JPS61175000A - Method for fermenting methane - Google Patents

Abstract

PURPOSE:To ferment in a low-temp. region with reduced energy by using a nonoceanic low-temp. ferment bacterium which is newly separated and belonging to Methanococcus. CONSTITUTION:A nonoceanic low-temp. ferment bacterium which is newly separated and belonging to Methanococcus is efficiently cultured at the low temps. of 20-25 deg.C, and fermentation is carried out. For example, when 1pts. seed culture soln. using a methane bacterium 8-G, a methane bacterium 8-P and a methane bacterium 16-11-L which are nonoceanic low-temp. ferment bacteria belonging to Methanococcus as the dominant species and 6pts. culture soln. for medium-temp. fermentation are used for the methane fermentation of excess sludge contg. 5.68% solid material, the generation speed of gaseous methane is increased by about 4 times at 20 deg.C and by about 6 times at 25 deg.C in comparison with the case where a usual culture soln. for medium-temp. fermentation is used.

Description

[Detailed description of the invention] It is related to.

More specifically, the present invention relates to a method of carrying out methane fermentation in a low temperature range while saving energy using a newly isolated non-marine low-temperature fermenting bacterium belonging to the genus Methanococcus.

In general, methane fermentation has attracted attention since the early 20th century because it is a treatment method for urban sewage sludge, saves on ghee, and recovers the methane produced. That is, from the viewpoint of industrial wastewater treatment, since it is an anaerobic fermentation method, it has advantages such as requiring less power and producing less surplus sludge than an aerobic treatment method. Furthermore, research is being actively conducted from the perspective of recovering methane from unused waste as an energy source.

Conventionally, methane fermentation has been carried out using a high temperature (50 to 55°C) fermentation method and a medium temperature (30 to 40°C) fermentation method. However, even with the medium-temperature fermentation method, the fermenter must be heated, and in particularly cold regions such as Hokkaido, a considerable amount of energy is required to heat it to the appropriate temperature for fermentation. This makes it difficult.

In order to enable methane fermentation in cold regions, the present inventors conducted an intensive search for low-temperature-fermenting bacteria, and found that all (4) non-marine low-temperature-fermenting bacteria, which are new and recognized as new species, were found.
They succeeded in isolating the strain.

The present invention was completed using the non-marine cold-fermenting bacteria isolated here. This is a methane fermentation method characterized by culturing non-marine cold-fermenting bacteria belonging to the genus Methanococcus.

Dai prison Shishiso Rui M a')n,%1"A wrathful a Kuwa Su Naeo 1
A C Dimensions = - Ram Kagliano (Methano)
This bacterium, known as Genium Cariaci, is a marine methane bacterium that cannot grow unless the salt concentration is 1.596" or higher. However, when performing methane fermentation for the purpose of industrial wastewater treatment or energy recovery from unused waste, non-marine methane bacteria that do not require salt must be used.

The present inventors isolated and searched for methane bacteria from fields, swamps, wetlands, etc. in the Hokkaido region with the aim of obtaining non-marine methane bacteria that can produce methane efficiently even at 20 to 25 degrees Celsius. We may have succeeded in isolating four strains that are fermentable at low temperatures, as these four strains are all cocci.
It is recognized as belonging to the genus Methanococcus. These four strains are Methanobacteria 8-G (Feikoken Bacteria No. 7873), Methanobacteria 8-M (Feikoken Bacteria No. 7874), and Methanobacteria 8-P.
(Feikoken Bacteria No. 7875), Methane Bacteria 16-11
-L (February Institute of Fine Arts and Science Deposit No. 7876). Next, the mycological properties of these strains will be shown.

The basic medium used here has the following composition, and using this medium, the gas phase is hydrogen/carbonic acid (80%/20%) 2
Each test was performed by culturing in a test tube (medium 15 mA) under atmospheric pressure.

Copy of specification (no changes to the contents) θ1θ Table Basal medium (per liter) Mineral 1” (Mineral 1”) (
ml) 50 Mineral 2' (Mineral
2″)) (ml) 50 trace minerals”
(ml) 10 (Trace m1ne
rals”) trace vitamin a+ (all) 1
0 (Trace vitamins) Ferrous sulfate (FeSOa-7HzO) (g)
0.002 sodium bicarbonate (NaHCOs)
(g) 5.0 Yeast Extract
ract) (g) 2.0 tripty case
(Trypticase) (g) 2. OL-cystine hydrochloride (g) 0.5 (L-C
ysteine hydrochloride -Hz
O) Sodium sulfide (NatS* 9HzO)
(g) 0.5 mm.

(Containings 6g of Ktl(PO,,
per 1 iter of distt-Engraving of specification (no change in content) +1ed water, ) 11) IA Distilled water contains the following substances in grams.

Potassium dihydrogen phosphate 6; ammonium sulfate 6; sodium chloride 12; magnesium sulfate 2.6; calcium chloride 0.16 (Containings+ in graIIsper
1 iter of distilled water:
KHzPOa+6; (NH4)! SO4,6; Na
C1,12; Mg504-7820.2.6. CaC
1,-2H,O,0,16) cl 11 Distilled water contains the following substances in grams.

Adjust the pH to 7.0 with potassium chloride) Nitrilotriacetic acid 1.5; Magnesium sulfate 3.0; Manganese sulfate 0.5; Sodium chloride 1.0: Iron sulfate 0.
1; cobalt sulfate or cobal chloride) 0.1; calcium chloride 0.1; anine nitrate 0.1; copper sulfate 0.01
; Potassium aluminum sulfate 0.01; Boric acid 0.01;
01; Sodium molybdate 0.01.

(Containings, in grams per
1 iter of distilled water (p
Hto7. Owith KOH): n1trilo
triace 1.5; Mg5Oa'7HzO,3,
0; Mn5Oa'2HzO,0,5; NaC1゜1
．． 0; FeSO44HzO,0,1; Co50a
or CoC1g, 0.1; engraving of specification (no change in content) CaC1z'2HzO+0.1: ZnSO4,0,1
;CuSO4”5Hz0,0.01AIK(SO4)
z, 0.01; HJOi+0. Of; NazMOO
n・2HzO.

o, oi> 4) Distilled water contains the following substances in units of ■.

Biotin 2; Folic acid 2i Pyridoxy hydrochloride 10: Thiamine hydrochloride 5; Riboflavin 5; Nicotinic acid 5; Calcium monopantothenate 5; Vitamin Bl! 0.1;
p-Aminobenzoic acid 5: Riboic acid 5° (Contai
ngs, in milligralls per
1 iter of still water:
biotin, 2; folic acid, 2; p
yridoxine hydrochloride+1
0; thiallIine hydro-chlor
ide, 5; riboflavin, 5; n1co
tinic acid, 5; DL-calcium p
antothenate, 5Hvitamine Bl
! 10.1: p-asinobenzoic acid
, 5; 1 ipoic acid, 5) Methanobacterium 8
The mycological properties of -G are as follows.

It is a1 coccus, and the size is 06-1.3μ.

b. No pen hair. No motility.

c. pH dependence is optimal at pH 7 to 8, and does not grow below pH 5.5. Moreover, it does not grow at pH 9 or higher.

d. Requires hydrogen and carbon dioxide for growth and methane production.

Acetic acid is not required for growth. When the gas phase is set to 80%/20% nitrogen/carbon dioxide, methane will not be produced and no growth will occur even if formic acid, acetic acid, etc. are present in the medium.

e, 1 at each temperature from 10 to 40 °C in the basal medium
Figure 1 shows the amount of methane generated during the cultivation of Kagetsu.

The optimal temperature is 30°C, and methane fermentation is possible at 20-40°C.

[When the culture solution of Kinoshita strain 1 is irradiated with light around 350 nm, it emits blue-white fluorescence, and when it is irradiated with light around 4201 m, it emits yellow-green fluorescence.

g, vancomycin 100 μg/me in the medium
Even when added, these strains do not inhibit growth or methane production.

h, AC medium for general anaerobic bacteria (manufactured by Difco)
, does not grow on thioglycolate medium, etc.

Next, the mycological properties of Methanobacterium 8-M will be shown.

It is a1 coccus, and the size is 06-1.2μ.

b. No pen hair. No motility.

c. pH dependence is optimal at pH 7 to 8, pH 5,
5, it will not grow (%).

Moreover, it does not grow at pH 9 or higher.

d. Requires hydrogen and carbon dioxide for growth and methane production.

Acetic acid is not required for growth. If the gas phase is nitrogen/carbon dioxide (80%/20%), methane will not be produced and no growth will occur even if formic acid, acetic acid, etc. are present in the medium.

e, 1 at each temperature from 10 to 40 °C in the basal medium
Figure 2 shows the amount of methane generated during the cultivation of Kagetsu.

The optimal temperature is 30°C, and methane fermentation is possible at 20-40°C.

When the culture solution of the f1 strain is irradiated with light around 350 nm, it emits blue-white fluorescence, and when it is irradiated with light around 42 Onm, it emits yellow-green fluorescence.

g, even if 100 μg/- of pancomine was added to the medium,
These strains do not inhibit growth or methane production.

h, AC medium for general anaerobic bacteria (manufactured by Difco)
, does not grow on thioglycolate medium, etc.

1. It is a feeding anaerobe.

j, -#-Methylbutyrate, coenzyme M, etc. l, V Next, the mycological properties of methane bacterium 8-P will be shown.

It is a1 coccus and has a size of 0.6 to 1.5μ.

b. No pen hair. No motility.

c. pH dependence is optimal between pH 7 and 8; pH 5,
It will not grow below 5. Moreover, it does not grow at pH 9 or higher.

d. Requires hydrogen and carbon dioxide for growth and methane production.

Acetic acid is not required for growth. When the gas phase is nitrogen/carbon dioxide (8096/20%), no methane is produced and no growth occurs even if formic acid, acetic acid, etc. are present in the medium.

The amount of methane generated is shown in Figure 3. The optimal temperature is 30°C, and methane fermentation is possible at 20-40°C.

When the culture solution of the f1 strain is irradiated with light around 350 nm, it emits blue-white fluorescence, and when it is irradiated with light around 4201 m, it emits yellow-green fluorescence.

g. Even when 100 μg/d of vancomycin was added to the medium, these strains did not inhibit growth or methane production.

h, General anaerobic bacteria culture medium AC@' (Di f co
(manufactured by S.A.), does not grow on thioglycollate medium, etc.

It is a1 coccus, and the size is 04-1.2μ.

b. No pen hair. No motility.

c, p) (dependency is optimal at pH 7-8, pH 5
, 5 or less, it will not grow. Moreover, it does not grow at pH 9 or higher.

d. Requires hydrogen and carbon dioxide for growth and methane production.

Acetic acid is not required for growth. If the gas phase is nitrogen/carbon dioxide (80%/20%), even if there is formic acid, acetic acid, etc. in the medium, it will not grow properly. The amount of methane generated during one month of culture at each temperature of 10 to 40°C is shown in Figure 4. The optimal temperature is 30°C, and methane fermentation is possible at 20-40°C.

When the culture solution of f8 strain is irradiated with light around 350 nm, it emits blue-white fluorescence, and when it is irradiated with light around 420 nm, it emits yellow-green fluorescence.

g. Pancomyrene 100μ strain does not inhibit growth or methane production in the medium.

h. It does not grow on general anaerobic bacteria media such as AC medium (manufactured by Difco) and thioglycollate medium.

1. It is an obligate anaerobic bacterium.

According to the 8th edition of Determinative Bacteriology, both are cocci and methanogens, so it is clear that they belong to the genus Methanococcus. However, currently, the only bacterial strain belonging to the genus Methanococcus is Methanococcus vannirei (Meth
anococcus vannielii), Methanococcus volta (Methanococcus vol.
tae), but the optimum growth temperature for both species is between 32°C and 40°C, and methane fermentation is almost impossible at 20-25°C. They are completely different in that both have an optimum growth temperature of around 30°C and are capable of methane fermentation at 20 to 25°C, and are recognized as new species belonging to the genus Methanococcus.

For methane fermentation in the present invention, four strains were isolated, namely, Methanobacterium 8-G, Methanobacterium 8-M1, and Methanobacterium 8.
-P, methane bacteria 16-11-L, a mixture of two or more strains, or a group of methane-fermenting bacteria to which one or two or more strains are added as main fermentation bacteria is used.

For the anaerobic culture of the inoculum, the above-mentioned basal medium or various food processing wastewaters are used as appropriate. Further, any wastewater containing organic matter, such as various food processing wastewaters and tap water wastewaters, can be used for methane fermentation at a temperature of about 2025°C.

Next, examples of the present invention will be shown.

Example 1 In order to perform methane fermentation on excess sludge with a solid content of 5.68%, compared to the case where a seed culture solution for normal meso-temperature fermentation was used, methane bacteria 8-G, methane bacteria 8-M, and methane Bacteria 8
-P, the seed culture solution with methane bacteria 16-11-L as the dominant species is 1 part, and the seed culture solution for medium temperature fermentation is 6 parts.
The methane gas generation rate increased approximately 4 times at 0°C and approximately 6 times at 25°C.

[Brief explanation of drawings]

-G (°C) -M 8-P (°C) Togoguchi 16-1l-L (C) Procedural amendment σt0 1, Indication of case 1985 Patent Application No. 16328 2
, Title of the invention Methane fermentation method 3, Relationship with the person making the amendment Patent applicant Address 1-3-1 Kasumigaseki, Chiyoda-ku, Tokyo Name (1)
14) Todoroki Tatsu 4, '4 Ikari Ijinyu rη12^, 7. Contents of the amendment ``Page 4, line 1 to page 5, line 8 of the specification originally attached to the application. As shown in the engraving and attached sheet. (No changes to the content).”

Claims (2)

[Claims] (1) A methane fermentation method characterized by culturing non-marine cold-fermenting bacteria belonging to the genus Methanococcus. (2) The methane fermentation method according to claim 1, wherein the culture is efficiently carried out at 20 to 25°C.