KR20120086182A - Method of hydrogen production using photosynthetic bacteria - Google Patents

Abstract

PURPOSE: A method for producing hydrogen using condition-optimized Rhodobacter sphaeroides KD131 is provided to maximize hydrogen productivity. CONSTITUTION: A method for producing hydrogen using Rhodobacter sphaeroides KD131 comprises a step of culturing Rhodobacter sphaeroides KD131 using succinate as a carbon source and (NH_2)_4SO_4 or glutamate as a nitrogen source. The initial concentration and pH of the bacteria is 0.45 g dcw/l-0.67 g dcw/l and pH 6-7.

Description

Hydrogen production method using photosynthetic bacteria {METHOD OF HYDROGEN PRODUCTION USING PHOTOSYNTHETIC BACTERIA}

The present invention relates to a method for producing hydrogen, and more particularly, Rhodobacter sphaeroides ) relates to a method for enhancing the hydrogen production of KD131.

The structure of modern industry has to rely on fossil fuels for most of its raw materials and energy sources. Since the 1970s energy crisis, research on hydrogen production began in earnest as an alternative to fossil fuels.

To date, commercially available hydrogen production technology is mainly produced by steam reforming of petroleum or natural gas, or is mainly obtained from crude oil refining process and steel mill by-product gas. However, due to serious problems such as international tension and the global environmental pollution caused by the absence and ubiquity of fossil fuels, the ultimate hydrogen production technology will utilize water or waste resources using clean technologies such as solar, hydro, wind and microorganisms. It is anticipated that hydrogen should be produced using environmentally friendly technologies.

Microbial technology that can use sunlight and produce hydrogen from water is a technology that can compensate for the disadvantages of such fossil fuel. The mechanism of hydrogen production of microorganisms varies depending on the type of substrate, the enzyme system, and the presence of a light source inherent in the microorganism.

Hydrogen-producing microorganisms are classified into photosynthetic bacteria, algal anaerobes and archaea, and the bacteria to be noted in this study are photosynthetic bacteria. Photosynthetic bacteria are further divided into purple non-sulfur bacteria, puple sulfur bacteria and green sulfur bacteria.

Among them, the red non-sulfur bacteria obtain electrons from organic materials and produce hydrogen by photosynthetic reaction, and generally live in lakes or ponds of fresh water containing organic compounds and containing little or no H ₂ S. The red non-sulfur bacterium has 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.

Rhodobacter genus Rhodobacter genus has its own red pigment called bacterio chlorophyll, and it receives long wavelengths of light and performs nitrogen fixation by electron transfer mechanism. The final enzyme involved in nitrogen immobilization is nitrogenase. When the concentration of nitrogen source is high, nitrogenase undergoes the nitrogen fixation process, which is the main reaction, and the cell growth is active, and nitrogen, ammonia and ammonium ion ( When no nitrogen source such as NH ₄ ⁺ ) is present, hydrogen gas is generated by reducing the proton (H ⁺ ) to hydrogen (H ₂ ).

Rhodobacter currently uses a variety of substrates and waste sphaeroides OU001, R. sphaeroides RV, R. sphaeroides ZX-5, R. sphaeroides NRLLB-1727, R. sphaeroides SCJ, R. sphaeroides SH2C, R. sphaeroides Research on hydrogen production of DSZM-158 is being actively conducted.

However, the hydrogen production capacity of microorganisms in Rhodobacter is known until now.

It is an object of the present invention to provide a method for producing hydrogen in photosynthetic bacteria with optimized culture conditions.

More specifically, an object of the present invention is to provide a hydrogen production method using Rhodobacter sphaeroides ( Rhodobacter sphaeroides ) KD131.

In order to achieve the above object, the present invention is Rhodobacter sphaeroides (Rhodobacter to enhance hydrogen production sphaeroides ) provides a method for producing hydrogen using KD131.

More specifically, the present invention relates to Rhodobacter using succinate as a carbon source and at least one of (NH2) 4SO4 and glutamate as a nitrogen source at an initial cell concentration of 0.40 g dcw / l to 1.14 g dcw / l and an initial pH of 6 to 8. sphaeroides (R. sphaeroides) provides a hydrogen production method using the bakteo sphaeroides (R. sphaeroides) also KD131 wherein KD131 culturing.

According to the present invention it is possible to maximize the hydrogen production capacity of Rhodobacter spheroroides ( R. sphaeroides ) KD131 through the optimization of the culture conditions.

Figure 1 shows the effect of the initial cell concentration on the hydrogen production of R. sphaeroides (D. sphaeroides ) KD131.
FIG. 2 shows the effect of (NH ₄ ) ₂ SO ₄ (a) and glutamate (b) on hydrogen production of R. sphaeroides KD131 in succinate 30 mM medium (• 0 mM, ◐ : 2 mM, Δ: 4 mM, ▼ 8 mM, ◇: 16 mM, ■: 32 mM, □ 64 mM).
Figure 3 shows the effect of (NH ₄ ) ₂ SO ₄ 4 mM (a) and glutamate 8 mM (b) on the hydrogen production of R. sphaeroides KD131 in succinate 30 mM medium (▨ : Cumulative hydrogen production, ●: pH, △: cell growth, ▼ NH ₃ -N).

Hereinafter, the configuration and operation of the present invention will be described in detail.

Hydrogen production method of photosynthetic bacteria of the present invention is characterized by optimizing the culture conditions such as initial cell concentration, initial pH, carbon source and nitrogen source.

More specifically, the method for producing hydrogen in photosynthetic bacteria of the present invention comprises succinate as a carbon source at an initial cell concentration of 0.40 g dcw / l to 1.14 g dcw / l, and an initial pH of 6 to 8, 1 in (NH 2) 4 SO 4 or glutamate. Rhodobacter speroroides ( R. sphaeroides ) KD131 is characterized in that the above as a nitrogen source.

When the initial cell concentration is less than 0.40 g dcw / l, hydrogen production is delayed, and when the initial cell concentration is above 1.14 g dcw / l, cell aggregation occurs, resulting in a self shading effect of the bacteria and thus photo-inhibition. This may reduce the availability of light and substrates, thereby reducing hydrogen production. More preferably, the initial cell concentration is 0.45 g dcw / l to 0.67 g dcw / l.

When the initial pH exceeds 8 in the hydrogen production method of photosynthetic bacteria of the present invention, since the productivity of nitrogenase is inhibited and the uptake hydrogenase is activated, hydrogen production may be reduced. More preferably, the initial pH is 6-7.

Rhodobacter spheroides KD131 used as a photosynthetic strain in the present invention obtains electrons from succinate, which is a carbon source, as a culture substrate, and produces hydrogen by photosynthesis.

In the present invention, (NH 2) 4 SO 4 or glutamate is added to the medium as a nitrogen source for the growth of Rhodobacter spheroides KD131.

In the present invention, the C / N ratio is maintained at 11.56 to 14.14 (w / w) during the culture of Rhodobacter spheroides K131.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are provided to illustrate the present invention and do not limit the content of the present invention.

Isolation and cultivation of bacteria

Photosynthetic strain Rhodobacter sphaeroides ) KD131 was collected from the coast of Daebudo Island and isolated. As a medium, a modified sistrom medium containing 4 mM (NH 2) 4 SO 4, 0.3 mM L-aspartic acid, and 30 mM succinic acid (pH 7) was used.

50 to 70 ml of the culture solution was put in a 150 ml serum bottle and inoculated with bacteria, and then replaced with argon gas by using a rubber stopper and an aluminum cap.

After substitution was incubated for 24 hours at 30 ℃ giving a light of 110 W / m ² with a halogen lamp, and stirred at 80 ~ 100 rpm using a magnetic stirrer.

Mirrors were placed at the rear of the incubator to make the best use of the reflected light.

Analysis method

The organic acid analysis was eluted at a flow rate of 0.6 μl / min using 0.01 MH ₂ SO ₄ as a mobile phase, and HPLC (Shimadzu LC-10AT) equipped with Aminex HPX-B7H at 30 ° C. using a UV detector at a wavelength of 210 nm. Measured.

Hydrogen content was analyzed by gas chromatography (Shimazu 14-B). Molecular sieve 5A (Supelco Inc.) was used as a filler and analyzed by a thermal conductivity detector (TCD). GC conditions for hydrogen gas quantification are column temperature 80 ° C, injector temperature 100 ° C, and detector temperature 120 ° C. The carrier gas maintained a flow rate of 60 ml / min with argon.

Experimental Example  1: Influence of initial cell concentration

The initial cell concentrations were set to 0.04, 0.27, 0.56, 1.14 g dcw / l (absorbance at 0.1 nm, 0.5, 1, 2 at 660 nm) to isolate bacteria in medium containing 30 mM malate and 8 mM L-glutamate. Was cultured.

Hydrogen production with time for each initial cell concentration is shown in FIG. 1. At the initial 0.56 and 1.14 g dcw / l, hydrogen production started after 2 to 2.5 hours of photosynthetic culture, while hydrogen production was delayed to 7 hours of photosynthetic culture at lower initial cell concentrations.

The cumulative hydrogen production reached its maximum within 33 to 48 hours of photosynthetic culture, and then gradually decreased.

As a result, the cumulative hydrogen production increased proportionally with the initial cell concentration up to 0.56 g dcw / l, but there was little difference in the hydrogen production between the initial cell concentrations of 1.14 g dcw / l and 0.56 g dcw / l.

Therefore, the high cell concentration was not suitable for hydrogen production, and was most efficient for hydrogen production when the initial cell concentration was 0.56 g dcw / l.

In subsequent experiments, the initial cell concentration was set at 0.56 g dcw / l.

Experimental Example  2: initial pH Influence of

In order to determine the effect of the initial pH to hydrogen production and cell growth, the experiment was adjusted to the initial pH of 6, 7, 8, 9 and the results are shown in Table 1. At 48 hours after photo-fermentation, the hydrogen production rate was the maximum, after which the hydrogen production rate decreased.

As shown in Table 1, when the initial pH was adjusted to 7 in a medium using maleate as a carbon source and glutamate as a nitrogen source, hydrogen was most produced by 1044 ml H ₂ / l- medium after 120 hours of photo fermentation.

Initial pH Effect Item Photo Fermentation Time (h) Initial pH 6.0 7.0 8.0 9.0
Cell Growth (OD ₆₆₀ ) 24 2.9 2.7 2.5 2.0 48 3.5 3.2 2.9 2.3 120 3.1 2.9 2.7 2.2 Cumulative Hydrogen Production
(ml H ₂ / l- medium) 24 364.0 608.0 432.0 300.0 48 506.0 942.0 442.0 314.0 120 552.0 1044.0 458.0 316.0
Degradation of Carbon Substrate (%) 24 54.0 67.3 48.0 27.0 48 81.2 86.7 59.0 31.0 120 91.0 93.1 67.0 38.0
Final pH 24 7.2 7.6 8.3 8.8 48 7.5 7.8 8.4 8.8 120 7.9 8.0 8.5 8.9

As a result, the hydrogen production was higher in the order of pH 7> 6> 8> 9 after 120 hours of photo-fermentation, and the hydrogen production at the initial pH 7 was about twice that of the initial pH 6 and 8.

In addition, the cell concentration was optimal after 48 hours of incubation, and the highest pH was in the order of 6> 7> 8> 9, and the highest value was the absorbance of 3.5 (measured at 660 nm wavelength) at the initial pH of 6.

In order for the cells to grow optimally, initial pH 6 was a suitable condition, and the optimum condition of hydrogen production was initial pH 7.0.

In addition, after 120 hours of photo-fermentation, more than 90% of the malate was decomposed at the initial pH of 6-7, compared to 38% at the initial pH of 9.

Experimental Example  3: Carbon source  effect

As the carbon source, maleate (octane sugar), succinate (octane sugar) and formate (monose sugar) were used, respectively, and for the carbon source, 8 mM glutamate was used for each carbon source. In addition, the initial cell concentration was 0.56 g dcw / l, the initial pH was set to 7. The maleate and succinate were 30, 60 mM, and formate were prepared in a concentration of 30, 60, 120, 240 mM, and the culture was performed for 48 hours according to the C / N ratio, and the results are shown in Table 2.

Impact of Carbon Sources temperament density
(mM) C / N ratio
(w / w) H ₂ Total amount
(ml / l-medium) Cell concentration
(g dcw / l) Substrate decomposition
(%) H ₂ production efficiency
(mol H ₂ / mol substrate) Substrate conversion
(%) Malrate 30 12.86 1098.6 2.80 83.20 1.97 32.7 60 25.71 364.8 2.39 31.00 1.75 29.2 Succinate
30 12.86 1482.0 2.25 67.00 3.29 46.2 60 25.71 1259.6 1.81 41.31 2.27 32.4 Formate 30 3.22 17.2 1.44 29.46 - - 60 6.43 14.8 1.45 18.01 - - 120 12.86 21.8 1.45 12.12 - - 240 25.71 25.4 1.40 7.42 - -

As a result, after 48 hours of photo-fermentation, cell growth was 1.94 and 1.56 times higher in the medium containing 30 mM maleate and succinate, respectively, than in the medium containing 30 mM formate.

The cell concentration was increased to the highest concentration of 2.80 g dcw / l after 48 hours of photo-fermentation, and 1.24 times higher than that of succinate.

On the other hand, cumulative hydrogen production after 48 hours of photo fermentation was 1.35 times higher in 30 mM succinate medium compared to 30 mM malate medium.

The hydrogen production efficiency in succinate medium was 1.67 times (30 mM addition experiment) and 1.30 times (60 mM addition experiment) higher than maleate medium, and the maximum hydrogen production efficiency was 3.29 mol H2 / mol substrate. (substrate conversion efficiency) was 46.2%.

Therefore, it was found that succinate is preferably included as a carbon source in the medium used for hydrogen production of Rhodobacter spheroides KD131.

Comparing the cumulative hydrogen production, hydrogen production efficiency, and SCE between 30 mM and 60 mM in succinate and malate, Rhodobacter spheroides KD131 showed optimal hydrogen production efficiency at 30 mM and C / N. The ratio was 12.85 (w / w).

Experimental Example  4: Influence of nitrogen source

A medium of pH 7 containing succinate as the carbon source and (NH 2) 4 SO 4 or glutamate as the nitrogen source was used.

The concentration of ammonium ion was measured using HS-NH 3 (N) -L and HS-NH 3 (N) -H (Humas Co.).

1) Influence of (NH2) 4SO4

The initial concentration of (NH2) 4SO4 was set to 0, 2, 4, 8, 16 and 32 mM, and the concentrations of ammonium ions and cumulative hydrogen production used by Rhodobacter spheroides KD131 during light fermentation are shown in FIG. .

At 48, elapsed 48 hours at the initial concentrations of 0, 2 and 4 mM (NH2) 4SO4, hydrogen was produced at 1.21, 1.16 and 0.81 l H ₂ / l- medium, respectively. In addition, when the initial concentration of 8 mM (NH2) 4SO4 was used as the nitrogen source, hydrogen was produced at 0.0003 l H2 / l- medium, and no hydrogen was produced at the initial concentration of 16 and 32 mM (NH2) 4SO4. As a result, the lower the initial concentration of (NH4) 2SO4, the higher the cumulative hydrogen production.

The higher the initial concentration of (NH2) 4SO4, the more prolonged hydrogen production is reversible. Once the ammonium ion is removed, its activity is restored to perform hydrogen production.

Given the initial concentrations of 2, 4, and 8 mM (NH2) 4SO4, hydrogen production began at 16, 24, and 70 hours, respectively, because hydrogen production begins after ammonium ion is used for both cell growth and other metabolic activity. to be.

2) Effect of Glutamate

Rhodobacter spheroides KD131 grows in glutamate medium and shows a 1.4-fold higher cell concentration when compared to (NH4) 2SO4 medium. However, the growth curves are similar, and after 48 hours the cells are optimal, and hydrogen production is slightly different.

When glutamate was used as a nitrogen source, the initial concentration was set to 0, 4, 8, 16, 32, 64 mM, and the concentration of ammonium ions used by Rhodobacter spheroides KD131 and the cumulative hydrogen production amount are shown in FIG. 2.

As a result, when the initial concentration of glutamate was 0, 4, 8, and 16 mM, hydrogen was produced at 2.65, 1.93, 1.2, and 0.6 l H ₂ / l- medium, respectively, after 120 hours. The phenomenon of inhibition appeared.

This is because the activity of nitrogenase is inhibited by ammonium ions generated by deamination of glutamate during photofermentation.

As a result of the experiment, when succinate was added as a carbon source and (NH 4) 2 SO 4 or glutamate was not added as a nitrogen source, hydrogen production was the best, but the cell growth was not good.

30 mM succinate and (NH2) 4SO4 Alternatively, hydrogen production, cell growth, and ammonium ion concentration of Rhodobacter spheroides KD 131 in a medium containing glutamate of 12.85 (w / w) by C / N ratio are shown in FIG. 3.

Cell concentration was 1.4 times higher when glutamate was used than when (NH 2) 4 SO 4 was used as the nitrogen source. However, the growth curves were similar for both nitrogen sources, with the highest cell concentration after 48 hours of culture.

On the other hand, in hydrogen production, the medium containing glutamate showed rapid hydrogen production up to 48 hours, and the hydrogen production up to 48 hours was 1.4 times higher compared to the medium containing (NH 2) 4 SO 4. On the other hand, the medium to which (NH2) 4SO4 was added as a nitrogen source was effective for long time incubation, and the hydrogen production up to 96 hours was 1.78 times higher than that in the medium containing glutamate.

Claims (4)

Rhodobacter spheroids ( R. sphaeroides ) Hydrogen production method using Rhodobacter speroroides ( R. sphaeroides ) KD131, characterized by culturing. The method of claim 1, wherein the initial cell concentration is 0.45 g dcw / l to 0.67 g dcw / l hydrogen production method using R. sphaeroides ( R. sphaeroides ) KD131. The method of claim 1, wherein the initial pH is 6 to 7, hydrogen production method using Rhodobacter spheroroides ( R. sphaeroides ) KD131. The method of claim 1, wherein the C / N ratio is 11.56 to 14.14 (w / w) hydrogen production method using R. sphaeroides ( R. sphaeroides ) KD131.

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