JPH04169178A - Bacterium capable of producing hydrogen gas - Google Patents

Abstract

NEW MATERIAL:A bacterium, capable of producing hydrogen gas,-derived from termites and having the following properties. Anaerobic Gram-positive bacillus having motility. Proliferating in a bouillon culture medium containing 0.3% glucose added thereto and a gum bouillon culture medium. Biochemical property; positive (+) to glucose, lactose, maltose, salicin, aesculin, cellobiose, mannose and raffinose, negative (-) to indole, urease, gelatin and catalase and partially negative (-) to mannitol, xylose, trehalose, etc. EXAMPLE:AM14A-2 strain (FERM P-11793). USE:Production of hydrogen gas, decomposition of organic substances (especially saccharides), etc. PREPARATION:Termites suffocated to death are solidified in an agar culture medium, cultured in an anaerobic gas atmosphere and separated.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a hydrogen gas producing bacterium, and more specifically, it provides a novel hydrogen gas producing bacterium isolated from termites.

[Background technology]

In modern industrial society, large amounts of fossil fuels such as oil, coal, and natural gas are consumed, and these fossil fuels emit large amounts of NOx, SOx, and CO2 when burned.
As a result, various problems have been caused such as environmental pollution, acid rain, and global warming.

Furthermore, its reserves are finite and are said to be depleted in the near future, making it an important social problem.

For these reasons, there is a worldwide need for new energy sources that do not pollute the environment in place of fossil fuels, and alcohol and methane gas are currently attracting attention as next-generation energy sources to replace oil. There is. However, alcohol and methane gas still have the problem of producing large amounts of 002 when burned, and furthermore, their inherent energy is not high enough to be used as fuel for filming or aircraft. It has its drawbacks.

By the way, hydrogen gas has three times as much exothermic energy per unit weight of combustion as oil, and the only by-products from smoking are H and O, so it is expected to be an ideal next-generation lean energy source. has been done.

However, current industrial methods for producing hydrogen gas rely on methods such as water electrolysis, liquefied propane (LPG), and high-pressure thermal decomposition of alcohol;
Since fossil fuels are consumed as the energy source, the problems such as environmental pollution mentioned above will not be fundamentally solved unless the energy source problem in the manufacturing method is resolved.

Research on the production of hydrogen gas by microorganisms has so far been attempted. If a method to produce hydrogen gas using microorganisms is established, the advantages of that method are that the reaction takes place at room temperature and pressure, so the system configuration is simple and energy consumption is extremely low. Moreover, renewable biomass is used as a raw material for hydrogen gas production, and since this biomass is originally converted from solar energy, it is an effective use of natural energy. Furthermore, the production of hydrogen gas by microorganisms has the advantage of solving the problem of environmental purification, since it is possible to use organic substances normally present in waste or waste liquid as a raw material.

Although the production of hydrogen gas by microorganisms has excellent advantages as a method for producing hydrogen gas,
Based on previous research achievements, it is still far from being established as an industrial manufacturing method. In particular, research conducted to date has not found any microorganisms that are highly productive of hydrogen moths and offspring to enable industrial production. Methods for industrially producing gas are completely undeveloped.

Microorganisms known to date that produce hydrogen gas can be roughly divided into photosynthetic microorganisms and non-photosynthetic bacteria. The former includes the photosynthetic bacterium Rhodobacillus
Ler 5pharoides, blue-green algae 0scilla
- toria sp, Miani BG7, the latter includes the nitrogen-fixing bacterium ^zotobacLer
Chroococum, Klebsi-ella
pneumonia, a facultative anaerobic bacterium Escheri
-chia coli, EnLerobacter
aerogenes, anaerobic bacteria CIosLridi
There are um butyricum, etc.

The production of hydrogen gas by photosynthetic microorganisms requires a culture tank with a large surface area and a large amount of water in order to utilize light energy.

On the other hand, the production of hydrogen gas by non-photosynthetic bacteria is also possible using small-scale fermenters, and has the advantage of having a wide range of installation locations, such as underground installation. It is believed that using photosynthetic bacteria is more advantageous than using photosynthetic microorganisms.

Among the microorganisms isolated to date, the microorganism that produces hydrogen gas most efficiently is Tan1sh.

Enterobacter aerogenes (En
terobacLer aerogenes) E82
It is said to be the 005 strain (Tanisho S,,
et al., Int. J., Hydrogen
Energy 12623.1987; Bioch
im, Biophys.

Acta, 97311989). However, this strain is a facultative anaerobic bacterium and grows even under anaerobic conditions;
Since it grows more actively under aerobic conditions, if a large amount of hydrogen is produced in the fermenter, it will be difficult to maintain the tank aerobically, making it unsuitable for industrial production of hydrogen gas. .

[Disclosure of the invention]

As a result of extensive research, the present inventors sought hydrogen gas-producing bacteria in insects, which had never been tried before, and found that they
We succeeded in isolating a new bacterium from Termites formosans that has an extremely fast reaction rate and produces large amounts of hydrogen gas.

That is, the present invention l) Hydrogen gas-producing bacteria belonging to the genus Clostridium.

2) Separate clothing 1-1. A hydrogen gas producing bacterium having the general properties, biochemical properties and enzymatic activity listed in Tables 1-2 and 1-3.

3) AM14A-2 strain, AM37E strain, AM21B
strain, M9A-2 strain, AM37F strain, AM40A strain, A
The present invention provides hydrogen gas-producing bacteria selected from M18B strain, AM38C strain, and AM42F strain.

The above AM14A-2 strain, AM37F strain, AM21B strain, AM9A-2 strain, AM37F strain, M40A strain, AM
18B strain, AM38C-1 strain, and AM42F strain were collected at the Institute of Microbiology, Agency of Industrial Science and Technology,
No. 793, No. 11794, No. 11795,
Same, No. 11800, Same, No. 11799, Same, No. 11
No. 798, No. 11796, No. 11797, and No. 11801.

The present invention will be explained in detail below.

The present inventors have discovered that Termites fo.
R-mosans) was suffocated to death while still alive.
Anaerobic bacteria were isolated through operations detailed later.

As a culture medium for bacterial isolation and culture, a culture solution (hereinafter abbreviated as 115ON) prepared by diluting ordinary bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.) to 1/50th of the usual amount was mixed with sodium acetate, which is a substrate for methane gas, and a substrate for hydrogen gas. A culture solution prepared by adding 2.5 g/(+) of certain sodium formate (hereinafter referred to as 115
(abbreviated as ON''') was devised and used.
Poor nutrition 11 with 1.5% agar added to 15ON''
We devised a 5ON agar medium.Next, we decided to use anaerobic culture at 35°C as the culture condition, considering that it would be applied to energy recovery type waste liquid treatment in the future.

In order to efficiently isolate and culture anaerobic bacteria from Forensic termites, it is important to avoid contact with oxygen as much as possible. To do this, live Forensic termites taken from the nest are placed in a petrie dish and placed in an anaerobic incubator ( It is stored in a glove box (manufactured by Forma, USA) and is equipped with an anaerobic mixed gas (Ha
The animals were suffocated to death in an atmosphere of (10%, Co, 10%, N, 80%). Without grinding these house termites, 10 termites were added to 115ON + agar medium 2011 (!
In addition, the mixture was thoroughly mixed and solidified. This operation created conditions in which the termites were buried in the agar and did not come into much contact with the mixed gas.

Ten plates of this agar were heated for 35 minutes in an anaerobic mixed gas atmosphere.
Culture was performed at ℃ for 3 weeks.

Next, 153 strains of bacteria were isolated by bacteriological separation, and the hydrogen gas production ability of the 153 strains was tested using a screening method.
The strain was found to produce hydrogen gas. Next, an oxygen demand test was performed, and as a result, 8 strains were facultative anaerobic bacteria. In the end, we succeeded in isolating 37 candidate bacterial strains suitable for our purpose.

Among the microorganisms isolated to date, the microorganism that most efficiently produces hydrogen gas is Ente.
robacLer aerogenes E820
05 strain, but this strain is l1m+1IoQ
Hz/Q-medium-hr or 246txQ H
z/f)-w+edium·hr, whereas nine of the strains isolated by the present inventors have a hydrogen gas production ability that far exceeds this.

The general properties, biochemical properties, and enzyme activities of these nine strains are shown in Tables 1-1, 1-2, and 1-3.

Table 1-3 ^M38C-1--out = ↓ -〒
-AM40A ---Samurai +-+-AM37F ---e
−e −AM42F −−−−+
−+ −AM14A-2−−± +
(9-e -AM21B
−−−−+ + + −AM37F
---+ e-+ -AM18
B −−−+ + −+
-Rap ANA II System (Su
bstance) LIRE Urea BLTS p-N1trophenyl-B, D-
disaccharideaARA p-N1tr
ophenyl-a, L-arabinoside OP
NG o-N1trophenyl-B, D-g
alactosideaGLU p-Ni1rop
henyl-a, D-glucoside BGLU
p-N1trophenyl-B, D-glucos
ideaGAL p-N1trophenyl-
a, D-galactosidea FUc p-
N1trophenyl-a, L (ucoside NA
G p-N1trophenyl-N-acety
l-B, D-glucosaIIIinidePO4p
-n1trophenylphosphate1, GY
Leucyl-glycine-B-naph
thylamideC;LY Glycine-
B-naphthylamide PROProline
-B-napt+thylamidePAL Ph
enylalanine-B-naphthylami
deARG Arginine-B-naph
thylamideSER5erine-B-naph
thyla+n1dePYRPpyrrolidonyl
-B-naphthylamide IND Tr
yptophaneNote: The e mark indicates extremely strong enzyme activity.
P history β-Galactosidase t -Glucosidase β-Glucosidase a -Galactosidase a -Fucosidase N-Acetylglucosaminidase Al
kaline phosphotase Leucylg
lycine aminopeptidase Gly
cine a+n1nopepLidaseProli
ne a+n1 no peptidase Phenyla
lanine aminopepticlaseArg
inine aminopeptidase Serin
e aminopeptidase Pyrrolido
ne aminopeptidase Tryptoph
anase (indole production) Properties of each of these strains (API 2OA, Rap A
According to NAU system), AM21B is C1
ostridiunbeiierinckii, A
M37F and eight M14^-2 are ClosLridium
It was identified as uLyricum. 8138C-1, AM
42F and AM18B 3m strains are C1ostridiu
fi6, which is thought to belong to the genus M, is a novel bacterium that has not been previously known as a tl. Furthermore, three strains, AM37F, AM9^-2, and HachiM40^, are completely new hydrogen-producing bacteria that cannot be identified. More details 1 = To say, AM37F, 8M9A-
2 and AM40A, which are Durham-positive, bacillus, and non-spore-producing anaerobic bacteria that produce hydrogen gas.
This has never been reported before and is unprecedented. These three strains are the first bacteria discovered in the world.

Table 1-4 shows the gas production performance of these strains in a high slant medium of gum agar.

Table 1-4 Gas production capacity AM 9A in high slope culture medium of gum agar
-2---+ + AM 14A-2+ + +AM 1
8B −+
10 +AM 21B -+
＋ ＋AM 37E
-half ＋ ＋
AM 37F −+ + +A
M38C-1-10 +
+AM 40A -+ →-+A
M 42E −+ + +mat
Table 1-Table 1-5 Gas production capacity of artificial sewage 8M 14A-2-± 10 ± + +++A
M 18B −± 10 ±
10 ++ To 18 21B -± 10 ± + +++AM 37E -± 10 ± + +++＾M 37F -± 10 ± ＋ +++＾M 38G-1-Sat
Shi Sat 10 ++ AM 40A
-Earth + Earth 10 +++AM
42E −± 10 ± + +++ Book (1) N: Ordinary bouillon (manufactured by Nippon Pharmaceutical) (2) NY
: 0.5% yeast extract (Tamizu Pharmaceutical If)
Added N(3) ND: Added 0.3% glucose N(4)
PY: 0.5% peptone and yeast extract (5) PYG: PY with 1% glufus (5)
PYS: PY with 1% starch (7) GAM: Gum bouillon (manufactured by Nippon Pharmaceutical) (8)
K-meet: K-meet 1-medium (manufactured by Nippon Pharmaceutical)
The amount of gas produced was about 1A in the Durham tube.The amount of gas produced was measured using the apparatus shown in FIG. 1 of the attached drawings.

That is, seal the mouth of the culture bottle; insert a No. 18 syringe needle through the rubber stopper, and connect a vinyl tube there.
This exhaust pipe was fitted with a Norindar S for gas measurement. After culturing, the cells were turned upside down/remained in a linder and the amount of gas produced was measured.

As mentioned above, En[erobacIer aero
The hydrogen gas production capacity of Genes E82005 strain is 246
1111/12-hr, on the other hand, the hydrogen production ability of the above-mentioned nine strains that the present inventors successfully isolated from the house termite is completely incomparable with that of the E82005 strain, as shown in Table 1-6. It produces a large amount of hydrogen gas.

Table 1-6 Hydrogen gas production capacity strain name Hydrogen gas production t@ CwaQ L/ (1
-hr) AM37F 2,790
11.3AM21B 2.515
10.28 138C-12,3969,7 AM42E 2,380 9.
7AM18B I, 898
7.7AM37E I, 815
7.4AM9A-21,5016,1 AM14A-21,478' 6.0^114
0^ 759 3.1 Next, when we looked at the glycolytic ability and glycolytic enzyme activity of the nine strains mentioned above, we found that they had a very wide variety of activities (Table 1-2, Table 1-2). (See 3).

In particular, the +r-glucosidase activity and tr-galactosidase activity of the four strains HachiM9^-2, Hachi137F, H1114^-2, and AM37F were found to be extremely strong. These research results indicate that these bacterial strains are particularly effective in treating wastewater containing natural products containing sugars, such as bulb fiber, starch, and polysaccharides. These strains play an extremely important role in environmental purification through the treatment of waste liquids and waste, and in the industrialization of hydrogen gas production.

The novel hydrogen sludge-producing bacteria discovered by the present inventors has the revolutionary characteristic of producing an extremely large amount of hydrogen gas per hour at an extremely fast reaction rate, and is useful in the food industry and paper manufacturing industry. It is an extremely effective bacterium for the treatment of wastewater originating from other sources, domestic wastewater, and all types of waste containing organic substances.

Next, examples of the present invention will be shown.

Example 1 AM21B bacteria was inoculated into ordinary broth (Tamizu Seiyaku) to which glucose was added, 0.3% and 1.0%,
Cultured in a 36°C water tank using the apparatus shown in Figure 1,
The amount of hydrogen gas generated was measured every hour. The results were as shown in Table 1-7.

Example 2 Production of hydrogen scum from artificial sewage Each plot was inoculated into 300 mQ each of 0.3% glucose-added ordinary broth (manufactured by Nippon Pharmaceutical Co., Ltd.) and gum bouillon medium (manufactured by Nippon Pharmaceutical Co., Ltd.).
One solution was statically cultured at 6°C. The amount of hydrogen gas produced was calculated from the amount of gas produced and the hydrogen gas concentration. The results were as shown in Table 1-8.

Table 1-8 Example 3 Gum bouillon (manufactured by Nippon Seiyaku) medium 90 as hydrogen gas production artificial wastewater by novel hydrogen gas producing bacteria
100+nf2 of each culture was added to 0 mC, stirred, and cultured. The amount of gas produced, the amount of bacteria (OD), pH, and sugar concentration were measured every hour, and the composition of the gas was analyzed when the amount of gas produced was 200 mQ or more. The amount of hydrogen gas produced was calculated from the amount of gas produced and the gas concentration, and the relationship between the culture time and the amount of hydrogen gas produced was examined.

'El-9 shows the results.

Table 1-9 (Continued) Table 1-9 (Continued) Table +-9 (J-X) As is clear from the above, the novel hydrogen gas-producing bacteria according to the present invention is suitable for the hydrogen gas industry. It has excellent activity that is useful for industrial production methods, wastewater treatment, and waste treatment.
It is an extremely valuable microorganism in industry.

[Brief explanation of drawings]

Attached FIG. 1 shows an example of an apparatus used to measure the amount of gas produced by the novel microorganism according to the present invention. Patent applicant 1) Oral text

Claims (1)

[Claims] 1) A hydrogen gas-producing bacterium belonging to the genus Clostridium. 2) Hydrogen gas-producing bacteria having the general properties, biochemical properties, and enzyme activity listed in Tables 1-1, 1-2, and 1-3. 3) AM14A-2 strain (Feikoken Bibori No. 11793),
AM37E strain (No. 11794), AM21B strain (
Id., No. 11795), AM9A-2 strain (Id., No. 118)
00), AM37F strain (same, No. 11799), AM
40A strain (No. 11798), AM18B strain (No. 11798),
No. 11796), AM38C-1 strain (No. 1179)
A hydrogen gas-producing bacterium selected from AM42E strain (No. 7) and AM42E strain (No. 11801).