JPH07194386A - Production of hydrocarbon using bacterium belonging to genus pseudomonas and new bacterium belonging to genus pseudomonas - Google Patents

Abstract

PURPOSE:To obtain a hydrocarbon useful as a fuel or a raw material for producing various organic compounds by culturing a bacterium which belongs to the genus Pseudomonas, capable of fixing a carbon dioxide gas and forming a hydrocarbon in an atmosphere containing a carbon dioxide gas and recovering a product after the culture. CONSTITUTION:A new bacterium [e.g. Pseudomonas anaerooreofila HD-1 (FERM-P 1,4035), ] which belongs to the genus Pseudomonas, is a bacillus, has a cell size of 0.5mumX1.2-1.5mum, is facultative anaerobic, is negative in Gram stain, has a thick cell membrane and waved surface, is hydrophobic and is capable of fixing a carbon dioxide gas and producing a hydrocarbon is cultured in an atmosphere containing a carbon dioxide gas. The cell is collected, ground, extracted with chloroform, etc., and the product is recovered to efficiently give the objective hydrocarbon such as n-tetradecane or n-hexadecane useful as a fuel or a raw material for producing various organic compounds from an unnecessary carbon dioxide gas.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing hydrocarbons using Pseudomonas bacteria and a novel Pseudomonas bacteria used therein.

[0002]

2. Description of the Related Art In recent years, the global warming phenomenon due to the accumulation of carbon dioxide gas has become an environmental problem, and as a practical carbon dioxide gas fixing method, a chemical method using a photocatalyst, a biological method using a photosynthetic bacterium, etc. Is being studied. However, bacteria that fix carbon dioxide gas to produce hydrocarbons are not known.

[0003]

An object of the present invention is to provide a method for immobilizing carbon dioxide gas to produce hydrocarbons, and a novel microorganism used in this method.

As a result of diligent research, the inventors of the present invention found a Pseudomonas bacterium that fixes a carbon dioxide gas and produces a hydrocarbon in the soil near a crude oil jet of an oil field.
The present invention has been completed.

That is, the present invention cultivates a bacterium belonging to the genus Pseudomonas and having the ability to immobilize carbon dioxide gas to produce hydrocarbons in an atmosphere containing carbon dioxide gas and recover the produced hydrocarbons. And a method for producing a hydrocarbon, comprising: The present invention also provides Pseudomonas anaerooleophyla, which is a new species of bacterium belonging to the genus Pseudomonas having the mycological properties described below.

The present invention will be described in detail below.

The inventors of the present invention, by the method described in detail in the following examples, grow anaerobically containing carbon dioxide and grow on a medium coated with crude oil from the soil near the crude oil ejection port of the Sagara oil field in Shizuoka prefecture. Gas (CO ₂ : H ₂ : N ₂ = 5:
5:90 v / v) Bacteria forming colonies were isolated by culturing under an environment, and one of the isolated bacteria was treated with H
It was named strain D-1.

The HD-1 strain has the following mycological properties. Morphology: Bacillus Cell size: 0.5 μm x 1.2-1.5 μm Oxygen requirement: Facultative anaerobic Gram stain: Negative Motility: Yes Sporulation: No Methanogenesis: No Catalase production: Yes Oxidase production: Yes Lipase Production: yes Amylase production: no Na ⁺ Requirement: none Glucose availability: none Aerobic degradation: n-decane: yes n-dodecane: yes n-tetradecane: yes n-hexadecane: yes Anaerobic degradation: n-decane : Yes n-dodecane: yes n-tetradecane: yes n-hexadecane: yes Affinity with petroleum: yes Growth temperature: 30 ° C: grows 37 ° C: grows 48 ° C: does not grow Cell membrane morphology and properties: cell membrane Thick, wavy surface and hydrophobic. Hydrocarbon production capacity: It fixes carbon dioxide gas and produces hydrocarbons.

The HD-1 strain is initially a liquid medium containing 1% n-tetradecane and mixed gas (CO ₂ : H ₂ : N ₂ = 5: 5).
Although it was grown under 5:90), it was confirmed that CO ₂ gas could be used as the sole carbon source as it was cultured under various conditions. Also, as a result of observation with an electron microscope,
The cell surface was found to be abnormally thick (about 0.02-
0.08 μm). Moreover, when high-concentration bacterial cells were suspended in sterile water, it was found that the cells were aggregated with their sides aligned, and the cell surface layer was extremely hydrophobic. Next, the cells were extracted with a mixed solvent of chloroform / methanol, and the fraction was quantified, which was about 30% per dry cell,
It was considerably larger than E. coli (7%) and Bacillus subtilis (4%). Further, when this hydrophobic fraction was developed by thin layer chromatography using a mixed solvent of benzene / hexane, spots were confirmed at places where the Rf value was considerably high.

When the above-mentioned mycological properties were searched by the API system, (1) no species with an identification probability of 100% was found ( Pseudomonas mesophilica with a maximum value of 76.3%), (2)
The feature of thick cell membrane and wavy surface is that there are no morphologically similar substances, and that (3) it has an affinity for petroleum (that is, it can grow on a solid medium coated with petroleum). Therefore, it was determined to be a new species of the genus Pseudomonas, not the existing species, and named Pseudomonas anaerooleophila .

Pseudomonas anaerooleophyla H
The D-1 strain has been deposited at the Institute of Biotechnology, Institute of Biotechnology, and the deposit number is FERM P-14035.
Is.

The Pseudomonas anaerooleophila of the present invention is, for example, BM liquid medium (Na ₂ S 0.1 g / l, (N
_{H 4) 2 SO 4 5.0 g} / l, MgCl 2 · 7H 2 O 0.5 g / l and KH ₂ PO ₄ 1.0
The pH of water containing g / l was adjusted to 7.2 with 2N NaOH)
In a mixed gas (CO ₂ : H ₂ : N ₂ = 1
(0: 5: 85 v / v) It can be cultivated by culturing under aeration.

By culturing under the above-mentioned conditions, carbon dioxide gas is fixed and aliphatic hydrocarbons such as n-tetradecane and n-hexadecane are produced. The produced hydrocarbon can be recovered by collecting the cells, crushing, and then extracting with an organic solvent such as chloroform. If necessary, the recovered hydrocarbon can be isolated into each component by a conventional method such as chromatography. The obtained hydrocarbon has applications as a fuel and a raw material for producing various organic compounds.

The present invention will be described in detail below based on examples. However, the present invention is not limited to the following examples.

Example 1 Isolation of Pseudomonas anaerooleophila HD-1 Strain A soil sample near the crude oil jet outlet of the Sagara oil field in Shizuoka Prefecture was suspended in sterile water, and then BM agar medium coated with crude oil ( in tap water _{(NH 4) 2 SO 4 5.0} g / l, MgCl 2 · 6H 2 O0.5 g / l
And pH of water containing 1.0 g / l of KH ₂ PO ₄ was adjusted to 7.0 with NaOH.
1.5% of purified agar (Agarose 1600, manufactured by Wako Pure Chemical Industries, Ltd.) was added to the product prepared in (1) and sterilized by autoclave (121 ° C., 15 minutes), and then spread on a solid). This agar medium is mixed with anaerobic gas (CO ₂ : H ₂ : N).
₂ = 5: 5: 90 v / v) At 37 ° C for 2 weeks, the growth of several colonies was confirmed. As a result of subculturing these several times under the same conditions, 3 strains that stably grow were obtained. One of these strains was named HD-1.

Example 2 Mycological properties of Pseudomonas anaerooleophila HD-1 strain. As a result of examining the bacteriological properties of the obtained HD-1 strain, the above results were obtained. For the above reasons, the HD-1 strain was determined to be a new species of Pseudomonas.
It was named Anaero Oleophyra.

Example 3 Analysis of Hydrocarbons Produced by Pseudomonas anaerooleophyla HD-1 Strain (1) Liquid Culture Pseudomonas anaerooleophyla HD-1 Strain
BM liquid medium (Na ₂ S 0.1 g / l, (N
_{H 4) 2 SO 4 5.0 g} / l, MgCl 2 · 7H 2 O 0.5 g / l and KH ₂ PO ₄ 1.0
The pH of water containing g / l was adjusted to 7.2 with 2N NaOH)
Mixed gas containing carbon dioxide gas as a carbon source (C
O ₂ : H ₂ : N ₂ = 10: 5: 85 v / v),
It was cultured at 37 ° C for 10 days. Aeration rate of mixed gas is 1
Min, 100 ml per liter of culture solution.

(2) Analysis of hydrophobic component Pseudomonas anaerooleophila HD-1 strain
The cells were collected by centrifugation at 0000G for 10 minutes. This was ultrasonically crushed, and 590 mg of the crushed cells was extracted with chloroform. The chloroform-soluble component was dried, and the obtained 20 mg fraction was dissolved in benzene. Since it was separated into a benzene-soluble fraction (9 mg) and a benzene-insoluble fraction (8 mg), these were again dissolved in chloroform and subjected to gas chromatography (GC). The results are shown in FIGS. 1 to 4.

FIG. 1 shows the results obtained by subjecting only the solvent (chloroform) to GC, and no peak derived from impurities was confirmed. FIG. 2 shows the results for the benzene-soluble fraction, and FIG. 3 shows the results for the benzene-insoluble fraction. Further, FIG. 4 shows aliphatic hydrocarbon standards having different chain lengths (carbon numbers 8, 9, 10, 12, 14, 15, 16, 2, 2).
The result about 0) is shown. In addition, GC is HiCap-CBP1
Using a column (manufactured by Shimadzu Corporation), the injection temperature is 250 ° C,
Temperature at the time of detection 250 ℃, development from 80 ℃ (5 minutes) to 240
Performed with a gradient of 8.5 ° C / min up to ° C (5 minutes).

As shown in FIGS. 1 to 4, peaks not found in the solvent-only control were detected in the bacterial cell-derived samples (benzene-soluble fraction, benzene-insoluble fraction), and some of them were detected. Standard aliphatic hydrocarbon (C
It was confirmed that the residence time was almost the same as that of ( ₁₄ , C ₁₆ ).

Then, further G for the two kinds of samples
C-Mass analysis was performed to examine whether or not the peak detected by GC was a hydrocarbon. The results are shown in FIGS.
Shown in.

FIG. 5 shows the results for the benzene-insoluble fraction and FIG. 6 shows the results for the benzene-soluble fraction. Further, in FIG. 7, n-hexadecane (molecular weight 226, a) is used as a standard.
Alternatively, 5-methylpentadecane (molecular weight 226, b) can be used as G
The result of having applied C-Mass is shown. It should be noted that a plurality of peaks are observed even when these preparations are used, because the preparations are cleaved to produce lower molecular weight hydrocarbons. FIG. 5 shows the peak 4 in FIG. 3 subjected to GC-Mass.
It is considered to be an aliphatic saturated hydrocarbon because it increases by H ₂ −) mass unit. Further, as is clear from comparison between FIG. 5 and FIG. 7, in FIG. 5, the fragment peaks are drawn in a smooth curve and decrease as in FIG. Since it has a molecular ion peak, it was identified as n-hexadecane. Further, in FIG. 6, peak 1 shown in FIG.
The results of subjecting 1 ′, 2, 3 to GC-Mass are shown. Of these, peaks 1 and 1 ′ are linear hydrocarbons because the fragment peaks decrease in a smooth curve, as in FIG. 7a.
It was identified as n-tetradecane and n-hexadecane, respectively, because they have molecular ion peaks of 8,226.

As described above, it was confirmed that Pseudomonas anaerooleophila HD-1 strain produces aliphatic hydrocarbons n-tetradecane and n-hexadecane by using carbon dioxide as the sole carbon source.

[0024]

The present invention provides a novel bacterium of the genus Pseudomonas capable of producing a hydrocarbon using carbon dioxide as the sole carbon source and a method for producing a hydrocarbon using the bacterium. According to the present invention, hydrocarbons useful as a raw material for producing fuels and various organic compounds can be produced from unnecessary carbon dioxide, and is expected to make a great contribution to industry.

[Brief description of drawings]

FIG. 1 is a view showing a result of subjecting chloroform, which is a solvent, to gas chromatography as a control.

FIG. 2 is a diagram showing the results of subjecting a benzene-soluble fraction of a crushed product of Pseudomonas anaerooleophila HD-1 strain of the present invention extracted with chloroform to gas chromatography.

FIG. 3 is a diagram showing the results of subjecting a benzene-insoluble fraction of a crushed product of Pseudomonas anaerooleophila HD-1 strain of the present invention extracted with chloroform to gas chromatography.

FIG. 4 is a view showing a result of subjecting an aliphatic hydrocarbon having 8 to 20 carbon atoms to gas chromatography as a standard.

FIG. 5 is a view showing a result of subjecting the benzene-insoluble fraction to GC-Mass.

FIG. 6 is a view showing a result of subjecting the benzene-soluble fraction to GC-Mass.

FIG. 7 shows the results of subjecting n-hexadecane (a) or 5-methylpentadecane (b) to GC-Mass as a standard.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1:38)

Claims (7)

[Claims] 1. A bacterium belonging to the genus Pseudomonas and having the ability to immobilize carbon dioxide gas to produce hydrocarbons,
A method for producing a hydrocarbon, which comprises culturing in an atmosphere containing carbon dioxide gas and recovering the produced hydrocarbon. 2. The method of claim 1, wherein the hydrocarbon is an aliphatic hydrocarbon. 3. The method according to claim 2, wherein the aliphatic hydrocarbon is n-tetradecane or n-hexadecane. 4. The Pseudomonas bacterium is Pseudomonas anaerooleophyla.
The method according to any one of 1. 5. The Pseudomonas anaerooleophyla HD-
The method according to claim 3, which is one strain (FERM P-14035). 6. Pseudomonas anaerooleophylla having the following mycological properties. Morphology: Bacillus Cell size: 0.5 μm x 1.2-1.5 μm Oxygen requirement: Facultative anaerobic Gram stain: Negative Motility: Yes Sporulation: No Methanogenesis: No Catalase production: Yes Oxidase production: Yes Lipase Production: yes Amylase production: no Na ⁺ Requirement: none Glucose availability: none Aerobic degradation: n-decane: yes n-dodecane: yes n-tetradecane: yes n-hexadecane: yes Anaerobic degradation: n-decane : Yes n-dodecane: yes n-tetradecane: yes n-hexadecane: yes Affinity with petroleum: yes Growth temperature: 30 ° C: grows 37 ° C: grows 48 ° C: does not grow Cell membrane morphology and properties: cell membrane Thick, wavy surface and hydrophobic. Hydrocarbon production capacity: It fixes carbon dioxide gas and produces hydrocarbons. 7. Pseudomonas anaerooleophyla H
D-1 strain (FERM P-14035).
Pseudomonas anaerooleophyla as described.