JPS60172289A - Preparation of oxygen-containing compound - Google Patents

Abstract

PURPOSE:To obtain an oxygen-containing compound suitable to fuel, by cultivating a thermostable anaerobic cellulolytic microorganism in a culture medium containing a material containing cellulose as a main carbon source at a specific temperature, and cultivating an anaerobic microorganism capable of producing butanol therein. CONSTITUTION:A themostable anaerobic cellulolytic microorganism, preferably clostridium thermocellum C-27 (FERM-P No.7451), is cultivated in a culture medium containing a material containing cellulose, e.g. pine or pulp, as a main carbon source at 50-70 deg.C. The temperature of the culture fluid is then adjusted to 20-40 deg.C, preferably 25-35 deg.C, and an anaerobic microorganism capable of producing butanol is then inoculated therein to carry out cultivation under anaerobic conditions. Thus, an oxygen-containing compound containing butanol is formed and recovered to give the aimed compound.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing oxygenated compounds, including butanol, from cellulose-containing materials.

Various methods are known for producing oxygen-containing compounds such as cyptanol by fermentation using cellulose-containing materials as raw materials. For example, a method for producing acetone and butanol in one step by simultaneously acting on a cellulose-containing substance with hepylulase and acetone/butanol producing bacteria (Japanese Unexamined Patent Publication No. 55-13558
No. 5) has been proposed. Although this method can be thought of as a one-step process, it requires a preparation step for heptalulase as a pre-step, and is essentially close to a two-step process.

Mata, Thermoan Aerobacter under anaerobic high temperature conditions
Method for producing ethanol by reacting Ethanolicus with Clostridium thermocellum (Japanese Unexamined Patent Publication No. 56-425
No. 82) is also known, but butanol cannot be obtained by this method. Furthermore, a method has been proposed in which ethanol is produced by culturing Clostridium thermocellum at 60°C for 3 days and then culturing Thymomonas anaerobia at 37°C for 3 days, but this method also does not yield butanol, and the production of ethanol It has drawbacks such as a low amount and the need to add cellobiase.

The purpose of the present invention is to provide a method for efficiently producing oxygenated compounds with a high butanol content from a cellulose-containing substance by a fermentation method. It is characterized by culturing heat-resistant anaerobic cellulose-degrading bacteria at 50 to 70°C, and then culturing anaerobic butanol-producing bacteria at 20 to 40°C to produce oxygenated compounds containing butanol, which are then recovered. This is a method for producing an oxygen-containing compound.

In the present invention, a cellulose-containing substance refers to cellulose and a substance having cellulose as a main component. Specifically, wood such as pine, cedar, beech, and poplar; hemp, mimmata, rice straw, and bagasse.

Stems, leaves and bark of rice husk, etc.; Seed hair of cotton, etc.; Waste paper such as newspapers, magazines, waste cardboard; Other fibrous waste; Pulp, cellulose powder, etc. It is desirable to use the carbon source as a carbon source after pretreatment. The cellulose-containing substance is used so that it is contained as cellulose in the medium at a rate of about 0.5 to 10%. The types and amounts of other components in the medium, such as nitrogen sources, inorganic salts, and other substances necessary for fermentation, may be determined as appropriate using conventional methods. In addition, the culture medium should be sterilized according to the usual method before use. ◎ Known heat-resistant anaerobic cellulose-degrading bacteria can be used, but
In particular, Clostridium thermoheptarum (Oloatri)
anm tbermocellum) AT O0
274o5 and 0-27 (TERM m'-7451
) etc. are preferred. Here, p Stridium thermo7arum C-27 is a new bacterium isolated from paddy soil by the present inventors, and its mycological properties are as follows.

(a) Growth status in each of the following media ■ Does not grow in meat juice ■ Does not grow in meat juice agar medium ■ Does not grow in gelatin medium ■ Does not grow in peptone water ■ Does not grow in lidmus milk ■ Medium with the following composition Table for fast growth Media composition Potassium monophosphate 1.51 Potassium diphosphate 2.9F Ammonium sulfate 1.5i Ferrous sulfate #I (heptahydrate) o, ool 25F Magnesium chloride (hexahydrate) ) 1.0) Calcium chloride 0.
15P yeast extract 2. o1 Cystine hydrochloride o, 51 Agar (solid medium only) 2 a, o f Distilled water 100
0m Carbon source (filter paper or hepta-robiose) 1o? pH 7, Q (b) Form■Size 0.5-0.8 X 2-10 pm■Shape Rod-shaped ■Spores present, oval 1.0-1.5X 1.2-2.
0 pfrL terminal growth ■Mobility: periflagellate ■Shape of colony: White, translucent, circular and extremely small.

(Q) Physiological properties ■ Optimum growth conditions pH 7.0+' 60°C, anaerobic ■ Conditions for growth pH 6-0 to 9.0. Temperature 50℃~70℃
℃ ■ Gram negative ■ No acid-fastness ■ Methyl red test positive ■ Voges-Proscalaer reaction negative ■ No indole formation ■ No hydrogen sulfide formation ■ No nitrate reducibility 0 No catalase formation ■ Liquefies gelatin/casein Does not hydrolyze zuO#l powder, ◎No citric acid utilization ■Kudzu does not coagulate milk @ uses ammonium salts and glutamic acid ■No urease activity Utilization of various carbon sources ■L-arabinose Not utilized■ D-Arabinose l ■D-7 Lactose Used ■D-Galactose Not used OD-Glucose Used ■D-Mannose Not used ■L-Rhamnose ■D-Li4C-Sl ■D-Xylose 〃 〇7 Marobiose Used O Lactose Not used O Maltose 0 Sucrose O Melipiose @ Trehalose [Phase] Raffinose l O Melezitose [Phase] Dextrin I O Glycogen 0 Starch 〃 ◎ Amygdalin O Aesculin O Salicin Not used [Phase] Erythritol @ Inositol 〃 [Phase] Mannitol l ◎ Glycerol [Phase] Dulcitol '0 Sorbitol Used [Phase] Adonitol Not used ■Cellulose Used in the home country of Purge Aids Manual Society of Determinated Bacteriology (Bergey's+)
random of Determinative B
According to Acteriology, 8th edition, it belongs to Clostridium thermocellum. Even when comparing the native bacteria with similar heat-resistant anaerobic cellulose-degrading bacteria, there was no suitable one, so the present inventors determined that it was a new bacterium and named it Clostridium thermocephum C-27. In addition, the home country and Clostridium thermocellum Too 274oS
Table 1 shows a comparison with stocks.

Table 1 Item 0-27 ATOO27405 Salicin Utilization 10 Yeast Extract Required 1i ff/l) a, 11. .

Cellulose decomposition power Strong Weak The home country has been deposited with the National Institute of Fine Technology, and its accession number is Fll.
! It is RM P-7451.

Next, known bacteria can be used as the anaerobic butanol-producing bacteria, and Clostridium satucaloperbutylacetonicum ATOO 27021 and Clostridium 27022 are particularly preferred.

In the present invention, first, 50% of heat-resistant anaerobic cellulose-degrading bacteria are added to a medium containing the above-mentioned cellulose-containing substance as a main carbon source.
Cultivate at a temperature of ~70°C, preferably 55-65°C.

Cultivation is performed under anaerobic conditions, and conditions other than temperature should be selected so that the microorganisms used can grow well and the cellulose-containing substance can be saccharified. The culture period is usually suitable for 2 to 10 days. This culture produces and accumulates ethanol, acetic acid, etc. in addition to reducing sugars.

After culturing the heat-resistant anaerobic cellulolytic bacteria, the temperature of the culture solution is adjusted to 20-40°C, preferably 25-35°C, and anaerobic butanol-producing bacteria are inoculated. Cultivation should be carried out under anaerobic conditions, and other conditions should be adopted that are suitable for the growth of the microorganisms used and that sufficiently generate and accumulate oxygen-containing compounds such as Ibutamele. The culture period is until the desired oxygen-containing compound is sufficiently produced and accumulated, and is usually 1 to 7 days. Cultivating bugol-producing bacteria produces butanol, acetone, ethanol, etc.

According to the present invention, oxygenated compounds including butanol can be efficiently produced from cellulose-containing materials, and the proportion of butanol in the product is high (usually 48-62%).
That's the characteristic. It also includes butanol, acetone, ethanol, etc., and these purpents can be produced in a single fermenter.

Moreover, the oxygen-containing compounds such as butanol obtained by the method of the present invention have excellent compatibility as mixed gasoline for automobiles, and are expected to be used as fuel. Furthermore, it is useful as a solvent for paints, adhesives, etc., and as a chemical raw material.

Next, one embodiment of the present invention will be explained in detail using examples.

Example 1 Culture medium A or B having the composition shown in Table 2 was prepared and sterilized by a conventional method.

On the other hand, Clostridium thermocellum ATOO27405 or Clostridium thermocellum ATOO27405 or 0-2 was added to the above medium 100- as a seed culture solution.
7 (FICIIIM P-7451) and 60
The cells were cultured anaerobically at °C for 5 days.

Inoculate 20 g of seed culture solution into the above medium 2 o am/, and add 6
The cells were cultured under anaerobic conditions at 0°C for 7 days.

After the completion of the culture, the temperature of the culture solution was adjusted to 30°C. Meanwhile, Clostridium saccharuba butylacetonicum AT0027022 was added to the medium containing glucose as the main carbon source.
was inoculated and cultured anaerobically at 50°C for 3 days to prepare a seed culture solution.

10 ml of this seed culture was inoculated into the above culture solution and heated to 30℃ for 6 hours.
Culture was performed under anaerobic conditions for days (13 days in total).

The culture solution was analyzed on the 78th day and 13th day after the start of culture, and then the products were analyzed. Alcohols and volatile fatty acids were detected by gas chromatography, reducing sugars by DNS reagent,
In addition, lactic acid was fixed using an enzymatic method. 3rd result
○ Shown in Table 2 pH Near, 8 Table 3 Procedural Amendment (Voluntary) October 8, 1980 Manabu Shiga, Commissioner of the Patent Office 1, Indication of Case Patent Application 1982-2611, Name of Invention Included Method for producing oxygen compounds & relationship with the case of the person making the amendment Patent applicant New Fuel Oil Development Technology Research Association 4, Agent 1-1-10-5, Kyobashi, Chuo-ku, Tokyo 104, Invention of the specification subject to amendment Detailed explanation field.

Brief explanation column of drawings and content of drawings and amendments (1) “Example” on page 11, line 6 of the specification has been changed to “Example 1”
1C Correct.

(2) Add the following sentence after Table 3 on page 13.

“Example 2 Phosphoric acid treatment as cell lease (Journal ofO
hemioal ]Cnginsering of J
The same medium as medium A in Example 1 was prepared, except that 2.5% cellulose (according to the method of APAN±4,550-535 (1981)) was added, and the medium was sterilized by a conventional method. .

On the other hand, Clostridium sirenchichirum CC-27 (FICB P-745) was added to 100d of the above medium as a seed culture solution.
1) was inoculated and cultured anaerobically at 60°C for 3 days.

20 M of the seed culture solution was inoculated into 20 ports of the above medium and cultured under anaerobic conditions at 60° C. for 8 days.

After culturing, adjust the temperature of the culture solution to 50°C, and add 1oau (5%) of the seed culture solution of Clostridium thermocellum and Satucaloperbutyla heptonicum ATOO27022.
The cells were inoculated and cultured under anaerobic conditions at 50°C for 4 days (12 days in total).

Figure 1 shows changes in the components of the culture solution over time.

4. Brief Description of the Drawings FIG. 1 is a graph showing changes over time in the components of the culture solution in Example 2. (3) Submit Figure 1.

(that's all)

Claims (1)

[Scope of Claims] 1) Thermostable anaerobic cellulose-degrading bacteria are cultured at 50 to 70°C in a medium containing a cellulose-containing substance as the main carbon source, and then anaerobic butanol-producing bacteria are cultured at 20 to 40°C. This produces oxygenated compounds including butanol.
A method for producing an oxygen-containing compound, characterized by recovering ◎
2) The manufacturing method according to claim 1, wherein the heat-resistant anaerobic cellulose-degrading bacterium is Clostridium thermocellum. 5) Claim 1, wherein the anaerobic butanol-producing bacterium is Clostridium satucaloperbutylacetonicum.
Manufacturing method described in section.