JP3224992B2 - Hydrogen-producing photosynthetic microorganism and method for producing hydrogen using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel hydrogen-producing photosynthetic microorganism capable of stably and efficiently generating hydrogen from anaerobic treated water after anaerobic treatment of a sugar wastewater discharged from a food factory or the like, and a novel hydrogen-producing photosynthetic microorganism. The present invention relates to a method for producing hydrogen by using hydrogen.

[0002]

2. Description of the Related Art As is well known, hydrogen (H ₂ ) has a large amount of energy per unit density and is a clean energy that does not emit pollutants after combustion. It is greatly expected as a future energy substance to replace fossil fuels. As such a method of producing hydrogen, a method of industrially obtaining a by-product during electrolysis of water or purification of natural gas is known, and in the laboratory, metals such as zinc, aluminum, and iron are used. Adding hydrochloric acid or diluted sulfuric acid to
Electrolysis of 0-20% sodium hydroxide solution or dilute sulfuric acid, 40% mixture of sodium formate and sodium hydroxide
It is generally known to heat the solution to 0 ° C. (HCOONa + NaOH → NaCO ₃ + H ₂ ).

[0003] However, such a conventional hydrogen production method requires a large amount of electric energy together with a metal or fossil fuel as a raw material, and is therefore a high-cost energy source that is not economically viable at present.

Accordingly, the present invention has been devised in order to effectively solve such a problem, and an object of the present invention is to provide a hydrogen-producing bacterium capable of utilizing a substrate such as acetic acid or ethanol which is often contained in anaerobic treated water. And a hydrogen production method capable of obtaining a large amount of hydrogen from a waste solution at low cost by using the hydrogen-producing bacteria.

[0005]

Means for Solving the Problems In addition to the above-mentioned conventional methods for producing hydrogen, a method using a hydrogen-producing photosynthetic microorganism that produces hydrogen by photosynthesis has been studied.

[0006] Hydrogen production by the photosynthetic microorganisms is based on industrial wastewater containing a substance in which the microorganisms grow and uses infinite solar energy. It has the feature that it can be implemented at low cost.

[0007] The sugar waste liquid obtained from some food factories, etc.
By performing an appropriate anaerobic treatment, anaerobic treated water having a composition suitable for the carbon-hydrogen-producing photosynthetic microorganism to produce hydrogen is obtained. However, the number of hydrogen-producing photosynthetic microorganisms that can utilize substrates such as acetic acid and ethanol, which are often contained in the anaerobic treated water, is low in known strains and extremely low in productivity.

Accordingly, the present inventors have searched for strains that can utilize these substrates, and have discovered and separated new hydrogen-producing photosynthetic bacteria from the natural world, thereby achieving the present invention.

That is, since sugar waste liquid obtained from a part of the food industry is rich in glucose (1 g / L), it can be anaerobically treated to produce hydrogen-producing photosynthetic microorganisms such as acetic acid, propionic acid, butyric acid, and ethanol. Anaerobic treated water containing many substances that can be a substrate (carbon source) for hydrogen production. Then, even if an attempt is made to produce hydrogen from the anaerobic treated water using a known hydrogen producing photosynthetic bacterium, the known hydrogen producing photosynthetic bacterium can efficiently remove acetic acid and ethanol contained in the treated water for hydrogen production. The present inventors have isolated Rhodopseudomonas Palustr, which was originally isolated by the present inventors.
is) When a cell of a novel hydrogen-producing photosynthetic bacterium named strain R-1 (FERM P-15615) is added to the anaerobic treated water, the R-1 strain becomes acetic acid, ethanol or the like in the anaerobic treated water. A large amount of hydrogen could be produced using the substrate effectively and using light energy such as artificial light or natural light.

The strain (R-1 strain) according to the present invention is isolated from relatively eutrophic river water in a sunny place near the mouth of the Sumida River in Koto-ku, Tokyo. It has such mycological properties. For various observations, sodium acetate 0.5 g, sodium pyruvate 0.5 g, sodium succinate 0.5 g, sodium malate 0.5 g, KH ₂ PO 0.4 g, K ₂ HPO
_{0.7g, MgSO 4 · 7H 2 O} 0.2g, (NH
₄ ) 1.0 g of ₂ SO ₄ , 0.1 g of NaCl, FeCl
_{3 · 6H 2 O 0.005g, CaCl} 2 · 2H 2 O
0.05 g, yeast extract 0.2 g, biotin 0.0
1 mg, thiamine hydrochloride 0.2 mg, agar 20, distilled water 1
An agar plate culture consisting of 000 ml was used.

(A) Morphological properties Shape Rod-like Size 0.5 × 3 μm Color Red-purple No spores present (b) Cultural properties The culture medium exhibits an orange to red-purple color. In addition, when culturing is performed for a long period of time, the cells may adhere to the wall of the incubator and exhibit a reddish brown color.

(C) Physiological properties Gram stainability Negative denitrification reaction No starch hydrolysis No inorganic nitrogen source: nitrate cannot be used. Ammonia salts can be used Pigment formation: produces water-soluble pigments Attitude to oxygen: facultative anaerobic (d) Photosynthetic bacteria Based on the above properties, "Photosynthetic bacteria" is described in Hiroshi Kitamura et al. As a result of a search based on the “Identification search table at the family and genus level of photosynthetic bacteria”, Rhodopseudomon of Rhodospirillaceae family, a red non-sulfur bacterium that is a kind of photosynthetic bacteria, corresponds to this strain
is determined to belong to the genus as, and the closest one is
Rhodopseudomonas P
alustris and Rhodopseu Monascapsulatus (Rhodop)
seudomonas capsulatua). However, as described above, these known strains cannot efficiently utilize substrates such as acetic acid and ethanol, but the present strain efficiently utilizes these substrates and uses light energy such as sunlight. Since this strain can generate a large amount of hydrogen, this strain was determined to be a new strain, and was named Rhodopseudomonas Palustris R-1 strain. Deposited with the National Institute of Advanced Industrial Science and Technology (FERM P-1561
5).

In the present invention, all strains having the above-mentioned properties are included, even if the strain is obtained by mutating the strain naturally or by artificial means.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a preferred embodiment of the present invention will be described.

[0015] In an environmentally conscious hydrogen production system assumed by the present inventors (Global Energy and Industrial Technology Research Institute (RITE) commissioned by New Energy and Industrial Technology Development Organization (NEDO)), a food factory Since a large amount of carbohydrates are contained in the sugar waste liquid obtained from such a method, first, the sugar waste liquid is subjected to anaerobic treatment in an anaerobic reactor or the like to which anaerobic microorganisms are added, whereby the carbohydrates are converted into hydrogen and carbon dioxide. And after being decomposed into organic acids, the anaerobic treated water flows into the photosynthetic reactor to which photosynthetic microorganisms such as red non-sulfur bacteria are added, where the remaining organic acids are reduced and a large amount of hydrogen is produced. Efficient collection is being considered.

In the anaerobic treated water flowing into the photosynthetic reactor, a relatively large amount (2000 to 4000 mg / L) of red non-sulfur bacteria such as acetic acid, butyric acid, propionic acid, and ethanol is used as an electron donor to generate hydrogen. Available organic substances are included (an ammonium salt of about 400 mg / L is also included). However, Rhodopse
Common cell components such as ethanol such as udomonas Palustris and Rhodopseudomonas capsulatua (CH
_Although there are many strains in nature that can efficiently utilize a substance that is considerably reduced in terms of 2O) _n as an electron donor for photosynthesis, known strains and strains of a deposited strain of a microorganism depositary institution can be used. For the most part, not only ethanol, but also acetic acid (CH ₂ O) _2, which is very close to the general cell component composition ratio, could not be effectively used. Therefore, the present inventors conducted a screening of a strain capable of stably producing a large amount of hydrogen from anaerobic treated water of an environment-friendly hydrogen production system using a red non-sulfur bacterium obtained by independently searching, and as a result, It was confirmed that the sulfur bacterium R-1 exhibited the highest hydrogen production rate and had a stable hydrogen production ability.

As a medium for storing the R-1 strain,
A synthetic medium or a natural medium containing a carbon source, a nitrogen source, an inorganic substance, and other nutrients as needed can be used. Any of these carbon sources and nitrogen sources may be used as long as the R-1 strain can be used. For example,
As a carbon source, acetic acid, sodium acetate, sodium pyruvate, sodium succinate, sodium malate,
Alcohol and the like can be used. On the other hand, as the nitrogen source, for example, yeast extract, ammonium sulfide and the like can be used. As the inorganic salt, various phosphates, magnesium sulfate, calcium chloride, iron chloride, biotin and thiamine hydrochloride can be used. Further, the pH of the medium for stably culturing the R-1 strain is around 7, preferably 6.8.
Is preferred.

The culture is performed under anaerobic conditions with an illuminance of 5,000 to 100.
It is performed by irradiating with incandescent light of 00 lux for about 7 days. The culture temperature is preferably room temperature, preferably 30 ° C. About 1 passage
Perform every month.

Next, as a method for producing hydrogen using the R-1 strain, first, the R-1 strain after the above-mentioned culture is separated into a lump by a centrifugal separator, and the hydrogen is produced in a photosynthetic reactor into which anaerobic treated water flows. And irradiate sunlight or artificial light to generate hydrogen. Since the gas generated in the photosynthetic reactor contains hydrogen, carbon dioxide, oxygen, hydrogen sulfide, etc., the extracted gas is first subjected to a coarse separation process to remove hydrogen sulfide, and then to a separation membrane process. Then, carbon dioxide and oxygen are removed, and the remaining hydrogen that has passed through the separation membrane process is recovered and stored by a hydrogen storage process using a metal hydride.

When hydrogen is produced using a large photosynthetic reactor, the most important problems are the maintenance of photosynthetic bacteria in a mixed culture system and the elimination of other microorganisms (mainly microorganisms contained in anaerobic treated water). Eliminate the effects as much as possible. For that purpose, first, the generation of hydrogen from the anaerobic treated water by the R-1 strain was tried for the case where glutamic acid was added as a nitrogen source for the growth of the R-1 strain and for the case where glutamic acid was not added, as shown in Table 1. In addition, when no glutamic acid is added, the amount of generated hydrogen is larger than when glutamic acid is added, and the amount is inversely proportional to the amount of glutamic acid added,
Stopped at 50 mg / l. From this, it was found that the addition of glutamic acid inhibited the hydrogen generation of the R-1 strain. This is because the addition of glutamate activates the cell nitrogen metabolism switch-off system, and a part of the reducing power obtained by the photochemical reaction is used for cell growth, greatly reducing the activity of nitrogenase activity. It is thought to be. In particular, this point is significantly different from previously known red non-sulfur bacteria. In addition, since the amount of carbon dioxide generated was less than the theoretical value, the R-1 strain could adjust and control the balance between oxidation and reduction in cells by fixing carbon dioxide as a means for discarding excess reducing power. It is considered possible.

When cultivation was continued for 3 weeks continuously with such a small amount of glutamic acid added, hydrogen generation was slightly continuously observed. From this,
It was confirmed that culturing for a relatively short period of time (within three weeks) did not provide an atmosphere for methane fermentation even without controlling the oxidation-reduction potential.

[0022]

[Table 1]

[0023]

Next, embodiments of the present invention will be described.

(Example 1) Eight kinds of known photosynthetic bacteria (red non-sulfur bacteria) A-1, K-2y, K-6 and K-
8, M-rad, N-1, S-2, T-1 and the R-1 strain according to the present invention, and the hydrogen generation rate of each of the nine types of photosynthetic bacteria were examined using equal amounts. The viable cell count of these samples was measured by examining the absorbance at 610 nm of the culture solution. And the absorbance is equivalent to the cell weight of about 0.35 mg / ml for all strains,
In the R-1 strain, the cell weight was 0.354 mg.

First, these nine photosynthetic bacteria were subjected to 0.5 g of sodium acetate, 0.5 g of sodium propionate and 0.5 g of sodium succinate, respectively.
g, sodium malate 0.5 g, ammonium chloride (NH ₄ Cl) 0.4 g, dipotassium phosphate (K ₂ HP
O ₄ ) 1.0 g, magnesium sulfide heptahydrate (MgSO ₄ )
₄ · 7H ₂ O) 0.2g, calcium chloride dihydrate (C
aCl ₂ .2H ₂ O) 0.05 g, iron chloride hexahydrate (F
_{eCl 3 · 6H 2 O) 0.005g} , sodium chloride (NaCl) 0.1 g, yeast extract 0.1 g, biotin 0.01 mg, thiamin hydrochloride 0.2 mg, medium containing agar 20g distilled water 1000 ml (0.5 N -Adjusted to pH 6.8 using NaOH) into the screw-in test tube,
After solidification, the strain was inoculated with a dose of 1 platinum (stab culture) and 30
Incandescent lamp (Toshiba Blue Soft Lamp: 60
w, 100v), and then cultivated at 10,000 lux under an illuminance, and then stored and cultured at room temperature under a luminous intensity of about 1,000 lux using a fluorescent lamp.

Next, each of these sample bacteria was cultured in a culture medium for hydrogen production consisting of the components shown in Table 2, and this was cultured for 3 hours.
After placing the flask in a flask at 0 ° C., the mouth of the flask was closed with a silicon stopper and irradiated with light of 10,000 lux to generate gas. The generated gas was taken out from a needle pipe pierced by a silicon stopper, and the hydrogen generation rate (μl / hr / mg dry cell) was measured by each gas analyzer.

As a result, as shown in Table 3, the highest hydrogen generation rate among known photosynthetic bacteria was S-2 (
43.2 μl / hr / mg dry cell), but the R-1 according to the present invention is about twice (84.8 μl / hr / mg dry cell) which is much higher than the hydrogen generation rate of S-2. Of hydrogen generation.

[0028]

[Table 2]

[0029]

[Table 3]

(Example 2) By using the R-1 strain of the present invention and appropriately changing each substrate constituting the medium,
Substrate specificity for hydrogen generation was investigated. The measurement was performed using sodium acetate, sodium propionate, sodium butyrate, sodium pyruvate, sodium succinate, sodium malate, sodium lactate, glucose (sugar), ethanol as a substrate, and the culture solution of Example 1 above. The R-1 strain cultured at 30 ° C. for 2 days was placed in a flask at 30 ° C., the flask opening was closed with a silicon stopper, and light was emitted at 10,000 lux to generate gas. Then, the generated gas is taken out from the needle pipe pierced by the silicon stopper, and each hydrogen generation rate (
μl / hr / mg dry cell).

As a result, as shown in Table 4, the highest hydrogen generation rate (126 μl / hr / mg) was obtained when succinic acid was used.
dry cell). In addition, acetic acid (81.1 μl / hr / m
g dry cell) or malic acid (71.1μl / hr / mg dry cel)
l) showed relatively high values, and ethanol (3), which was hardly available in conventional photosynthetic bacteria,
It was confirmed that a large amount of hydrogen was produced from 4.9 μl / hr / mg dry cell).

[0032]

[Table 4]

(Example 3) Using the R-1 strain according to the present invention, a change in substrate concentration accompanying hydrogen production was examined. As a measuring method, a medium simulating anaerobic treated water obtained by anaerobically treating a sugar waste liquid containing 1% glucose, that is, sodium acetate 800 mg / l and sodium butyrate 800 mg
/ L, 800 mg / l of sodium propionate, 400 mg / l of ethanol, the R-1 strain according to the present invention was cultured on a hydrogen generation medium, and exposed to 10,000 lux of light.
After generating hydrogen for 5 days, the consumption of each substrate was measured.

As a result, as can be seen from Table 5, the concentration of each organic acid and alcohol was greatly reduced to 1/10 or less.

[0035]

[Table 5]

[0036]

In summary, according to the present invention, the R-1 strain discovered by the present inventors can utilize substances having low potential energy such as acetic acid and ethanol as substrates for hydrogen generation. A large amount of hydrogen can be stably and efficiently generated from the anaerobic treated water containing sugar substrate containing various substrates. Therefore, it is possible to exhibit excellent effects such as greatly contributing to the realization of an environment-friendly hydrogen production system which the present inventors are promoting.

[Brief description of the drawings]

FIG. 1 is a photomicrograph showing a Rhodopseudomonas pulse Tris R-1 strain according to the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:01) C12R 1:01) (C12P 3/00 (C12P 3/00 Z C12R 1:01) C12R 1:01) ( 72) Inventor Masya Motoya 2-81-11 Nishi-Shimbashi, Minato-ku, Tokyo The Institute for Global Environmental Research Institute, CO2 Fixation Project Room (72) Inventor Toshi Otsuki 2--8 Nishi-Shimbashi, Minato-ku, Tokyo No. 11 Office of Global Environmental Technology Research Institute CO2 fixation project room (72) Inventor Sakae Fukunaga 2-8-11 Nishishinbashi, Minato-ku, Tokyo Office of Global Environmental Technology Research Institute CO2 fixation room (56) References Hydrogen Energy Prog. , 11th, Vol. 3 (1996. Jun.) P. 2607-2611 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 3/00 C12N 1/20 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. Rhodopseudom pulse tris capable of generating hydrogen by photosynthesis
onas Palustris) R-1 strain (FERM P-1561)
A hydrogen-producing photosynthetic microorganism designated 5). 2. The hydrogen-producing photosynthetic microorganism according to claim 1 is cultured, and the culture is added to a sugar wastewater anaerobic treatment water containing acetic acid, ethanol, and the like, and then irradiated with light to generate hydrogen. A method for producing hydrogen using a hydrogen producing photosynthetic microorganism, wherein the hydrogen is collected.