.765 g 0.1ml ZnSO ₄ ㆍ 7H2O 10.95 g MnSO ₄ ㆍ H2O 1.54 g CuSO ₄ ㆍ 5H2O 0.392 g Co (NO ₃ ) ₂ ㆍ 6H2O 0.248 g H ₃ BO ₃ 0.114 g Na ₂ MoO ₄ 2H ₂ O 0.75 g FeSO _{4 7} H ₂ O 5 g Vitamin Solution / 100ml Nicotinic acid 1 g 0.1ml Thiamine HCl 0.5g Biotin 0.01 g Final volume 1L

* Adjust to pH 6.8 ~ 7.0 with KOH and sterilize.

Table 3 Cystrom Medium (for Hydrogen Production)

DL-maleate 4.023 g KH ₂ PO ₄ 2.72g L-glutamate 1.2 g Nitrilotriacetate 0.2 g MgCl ₂ 6H ₂ O 0.244 g 5% CaCl ₂ ㆍ 2H ₂ O (5g / 100ml) 0.668ml 2% FeSO ₄ 7H ₂ O (2g / 100ml) 0.1ml Trace Element Solution / 100ml EDTA 1.765 g 0.1ml ZnSO ₄ ㆍ 7H ₂ O 10.95 g MnSO ₄ H ₂ O 1.54 g CuSO ₄ ㆍ 5H ₂ O 0.392 g Co (NO ₃ ) ₂ ㆍ 6H ₂ O 0.248 g H ₃ BO ₃ 0.114 g Na ₂ MoO ₄ 2H ₂ O 0.75 g FeSO _{4 7} H ₂ O 5 g Vitamin Solution / 100ml Nicotinic acid 1 g 0.1ml Thiamine HCl 0.5g Biotin 0.01 g Final volume 1L

* KOH pH 6.8 ~ 7.0 After sterilization, use it after sterilization.

TABLE 4 GL and aSY medium composition

    aSY badge (per 1ℓ) Ammonium sulfate 1.25 g Sodium Succinate 9.8 g Yeast extract 1.0 g     GL medium (per 1ℓ of basic medium) L-Glutamate Monosodium 1.872 g Sodium DL-Lactate (or Lactate) 9.34g (7.15g) Sodium hydrogencarbonate 1.5 g    ※ Basic Badge K ₂ HPO ₄ 0.75 g KH ₂ PO ₄ 0.85 g H ₃ BO ₃ 2.8 mg Na ₂ MoO ₄ 2H ₂ O 0.75mg ZnSO ₄ ㆍ 7H ₂ O 0.24mg MnSO ₄ 4H ₂ O 2.1mg Cu (NO ₂ ) ₂ ㆍ 3H ₂ O 0.04mg CaCl ₂ 2H ₂ O 0.75mg EDTA 2.0mg MgSO ₄ ㆍ 7H ₂ O 0.2mg FeSO _{4 7} H ₂ O 10mg Vitamin B1 3.783mg 3.566 mg Biotin P-aminobenzoate 5.25mg Nicotinamide 6.48mg  Finally, adjust the pH to 6.8 with 2N NaOH.

I. Bioculture

(1) Serum bottles of 50 ml and 150 ml volumes

50 ml and 150 ml volume incubators were incubated in a 30 ° C. thermostat in a cylindrical form made of pyrex. Cells were seeded at 30 ° C. for 3 to 4 days to inoculate so that the absorbance was 0.5 at 660 nm, closed with a rubber stopper, sealed with an aluminum cover, and replaced with argon for 10 minutes to make anaerobic conditions. The initial pH was adjusted to 6.8 using sterile 2N KOH and incubated while irradiating a halogen lamp to 8-10 klux / m 2. The generated gas was measured with a syringe, and the hydrogen content was analyzed by GC by collecting 100 µl of a syringe head space with a gas tight syringe.

(2) 1 L column type photosynthetic bioculture

The columnar reactor is a cylindrical jacketed incubator with an inner diameter of 3.5 cm and a height of 26 cm, with a total volume of 1 liter and a practical volume of 0.9 liter. 10% seed cultures were inoculated for hydrogen production, and the culture temperature was maintained at 30 ° C. and the pH was maintained at 6.8 to 7.0. After inoculating the cells with 0.8 L of the culture medium, the cells were replaced with argon for 30 minutes to make the inside of the incubator anaerobic. The light source was irradiated from only one side with halogen, and the illuminance was maintained at 8 to 10 klux. During the incubation period temperature was 30 ℃, the initial pH was adjusted to 6.8 ~ 7.0, the culture was stirred with a magnetic stirrer. Gas generation was measured using a measuring cylinder and hydrogen content was measured by GC.

(3) 3.6 liter capacity flat photosynthetic bioreactor

The 3.6 L flat panel biocultivator is made of 20 × 30 × 6 cm (width × length × depth) stainless steel, but both sides are made of glass, but due to the nature of the glass, both sides are made of stainless steel so that only the front side can receive light. It became. The 3.6L flat panel bioincubator was made of transparent acrylic to increase light transmittance. Both incubators had a total volume of 3.6 L and a practical volume of 3 L. After inoculating the cells with 3 L of the culture solution, the cells were replaced with argon for 30 minutes to prepare anaerobic conditions. The light source was irradiated from only one side with halogen, and the illuminance was maintained at 8 to 10 klux. During the incubation period temperature was 30 ℃, the initial pH was adjusted to 6.8 ~ 7.0, the culture was stirred with a magnetic stirrer. Gas generation was measured using a measuring cylinder and hydrogen content was measured by GC.

All. Analysis method

(1) gas analysis

The total gas generated during the culture was collected in a mass cylinder containing water or measured by a gas meter. Hydrogen content was analyzed by gas chromatography (Shimazu 14-B) by taking 100 μl of head space gas in the fermenter with a gas tight microsyringe. The column used was 300 mm × 2 mm (length × diameter) glass, and molecular sieve 5A (Supelco Inc.) was used as a packing material and analyzed by a thermal conductivity meter (TCD). The conditions of GC for quantifying hydrogen gas in the generated gas were a column temperature of 80 ° C, an injector temperature of 100 ° C, and a detector temperature of 120 ° C.

(2) organic acid analysis

The fermentation broth was taken at regular intervals, centrifuged at 10,000 g for 5 minutes to separate the cells and the supernatant, and the supernatant was filtered using a 0.45 μm filter. 20 µl of the culture supernatant was injected and eluted at a flow rate of 0.6 ml / min using 0.01 NH ₂ SO ₄ as the mobile phase. Organic acid analysis was performed at 30 ° C. using Aminex HPX-87H, HPLC equipped with 300 × 7.8 mm (length × inner diameter) (Shimadzu LC-10AT) and measured at 210 nm using a UV detector.

To calculate the decomposition and production of organic acids (formate, acetate, propionate, butyrate, lactate) a standard curve was prepared with a solution of known concentration. The degradation rate and production rate were calculated by applying the analysis result of the culture solution to this standard curve.

la. Characterization of strains and hydrogen production capacity

(1) Comparison of spawn culture conditions and hydrogen productivity

Cell growth and hydrogen production were investigated according to the culture conditions of Rhodobacter spheroides KD131, an anaerobic strain. As the medium, cystrome synthetic medium was used. Spawn culture was divided into anaerobic / light and aerobic / cancer conditions, respectively. First, anaerobic / light condition seed culture was inoculated with 10% seed in a serum bottle, covered with a rubber stopper and an aluminum cover, and then replaced with argon to form anaerobic conditions, and incubated for 24 hours at 30 ° C. at about 8 klux under a halogen lamp. Aerobic / dark conditions Species were inoculated with a 10% seed in a Erlenmeyer flask and incubated for 24 hours at 30 ° C. under aerobic conditions and darkness with a cotton plug. The spawns incubated by two methods were used for hydrogen production, respectively, inoculated by 10% in a 50ml serum bottle, replaced with argon for 5 minutes in 20ml of practical use, and then made in anaerobic conditions. It was. The culture was shaken twice a day to mix the cells. The initial pH was adjusted to 7.0 and the pH change over time and the total gas volume in the headspace were measured and hydrogen production was calculated using GC.

In the case of anaerobic / cancer species, the cells showed a high color (green in fluorescent lamps) and growth was good, whereas in aerobic / cancerous cells, the cells were red in color and slowed down compared to the same incubation time. Anaerobic / light culturing was therefore suitable as a seed culture condition for hydrogen production.

Table 5 shows the results of the main culture under anaerobic / light conditions using the seed culture under anaerobic / light and aerobic / cancer conditions. For anaerobic / light seed, 2.6 ml H ₂ / ml-culture hydrogen was produced after 72 hours of cultivation, while for aerobic / dark seed, initial hydrogen production rate was also slow and 2.2 ml H ₂ / ml- after 72 h. The cultures were less suitable for hydrogen production than anaerobic / light seed.

(2) Cell growth curve of strain

Rhodobacter spheroides KD131 is 8 Klux using halogen, etc. in cystrome synthetic medium containing 30mM of malate as carbon source and L-glutamate and (NH ₄ ) ₂ SO ₄ as nitrogen source. Cells grew and produced hydrogen under photosynthetic conditions of luminosity (FIG. 5).

The logarithmic growth phase of Rhodobacter spheroides KD131 lasted up to about 40 hours of culture, and hydrogen production began about 24 hours in the middle of the log phase and continued even after cell growth was stopped. The initial pH of the culture was 7.0, and it started to increase as the cells grew, and increased from 8.6 to 8.8 after about 20 to 24 hours, and decreased to 7.4 to 7.7 after 20 hours of culture.

(3) light intensity comparison

20 ml of cystrome synthetic medium containing initial malate (initial pH 6.8 to 7.0) were placed in a 50 ml serum bottle, and the cultured strains were added to the cell so that the cell concentration was 0.5 (660 nm). It was. Light was irradiated on one side of the syrup bottle containing photosynthetic strains by dividing into 3 to 3.5 klux, 7 to 8 klux, and 15 to 16 klux, respectively using a halogen lamp. Two to three times a day of incubation was inoculated with the cells and cultured for two days, and cell growth and hydrogen production are shown in FIG. 6.

Cell concentration was highest when the light intensity was 3 to 3.5 klux and 7 to 8 klux. The strain was inhibited by cell growth when irradiated at 100-110 klux or higher, and showed about 50% growth rate compared to 3-3.5 klux and 7-8 klux. Cells showed relatively similar growth rates at other light intensities except when the dose was above 100-110 klux. However, when the incubation time was over 30-32 klux, the inherent index reddish-brown color lost the pigment and the culture was bleached. It showed a phenomenon.

This phenomenon affects the hydrogen production rate, and the hydrogen production amount was reduced by about 30% in the case of 50 to 52 klux than in the case of 7 to 8 klux. In the case of 100 to 110 klux, the cell growth was also low and at the same time, the hydrogen production was reduced by 70 to 90% compared to the 7 to 8 klux.

TABLE 5 Effect of Light Intensity on Rhodobacter spheroides KD131 Cell Growth and Hydrogen Production

 Light intensity (Klux) pH Cell concentration (abs. 660nm) H ₂ produced (ml H ₂ / ml-culture) 0 6.84-6.88 0.32 to 0.35 0 3 to 3.5 7.43-7.53 3.02-3.39 0.65 to 0.76 7-8 7.51 to 7.75 2.90-3.39 0.94-1.00 15-16 7.70-7.79 2.62-3.12 0.59 to 0.90 30-32 7.80-7.85 2.66-3.02 0.61 to 0.78 50-52 7.84-7.96 2.82-2.83 0.64 to 0.68 100-110 7.50-7.72 1.64-1.74 0.11 to 0.28

(4) Effect of pH on Cell Growth and Hydrogen Production

When the initial pH of the Rhodobacter spheroides KD131 was adjusted to 5.2 to 7.08, the cells were aggregated in the liver, but when the pH was above 7.22, the aggregation was not observed. When the initial pH range is 7.1 to 7.3, cell growth is active, and the initial cell concentration is Abs. 1.5 to 1.6 at 660 nm increased to 4.44 to 4.75 after 19 hours of incubation, at which time the pH rose to 7.3 to 7.4. When the initial pH was adjusted to 5.73, the pH increased to 6.72 after 19 hours at which time the cell concentration was 3.5. Hydrogen production was best with about 1.4-1.5 mL H ₂ / mL culture medium at pH range of 7.4 to 7.5, and pH 7.4 to 7.5 days with 0.54-0.83 mL H ₂ / mL culture medium at pH range of 6.7 to 7.0. Although lower than the amount produced, the cell concentration was also relatively low at this time. In this experiment, although the initial pH range was adjusted to 5.3 to 7.6, hydrogen generation could not be measured at all pH ranges at 0 hours of cultivation, but hydrogen production was measured after 6 hours of cultivation while the cells were photosynthetic. Synthesis medium for hydrogen production continued cell growth because L-glutamate was added as a nitrogen source, hydrogen was also generated after the nitrogen source was exhausted.

(5) Influence of nitrogen source concentration

When the concentration of the carbon source is constant at 30 mM, Rhodobacter spheroides KD131 has similar results to organic acid decomposition, pH change, cell growth, and hydrogen production depending on the nitrogen source and L-glutamate concentration (2 mM, 5 mM, 8 mM) added. Table 6 shows. In other words, when the concentration of nitrogen source was 5mM and 8mM, the pH increase was the lowest during 48 hours of culture at about 2,27, which was about 7.27, and the absorbance measured at 660 nm was 8-mM. It was about 1.7 times lower than when. However, the hydrogen produced per unit volume of culture was about 1.4 times higher than with 8 mM addition. This is because hydrogen is produced by the nitrogenase of Rhodobacter spheroides. When the nitrogen source is abundant, the cells undergo growth and nitrogen fixation by nitrogenase, and when the nitrogen source is insufficient, the cells undergo hydrogen production using nitrogenase.

<Table 6> Effect of Nitrogen Concentration on Cell Growth and Hydrogen Production of Rhodobacter spheroides KD131

L-glutamate (mM) DL-maleate (mM) Incubation time (hr) pH Cell concentration (Abs660) H ₂ (ml.ml-fermentation solution) Organic acid decomposition 2 30 0 6.70 0.51 0 0 24 7.24 1.99 0.69 38 48 7.27 2.29 0.98 38 72 7.33 2.24 1.38 61 90 7.30 2.23 1.36 65 5 30 0 6.67 0.52 0 0 24 7.27 2.41 0.79 37 48 7.32 3.02 1.37 62 72 7.50 3.01 1.42 76 90 7.80 2.91 0.96 83 8 30 0 6.80 0.51 0 0 24 7.32 2.21 0.77 37 48 7.55 3.18 1.09 67 72 7.83 3.12 0.93 75 90 7.96 3.03 0.87 79

(6) Use of various carbon sources

Rhodobacter spheroides KD131 cell growth and hydrogen production were compared and analyzed by adding various carbon sources to the cystrom medium (Table 7).

All carbon sources were added 30 mM and starch was added 1%. Cultivation was carried out in an 8 klux halogen lamp for 48 hours in a 30 ℃ thermostat after adjusting to an initial pH of 6.8 in a 20 ml volume in a 50 ml bottle. Glycerol, sucrose, and starch were not used by this strain. When glucose was used as a carbon source, the cells grew until pH 6.0, but when glucose was consumed, the pH was lowered to stop cell growth and hydrogen production was low. By using DL-maleate as a carbon source, maleate was degraded by 95% or more during 48 hours of culture, and the hydrogen production rate was higher than that of lactate or acetate in 2.1 ml H ₂ / ml-culture. Acetate had a high pH above 9 at 48 hours after incubation, resulting in low substrate degradation and hydrogen production. This increase in pH of 9 or more occurred simultaneously with the accumulation of β-D-hydroxybutyrate in the culture medium. Acetate is a precursor for the synthesis of polyhydroxybutyrate (PHB) in Rhodobacter spheroides KD131 wild type cells, and in this experiment, PHB accumulated in the cells over time. PHB is a macromolecule that accumulates in cells, but it is analyzed that β-D-hydroxybutyrate, an intermediate in synthesis, is discharged out of cells to raise the pH of the culture solution.

<Table 7> Cell growth and hydrogen production of Rhodobacter spheroides KD131 using various carbon sources in cystrom medium

pH Cell concentration ^a H ₂ produced (ml H ₂ / ml culture) Substrate Hydrolysis (%) Glucose 6.26-6.30 3.64 0.36 49.1 Glycerol 7.89-8.19 1.35 0 ND ^b Sucrose 7.47-7.71 1.83 0.14 ND ^b Starch 7.44-7.53 0.81 0 <1.8 Malate 7.51 to 7.75 3.10-3.36 2.10 > 95.0 Lactate 7.19-7.48 2.77 0.80 60.9 acetate 9.22-9.93 3.10 0.24 55.2

^a Cell concentration means absorbance at 660 nm.

^b ND means no measurement.

(7) the impact of minerals

To compare the effects of minerals, 30 mM of various carbon sources were added to the GL base. Set the initial pH to 6.8 with 20ml volume in each 5ml bottles and 8klux Halogen lamps were incubated at 30 ° C. for 48 hours and are shown in Table 8. Hydrogen production by Rhodobacter spheroides KD131 was also influenced by types of trace elements such as inorganic substances and vitamins. GL medium (Table 4) has a composition very similar to that of cystrome medium, but differs in the type and content of trace elements minerals and minerals. When using GL synthetic medium, lactate was degraded by about 73.7% in 48 hours of culture under the same photosynthetic culture conditions, and hydrogen was generated in 2.79-2.94 ml H ₂ / ml-culture.

Table 8 Cell growth and hydrogen production of Rhodobacter spheroides KD131 using various carbon sources in GL medium

pH Cell concentration ^a H ₂ produced (ml H ₂ / ml culture) Substrate Hydrolysis (%) Lactate 7.79-7.87 3.41-3.96 2.79-2.94 73.7 acetate 8.46-8.74 3.77-4.15 1.28-1.43 72.9 Malate 7.35-7.49 2.21-2.62 2.41 95.9 Butyrate 7.67 4.12 2.13 32.9 Butyrate ^b 7.34 4.02 4.11 42.3 Butyrate ^c 7.20 3.80 6.76 64.3

All 30 mM organic acids were added and 70 mM for butyrate.

^a Cell concentration means absorbance at 660 nm.

^{b, c} results from 96 and 144 hours of incubation, respectively.

This occurred about 3.6 times more at the same incubation time than the amount of hydrogen generated in the cystrom medium, and 1.2 times higher lactate degradation. Acetate showed the same trend of pH increase as in the cystrom medium, but substrate degradation rate and hydrogen productivity increased 1.3 and 5.7 times with GL medium, respectively. Butyrate was slower in decomposition than other organic acids, while hydrogen production was good. 70 mM butyrate degraded about 33% during 48 hours of culture, 64.3% after 144 hours, and hydrogen production was best with 6.76 ml H ₂ / ml-culture.

(8) Influence of carbon source concentration

Rhodobacter spheroides KD131 cell growth and hydrogen production were compared when the concentration of nitrogen source was fixed to 12.7 mM L-glutamate in GL medium and the concentration of carbon source was changed (Tables 9 and 10). The lactate concentrations were changed to 30 and 70 mM and the acetate concentrations were changed to 30, 50 and 70 mM. The higher the lactate concentration, the better the cell growth and hydrogen production. The pH change during the culture did not change significantly between 6.8 and 7.6. When the concentration of the carbon source was low, the cell aggregates were observed during the culture. This phenomenon lowered the photosynthetic efficiency of the cells and inhibited proliferation and hydrogen production. The agglomeration of cells in the culture of photosynthetic bacteria has been observed by several researchers, but the direct cause of this is unknown. In the case of acetate, the higher the concentration of carbon source, the better the cell growth and the smaller the hydrogen production. The pH change during the cultivation ranged from 7.2 to 9.6. The higher the concentration of acetate, the larger the pH increase. In comparison with the lactate and acetate organic acid degradation rate, the lactate produced 2.21ml H ₂ / mL culture at 91.43% of the organic acid degradation rate at 72 hours of culture, and acetate at 95.84% at 24 hours of culture. When 1.02ml H ₂ / ml culture was produced and lactate was used as the carbon source, hydrogen production was about 2 times higher than that of acetate.

<Table 9> Cell growth and hydrogen production of Rhodobacter spheroides KD131 by lactate concentration in GL medium

Lactate (mM) Incubation time pH Cell concentration (Abs. 660nm) H ₂ produced (ml H ₂ / ml culture) Organic acid decomposition rate (%) 30 0 6.84 0.50 0 0 24 7.44 1.85 0.83 44.86 48 7.66 3.10 1.98 85.87 72 7.56 2.83 2.21 91.43 70 0 6.55 0.47 0 0 24 7.25 2.12 0.66 14.85 48 7.3 4.39 2.10 50.14 72 7.52 4.27 3.89 87.65

Table 10 Cell growth and hydrogen production of Rhodobacter spheroides KD131 by acetate concentration in GL medium

Acetate (mM) Incubation time pH Cell concentration (Abs. 660nm) H ₂ produced (ml H ₂ / ml culture) Organic acid decomposition rate (%) 30 0 7.41 0.52 0 0 24 8.85 3.97 1.02 96.44 48 8.60 3.95 1.29 94.47 72 8.14 3.43 1.66 95.58 50 0 7.39 0.51 0 0 24 8.64 3.70 0.95 49.27 48 8.75 5.31 2.02 97.98 72 8.21 5.50 2.69 97.77 80 0 7.25 0.50 0 0 24 9.38 4.71 0.92 42.76 48 9.56 5.86 1.67 73.71 72 9.33 6.40 3.06 92.38

(9) Influence of optical isomer carbon source

Among the carbon sources, the influence of the optical isomers was compared with DL-, D-, and L-maleate as the carbon source based on the cystrom medium. All three organic acids were used at about the same rate as the substrate by the wild type strain (FIG. 7). In the HPLC organic acid analysis, maleate eluted at 9.16 minutes and unknown material eluted at 14.93 minutes. The material eluting at 14.93 minutes was estimated to be β-D-hydroxybutyrate (β-D-HB) and was confirmed to have the same retention time in HPLC as the β-D-HB standard. In the case of D-maleate, the peak of 9.16 component was reduced by the consumption of maleate, a carbon source, and a small amount of 14.93 component was accumulated (Table 11). In the case of L-maleate, the 9.16 component peak was also reduced by consuming the maleate, a carbon source, over time. However, unlike the case of D-maleate, 14.93 components were accumulated. In the case of DL-maleate, 14.93 components were included from the beginning of the culture, and this material decreased with the incubation time with D, L-maleate. It is believed that this is different from the form of conversion to β-hydroxybutyrate depending on the D- and L-forms, and thus also related to the synthesis of PHB polymer material.

Table 11 Changes in Maleate and Hydroxybutyrate Amounts in Rhodobacter spheroides KD131 Culture

Malate Incubation Time (hrs) Peak Area (Area × 10 ^-3 ) Maleate (9.16 minutes) Hydroxybutyrate (14.93 minutes) D, L-maleate 0 4,513 4,560 24 2,850 4,870 D-maleate 0 4,835 0 24 2,923 101 L-maleate 0 5,041 604 24 2,605 11,946

(10) incubator effect

The hydrogen productivity was compared using the 50 ml bottle, 1 L column incubator, 3.6 L stainless side upright glass incubator, and 3.6 L front acrylic upright incubator introduced above.

50 ml capacity glass serum bottle, flat vertical glass incubator made of 3.6 l capacity stainless steel side / glass both sides, vertical flat incubator made of 3.6 l capacity acrylic, 1 l glass column incubator and glass The hydrogen productivity was compared using a coil incubator. In order to maximize hydrogen production by photosynthetic bacteria, it is necessary to improve the genetic factors of microorganisms themselves, such as improving light conversion efficiency of microorganisms, light inhibition at high light intensity, and hydrogen consumption under dark conditions. Research is needed to continuously produce hydrogen by producing and designing a reactor, that is, an incubator in which light capable of inducing optimal cell growth and hydrogen production is evenly dispersed in all parts of the incubator in an economical material and form. Although this research is still in the early stages of the world, green algae cultured open pond incubators for the production of raw biomass for the extraction of some high-value substances such as beta-carotene have been commercialized. The internal lighting type, bioculture facility, vertical module and shape, and floating type floating on the lake are being considered on a laboratory scale in each country. In the present invention, the photosynthetic incubator in columnar, vertical rectangular, and coil form was examined for hydrogen production and cell growth under similar culture conditions.

The hydrogen generation rate was columnar with the same diameter, but the 50 ml serum bottle incubator showed about 2 times higher hydrogen production rate. It is thought that it is difficult to disperse the light evenly when the size is enlarged, and some dark places occur and the hydrogen production is reduced. It is reported that the depth of sunlight can pass through the incubator in which cells are present is about 3 cm, and the preliminary experiments show that the flat form of the photosynthetic incubator has a uniform light dispersion compared to the column type, and thus the intensity of light received inside the incubator. Was relatively constant. Vertical flat glass incubator made of 3.6L capacity stainless steel side / glass both sides Vertical flat glass incubator made of 3.6L capacity acrylic is a vertical flat type photosynthetic incubator with flat light It was produced using transparent acrylic as a material.

Rhodobacter spheroides KD131 is a flat vertical incubator (3.6 L total volume, 3 L culture volume) made of transparent acrylic with 30 mM malate at 30 ° C without pH adjustment at 8-9 klux When incubated in cystrom medium, more than twice as much hydrogen was produced as in the same size incubator made of glass on both sides. Both glass and acryl were transparent materials, and the comparison of light transmittance was the same. However, the glass reactor was not made of glass on the front and stainless steel was supported on the side.

The coiled photosynthetic bioincubator made of Pyrex glass was wound around a glass tube (outer diameter of 1.94 cm, inner diameter of 1.74 cm) in the form of a cylinder of about 20 cm in diameter, 55-57 cm in height, and about 3.0 l in volume.

The use of the material thickness of the incubator made of acrylic 0.4 and 0.8cm, respectively, affected the production of the cells (Fig. 8). That is, when produced in 0.4cm thickness, cell growth by photosynthesis increased about 1.4 times during 48 hours of incubation.

However, hydrogen production did not change within the margin of error. Increasing the cell mass may lead to a proportional increase in hydrogen production, but in a sub-experiment, a sufficient concentration of cells has already been secured. Rather, a higher cell concentration may decrease the light transmittance and increase the hydrogen production. As time passes during incubation, even when the culture medium is stirred, the cells often aggregate or adhere to the incubator wall. This phenomenon shows a phenomenon in which the final cell concentration is finally lowered (FIG. 8).

When the coiled bioincubator was stirred while circulating cells under the same culture conditions as compared with other bioreactors, Rhodobacter spheroides KD131 reached more than twice the cell growth after about 24 hours. However, hydrogen production was lower than other types of incubators. The reason for this is that because the circulating medium is not released all of the hydrogen is released to the outside and some are trapped in the coil incubator or circulated with the medium. This phenomenon was lowered the pH in the culture medium was a large accumulation of formic acid, cells after about 96 hours showed a bleaching phenomenon by the pH decrease. Among the columnar, vertical plate and coil type incubators examined in the present invention, the vertical plate incubator was most suitable for Rhodobacter spheroides cell culture and hydrogen production, and the transparent acrylic material was relatively easy to process and inexpensive. It was suitable for the enlargement of the photosynthetic biological incubator.

Table 12 Influence of reactor morphology

Incubator shape A rum bottle Flat glass Flat Slope Acrylic Columnar Coil type Diameter X height (cm) 3.5 × 5.5 - 3.5 × 26 Tube diameter, 1.74 coil diameter, 20 height, 55 Width X height X thickness (cm) - 20 × 30 × 6 20 × 30 × 6 - Total volume (ml) 50 3,600 3,600 1,000 3,000 Working volume (ml) 20 3,000 3,000 900 3,000 Hydrogen generation rate (ml H ₂ / ml-fermentation solution) 1.0 to 1.6 0.37-0.41 0.8 to 1.0 0.51 to 0.62 0.1 to 0.15

(11) Comparison of Hydrogen Productivity

Table 13 compares Rhodobacter spheroides KD131 and Rhodobacter spheroides RV known to have the highest hydrogen productivity to date. The seed was inoculated with 30 mM maleate and the nitrogen source with 8 mM L-glutamate, and seeded so that the initial cell concentration was 0.5 in absorbance at 660 nm in cystrome and GL medium, respectively. Comparative analysis.

The cell culture was carried out in a 50 ml serum bottle to a practical volume of 20 ml, and substituted with argon for 5 minutes to make anaerobic conditions. Incubated with a halogen lamp. As a result, KD131 had both good cell growth and good hydrogen production, and the hydrogen production was about 2 times higher in the Sistrom medium. On the other hand, cell growth was slightly delayed in KD131 in GL medium, while hydrogen production was high. Since KD131 has excellent hydrogen productivity along with RV strains, higher hydrogen production can be expected through various gene mutations.

<Table 13> Cell growth and hydrogen production of Rhodobacter spheroides KD131 and Rhodobacter spheroides RV in cystrom and GL medium

Strain badge Incubation time (hr) Cell concentration (Abs. 660) Hydrogen Production (mL H ₂ / mL-culture) KD 131 GL 72 2.12 2.63 Cystrom 72 3.19 1.02 RV GL 72 2.56 2.59 Cystrom 72 2.36 0.45

The photosynthetic bacterium according to the present invention can survive in a high salt environment, and has the ability to produce four times or more hydrogen with respect to a conventionally known strain by photosynthesis from organic wastewater. In addition, the photosynthetic bacterium according to the present invention may also be used for the purpose of removing heavy metals from the waste water because it functions to accumulate heavy metals in its body during the process of photosynthesis.

As described above, the present invention has been described with reference to the preferred embodiments, but those skilled in the art to which the present invention pertains vary the present invention without departing from the spirit and scope of the present invention as set forth in the claims below. It will be understood that modifications and changes can be made.

Figure 1 is a graph of the measurement results of the absorption wavelength by the photosynthetic apparatus for the nine new photosynthetic bacteria isolated through the embodiment of the present invention.

Figure 2a shows the chromosomal DNA structure of Rhodobacter spheroides 2.4.1 1) hupSL gene region, 2) phbC gene region.

2b shows 1) hydrogenase (hupSL) and 2) PHB (phbC) as a result of Southern analysis.

Figure 3 is a growth curve according to the salinity concentration of the photosynthetic bacteria Rhodobacter spheroides KD131 and Rhodobacter spheroides 2.4.1 according to the present invention.

Where ◆: Rhodobacter spheroides 2.4.1 was cultured in a normal cystrom medium containing 0.05% NaCl, and: Rhodobacter spheroides KD131 was cultured in a normal cystrom medium containing 0.05% NaCl. , ▲: When Rhodobacter spheroides KD131 was cultured in normal cystrom medium containing 3% NaCl, ○: When Rhodobacter spheroides KD131 was cultured in normal cystrom medium containing 5% NaCl, ◇ : When Rhodobacter spheroides 2.4.1 was incubated in normal cystrom medium containing 3% NaCl, When Rhodobacter spheroides 2.4.1 was incubated in normal cystrom medium containing 5% NaCl

4 is a graph comparing the amounts of photosynthetic apparatus of Rhodobacter spheroides KD131 and Rhodobacter spheroides 2.4.1, which are photosynthetic bacteria according to the present invention.

A) represents the amount of B800-850 complex and B) represents the amount of B875 complex, respectively, wherein 1. Rhodobacter spheroides 2.4.1 was cultured in a normal cystrom medium containing 0.05% NaCl, 2. When Rhodobacter spheroides KD131 was cultured in normal cystrom medium containing 0.05% NaCl, 3.Rhodobacter spheroides KD131 was cultured in normal cystrom medium containing 3% NaCl, 4. The case where Rhodobacter spheroides KD131 was incubated in the normal cystrom medium containing 5% NaCl is shown, respectively.

5 is a graph showing the cell growth and hydrogen production degree of Rhodobacter spheroides KD131 according to the present invention.

Where △: total production hydrogen, □: cell concentration, ○: pH of culture

Figure 6 is a graph of the culture pigment changes according to the change in light intensity during the culture of Rhodobacter spheroides KD131 according to the present invention.

(A) matt, (B) 3 to 3.5 klux / m 2, (C) 7 to 8 klux / m 2, (D) 15 to 16 klux / m 2,

(E) 30 to 32 klux / m 2, (F) 50 to 52 klux / m 2, (G) 100 to 110 klux / m 2

Figure 7 is a graph comparing the decomposition rate of D-, L-, D, L-maleate of Rhodobacter spheroides KD131 according to the present invention

8 is a graph showing the effect of the acrylic thickness of the vertical plate reactor used in the present invention on the hydrogen production (30 mM lactate as a carbon source, D, L- glutamate was used as a nitrogen source).

Claims (9)

Irradiated with 7-8 klux light in a vertical plate incubator made of a transparent medium material containing GEL medium containing one organic acid selected from the group consisting of maleate, acetate, lactate, and butyrate. Method for producing hydrogen from the Rhodobacter spheroides KD131 KCTC 12085 capable of photosynthesis at a salt concentration of% NaCl delete delete delete delete delete delete delete delete