KR20010011333A - Rhodopseudomonas palustris p4 and preparation for hydrogen by using the same - Google Patents

Abstract

PURPOSE: A microorganism, Rhodopseudomonas palustris P4(KFCC-11086) and a method for producing hydrogen by using the same are provided. Thereby, carbon monoxide can be converted into hydrogen, then the hydrogen is used as a source of energy, so that the environmental pollution can be inhibited. CONSTITUTION: The microorganism, Rhodopseudomonas palustris P4(KFCC-11086) is capable to rapidly convert carbon monoxide into hydrogen and its growth rate is fast. The microorganism, Rhodopseudomonas palustris P4(KFCC-11086) is isolated from the sludge in an anaerobic waste water vessel by incubating and screening procedures and is identified by morphological analysis and 16S rDNA analysis. The hydrogen is produced by incubating the microorganism, Rhodopseudomonas palustris P4(KFCC-11086) in a medium containing carbon monoxide as a sole carbon source under illuminating light, then blocking the light.

Description

RHODOPSEUDOMONAS PALUSTRIS P4 AND PREPARATION FOR HYDROGEN BY USING THE SAME}

The present invention relates to a novel microorganism which is very useful for producing a large amount of hydrogen, a useful substance using carbon monoxide, separation and cultivation thereof, and a method for producing hydrogen using the same.

Waste gases from industrial processes contain very large amounts of carbon monoxide, carbon dioxide, hydrogen, sulfur and nitrogen oxides. Among them, carbon monoxide is simply abandoned even though it is a useful energy source, and thus adversely affects the atmospheric environment as well as economic losses. In particular, COG (coke oven gas), BFG (blast furnace gas), LDG (converter gas), and waste gas generated in carbon black production in steel mills contain large amounts of carbon monoxide and carbon dioxide. Pohang Works alone reaches 3 X 10 ¹⁰ m ³ per year, and their carbon monoxide ratio is up to 70%. By-product gas of steelworks, a national infrastructure industry, is poorly recycled and only a part of it is recovered and the rest are discarded. In particular, hydrogen can be used not only as an energy source but also as a fuel cell, a raw material of a chemical industry, and a reducing agent, and thus its use range is very wide. Therefore, the development of this technology is urgent and very useful in terms of efficient use of resources in our country which lacks resources.

The process of producing hydrogen from carbon monoxide and water can be divided into chemical and biological processes. Chemical processes require high pressures and temperatures, low yields, and easy side reactions. On the other hand, the biological process uses a microorganism that converts carbon monoxide to hydrogen, and like other biological processes, there is an advantage that a high conversion rate can be obtained under mild conditions of room temperature and pressure.

Carbon monoxide conversion strains known to date can be divided into photosynthetic bacteria, anaerobic bacteria and the like, including photosynthetic bacteria such as Rhodopseudomonas gelatinosa, Rhodospirillum rubrum, and meta Absolute anaerobic microorganisms such as Methanosarcina barkeri (O'Brien, JN, Wolkin, RH, Moench, TT, Morgan, JB, and Zeikus, JG (1984) J. Bacteriol. 158, 373-375) Are known. Of these, photosynthetic bacteria, Rhodospirilum rubrum, are known to have the highest conversion rate (Klasson, KT, Lundback, KMO, Clausen, EC, and Gaddy, JL (1993) J. Biotechnol., 29, 177 -188), very low growth rates and low cell concentrations limit their commercial applications (Cowger, JP, Klasson, KT, Ackerson, MD, Clausen, EE, and Gaddy, JL (1992) Appl. Biochem. Biotechnol., 34/35, 613-624). Thus, in the development of biological processes that efficiently produce hydrogen from carbon monoxide, the development of new microorganisms with high conversion and growth rates is essential.

These photosynthetic bacteria have slow growth and conversion rates, and severe substrate inhibition occurs in the presence of high partial pressure substrates. In addition, light acts as an important factor for stability, hydrogen production ability can be long lasting only in the presence of light, growth and conversion at the same time, productivity is reduced.

In order to solve the above problems, the present invention is to provide a novel microorganism having a very high conversion rate from carbon monoxide to hydrogen, a fast growth rate. In another aspect, the present invention is to provide a method for converting carbon monoxide to hydrogen by culturing under light irradiation for microorganisms having a very high conversion rate from carbon monoxide to hydrogen, and then blocking light.

1 shows a transmission electron micrograph (X 40,000) of Rhodopseudomonas palustris P4.

FIG. 2 shows the effect of light on the growth and hydrogen production of Rhodoschudomonas palustris P4. The other conditions were the same and the irradiation time of the light was 48 hours, 24 hours, and 0 hours, respectively. 1, 2-2 and 2-3 are shown.

FIG. 3 shows the effect of light cutoff time on carbon monoxide consumption and hydrogen production by Rhodoschudomonas palustris P4, with cutoff times of 48 hours and 300 hours, respectively, shown in FIGS. 3-1 and 3-2. The mark shows the amount of carbon monoxide consumed for 12 hours per liter of culture, and the mark ○ shows the amount of hydrogen produced for 12 hours per liter of culture.

The present invention relates to a novel microorganism producing hydrogen from carbon monoxide, its culture and a method of producing hydrogen. More specifically, the microorganism having a high conversion rate from carbon monoxide to hydrogen and a rapid growth rate is related to the strain Rhodoschudomonas palustasis P4 and a method for producing hydrogen from carbon monoxide using the same. The present invention relates to a strain of Rhodopseudomonas palustris, which is a hydrogen conversion strain using carbon monoxide as a carbon source. This strain was deposited with the Korean spawn association and accession number is KFCC-11086.

For this strain, morphological analysis and 16S rDNA were analyzed. According to the morphological analysis, it has a lamellar inner membrane structure, a short rod, and a Gram-negative bacterium. It was also an anaerobic bacterium, a photosynthetic independent nutrient, and grew in chemically dependent nutrients. However, purple pigments were produced only under photosynthetic autotrophic and anaerobic conditions. This property is typical of the genus Rhodoschdomonas (Truper H. G. and Imfoff, J. F. (1989) Arch. Mikrobiol., 55: 245-256).

In addition, for the analysis of 16S rDNA, the analysis using the Sequence_Match program showed a lot of similarities with Rhodoschudomonas palustasis. Analysis by the BLAST program showed a high similarity with the Rhodoschudomonas paluristiss in the database of strains of the present invention and GenBank. An electron micrograph of the microorganism is shown in FIG. 1.

Growth and hydrogenation medium composition of the microorganism is as follows.

Medium Medium content Baktoeast Extract 1 g / l Trace minerals 50 ml / l Trace metal 1 ml / l Complex vitamins 5 ml / l

The trace minerals are KH ₂ PO ₄ , MgCl ₂ , NH ₄ Cl, CaCl ₂ · 2H ₂ O, and the trace metals are ZnSO ₄ · 7H ₂ O, MnCl ₂ · 4H ₂ O, H ₃ BO ₃ , CoCl ₂ · 6H ₂ O, CuSO ₄ · 5H ₂ O, Na ₂ MO ₂ · 2H ₂ O, NiCl ₂ · 6H ₂ O, FeSO ₄ · 7H ₂ O, and the complex vitamins include biotin, folic acid, pyridoxalic acid, lipoic acid, riboflavin, Thiamine hydrochloride, calcium D-pantothenate, cyanocobalamin, p-aminobenzoic acid and nicotinic acid.

The present invention also follows a method of producing hydrogen using carbon monoxide as a conversion substrate by culturing the microorganisms of Rhodoschudomonas palustris P4 using carbon monoxide as the sole carbon source under light irradiation, and then blocking the light. In other words, light grows as an energy source, but light irradiation is not necessary to maintain hydrogen production activity, and hydrogen production is possible for a long time only with energy generated when hydrogen is produced from carbon monoxide. Therefore, light was used for the microbial growth, and a two-stage culture method for blocking the light for the hydrogen generator was used.

Effect of light on growth and hydrogen production-If light is irradiated, it can grow at a high rate. From FIG. 2-2, if light is blocked at 24 hours in the middle of logarithmic growth, growth stops at the same time as light is blocked. From Fig. 2-3, the cells do not grow when no light is supplied from the beginning. From this fact, unlike Rhodoschodomonas gelatinosa (Uffen, R. L. (1976) Proc. Natl. Acad. Sci. USA, 73, 3298-3302), Rhodoschodomonas palustris indicates that light is essential for growth.

In order to investigate the effect of light on the growth and hydrogen production of the strain, Rhodoschdomonas palustris under the conditions of irradiating with light and supplying CO-Ar mixed gas (20:80, v / v) every 12 hours After cultivation, after 48 hours (end of logarithmic growth) and 300 hours (normal phase), the light is blocked and hydrogen production ability is investigated for 400 hours while supplying 100% carbon monoxide every 12 hours. In the case of transition to the hydrogen production step at 48 hours as shown in Figure 3-1, the high hydrogen production capacity up to 400 hours and the carbon monoxide consumption rate coincides with the hydrogen production rate, In the case of the transition to the hydrogen production step, the carbon monoxide consumption capacity lasts only 100 hours and there is no accumulation of hydrogen. After the light is shut off, there is no carbon monoxide consumption and no hydrogen accumulation. This indicates that after stopping growth, light irreversibly suppresses carbon monoxide consumption.

The present invention will be described in more detail with reference to the following examples, but the following examples are not intended to limit the protection scope of the present invention.

Example

Example 1 Isolation and Culture of Strains

Samples obtained from granular sludge in an anaerobic wastewater tank located in Busan were inoculated into 45 ml of the selective medium shown in Table 1. And integrated culture at 30 ℃ in a continuous anaerobic culture under CO-Ar mixed gas (20:80, v / v). After turbid culture, 5 ml of the culture solution was inoculated into 45 ml of the selective medium having the above composition. After repeating this incubation several times, an appropriate amount of culture was spread on agar medium containing the selective medium and incubated under light irradiation. Fast growing colonies were isolated after 2-3 days. The isolated strain was named Rhodoschudomonas palustasis P4, and was deposited with the Korean spawn association on April 30, 1999 (Accession No .: KFCC-11086). The transmission electron micrograph of the present invention is shown in FIG.

Example 2: Identification of Strains

Morphological analysis, quinone analysis, and 16S rDNA partial sequencing were performed to identify the strains isolated in Example 1. The same culture medium as in Example 1 was cultured.

Through microscopy, gram staining, and the like, the strain had a lamellar inner membrane structure, was in the form of a short rod, and was a gram negative bacterium. In addition, it was an anaerobic bacterium and was a photosynthetic autotrophic bacterium. However, purple pigments were produced only in photosynthetic autotrophs and anaerobic conditions. This feature is typical of the genus Rhodoschdomonas (Truper H. G. and Imfoff, J. F. (1989) Arch. Mikrobiol., 55: 245-256). For more accurate identification, the 16S rDNA of the Rhodoshu domonas genus strain was amplified with the 16S rDNA gene by TaKaRa PCR amplification kit (Takara Shuzo Co., Ltd. Shiga, Japan). The genomic DNA of the strain of the present invention is a template, and the primers are Lane, D. J. 16S / 23S rRNA sequencing, p. About 1.5 kb of 16S rDNA was amplified using 27f and 1492r described in 115-147 In Stackebrandt, E. and Goodfellow, M. (ed.). Reaction conditions were PCR in 30 cycles at 94 ° C (1 minute), 55 ° C (1 minute) and 72 ° C (3 minutes) for 2 minutes. After 30 cycles were completed, the final chain extension was performed at 72 ° C. for 5 minutes. The size of the amplified product was confirmed by agarose (1%, w / v). PCR products were cloned into pGEM-T Easy vector system II (Promega, Madison, Wis., USA). Cloning was confirmed by electrophoresis after digestion with EcoRI (promega). The cloned 16S rDNA was partially sequenced and analyzed with a DNA sequence analyzer (Applied Biosystems model 373A, Foster City, CA, USA). Sequences obtained using the Sequence_Match and BLAST programs were screened for the Ribosomal Database Project (RDP) and GenBank databases.

The sequence_Match analysis showed much similarity with Rhodoschudomonas palustasis, and the result of analysis by BLAST program also showed very high similarity with Rhodoshudomonas palustasis. Thus, the isolated microorganisms were identified as Rhodoschudomonas Fallulus.

Example 3: Strain Culture and Conversion to Hydrogen Production

For cultivation of the strain, Rhodoschudomonas paluristris P4 was incubated in a culture medium consisting of the components of Table 1 under an incandescent lamp (1,500 lux) and CO-Ar (20:80, v / v) mixed gas. The conversion reaction from carbon monoxide to hydrogen was carried out under various carbon monoxide partial pressures under light shielding. Cultures were incubated in 50 ml volumes in 150 ml serum bottles. The gas was analyzed using a gas chromatography with a thermal conductivity measuring device and a column filled with Molecular Sieve 5A.

Example 4 Effect of Light on Cell Growth and Hydrogen Production

According to the medium composition of Example 3, Rhodoshu domonas paluristris P4 was cultured. When light was supplied, the cells grew logarithmically at a rate of 0.3 h ⁻¹ at the maximum specific growth rate. Final cell concentration was about 0.95 g / l after 48 hours (FIG. 2-1). When light was blocked at the middle of the logarithmic growth phase at 24 hours, growth was stopped almost simultaneously (FIG. 2-2), and when the light was blocked from the beginning, little growth was observed (FIG. 2-3). From these facts, Rhodoschudomonas Fallustris P4 indicates that light is essential for growth.

Example 5 Effect of Light Cutoff Time on Cell Growth and Hydrogen Production

Rhodo Pseudomonas Fallusris P4 was cultured under light irradiation under the medium condition of Example 4. Afterwards, to investigate the effect of the light blocking time on the hydrogen production performance of Rhodoschudomonas palustris P4, the light was cut off at 48 hours (late logarithmic) and 300 hours (normal phase) and investigated for 400 hours. The CO-Ar mixed gas was refilled in the upper space of the serum bottle every 12 hours. The CO-Ar mixed gas (20:80, v / v) was supplied during the growth period under light irradiation, and hydrogen The generator was fed with various concentrations of carbon monoxide at 20-100%. After culturing under light irradiation for 48 hours, when light was blocked, high hydrogen production capacity was maintained up to 400 hours, and the maximum hydrogen production rate reached 23.2 mmol / g cell · h. The amount of carbon monoxide consumed was consistent with the amount of hydrogen produced (FIG. 3-1). When irradiated with light up to 300 hours, after 100 hours did not show carbon monoxide consumption activity and hydrogen accumulation (Fig. 3-2). In addition, after 300 hours, even when the light was blocked, carbon monoxide consumption and hydrogen production were no longer observed. These results indicate that light stops growing irreversibly after carbon monoxide consumption.

Cell Growth and Carbon Monoxide Partial Pressure Carbon monoxide partial pressure (atmospheric pressure) Specific growth rate (h ^-1 ) 0.06 0.02 0.07 0.02 0.10 0.05 0.15 0.1 0.20 0.3 0.32 0.3 0.41 0.26 0.52 0.25 0.59 0.1 0.67 0.07 0.78 0.03 0.89 0.02 0.95 0.01 1.02 0.01

Carbon monoxide partial pressure and hydrogen production rate Carbon monoxide partial pressure (atmospheric pressure) Hydrogen Specific Production Rate (mmol / g cell · h) 0.06 4.60 0.07 5.24 0.20 8.70 0.22 10.80 0.23 11.31 0.24 11.52 0.25 16.49 0.27 17.80 0.29 16.00 0.32 19.44 0.34 16.23 0.35 21.53 0.37 23.20 0.38 21.09 0.41 22.0 0.45 21.0 0.46 20.18 0.52 19.24 0.53 16.02 0.59 15.87 0.67 14.02 0.78 13.24 0.89 11.02 0.95 11.0 1.0 10.14

The present invention provides a novel microorganism having a high growth rate and a high hydrogen conversion rate, Rhodoschudomonas palustris P4, and a method for producing hydrogen by this, wherein the growth step and the conversion step are separated and grow at high speed using light In addition to producing hydrogen at high rates in the absence of these conditions, there is no substrate inhibition even under high partial pressure carbon monoxide, which is very useful industrially. In addition, by converting carbon monoxide, which is now discarded, into useful hydrogen, economic benefits can be increased by recycling waste, and environmental pollution can be prevented, and hydrogen produced can be used as a clean energy source.

Claims (2)

Rhodopseudomonas palustris, a hydrogen conversion strain containing carbon monoxide as a carbon source and hydrogen conversion substrate, P4. (Accession No. KFCC-11086) A method of producing hydrogen from carbon monoxide using the microorganism of claim 1, wherein the microorganism of claim 1 is cultured using carbon monoxide as the sole carbon source under light irradiation, and then hydrogen is produced using the carbon monoxide as a conversion substrate by blocking light.