JP2000125849A - New microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new microorganism capable of decomposing hydrocarbons under a non-oxygen condition (under an anaerobic condition), also having a resistance to the hydrocarbons and being a facultative anaerobe in an operational viewpoint. SOLUTION: This microorganism belongs to the genus Klebsiella, and produces alkanes or decomposes the alkanes under an anaerobic condition. Further, the microorganism of the present invention is Klebsiella anaerooleophila TK-122 deposited as FERM P-16920. Further, the present invention is a method for decomposing the alkanes contained in petroleum, and can decompose the petroleum under an anaerobic condition. Furthermore, the present invention can fix carbon dioxide in atmosphere to produce the alkanes and alkanes by using the above microorganism.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to novel microorganisms, and more particularly, the present invention relates to Klebsiell
a) a microorganism belonging to the genus, which decomposes alkanes under anaerobic conditions or produces alkanes from carbon dioxide, and decomposes petroleum under anaerobic conditions using said microorganisms, or decomposes petroleum using said microorganisms; It relates to a method of manufacturing.

[0002]

2. Description of the Related Art With respect to the action of microorganisms on hydrocarbons, many studies have been made on the decomposition of these compounds from the viewpoint of preventing environmental pollution. In addition, many studies on fermentation using hydrocarbons as a raw material using microbial cultivation methods or studies on microbial conversion reactions have been reported, and these studies are also very useful as techniques for producing useful substances using microorganisms. It is considered important ("Petroleum Fermentation" edited by Petroleum Fermentation Research Society, published by Koshobo 1970).

[0003] Specific examples include environmental measures such as microbial decomposition of coastal contaminated crude oil, activated sludge treatment of hydrocarbon-containing wastewater, and microbiological methods of dibasic acid production using n-paraffin as a raw material. Manufacturing technologies and the like have been put to practical use.

Further, there are reports on application to petroleum refining processes and research on the production of aromatic carboxylic acids by microbial oxidation of aromatic hydrocarbons. (1) "Conversion of organic compounds by microorganisms" (G.K. L.A.
M. Gorobreaver (Academic Publishing Center)) and (2) R.G. M. Atlas, Microbial Degradation of Petr
oleum Hydrocarbons. Microbiological Reviews, 45, 1
80-209 (1981)]. In these studies, the genus Arthrobacter, the genus Brevibacterium, the genus Corynebacterium, the genus Micrococcus, the genus Mycobacterium, the genus Nocardia. , Pseudomonas (Pseu
domonas, Rhodococcus
cus), Candida, Bacillus, Acinetobacter (Ac)
It has been clarified that microorganisms such as the genus inetobacter have hydrocarbon-assimilating properties.

[0005]

However, most of these hydrocarbon-assimilating bacteria are aerobic bacteria, which require nutrient replenishment, large amounts of water, and continuous ventilation. There are drawbacks such as the need for

[0006] For this reason, the use of aerobic bacteria in the microbiological application of hydrocarbons has many physical limitations, such as under conditions that permit ventilation, or economic limitations, such as the provision of large ventilating equipment. In addition, there is a problem that there is a danger of fire, explosion, and the like, because the condition is such that a dangerous substance such as hydrocarbons and oxygen coexist.

Further, hydrocarbons generally show strong toxicity to microorganisms, and therefore, when a microbial reaction using hydrocarbons as a substrate is carried out, these compounds are in gaseous form so as not to come into direct contact with the microorganisms. It is common practice to supply at low concentration or contact at low concentration (0.1% or less).
Therefore, the decomposition rate and productivity are often low, and there is a problem that the concentration of hydrocarbons as substrates must be controlled to a low concentration.

[0008] In recent years, in order to overcome these drawbacks, researches for broadly searching for microorganisms resistant to organic solvents have been actively conducted, and novel microorganisms resistant to hydrocarbons have been identified ( JP-A-63-216473,
JP-A-63-216474, JP-A-5-2770
No. 33). Many of these resistant microorganisms are also aerobic bacteria, and some of them have the ability to assimilate hydrocarbons. However, at present, there are various restrictions such as the need for molecular oxygen for assimilation. The microorganism HD- described in JP-A-5-276933.
1 has the ability to assimilate under anaerobic conditions, but its ability is very weak. There are also reports of assimilative decomposition of hydrocarbons using anaerobic bacteria under anaerobic conditions (eg, Nature, 372, 455-458 (1994)), but the microorganisms do not tolerate oxygen. Therefore, the operation is very difficult at present.

Under the above circumstances, hydrocarbons can be assimilated under anoxic conditions (anaerobic conditions), have a resistance to hydrocarbons, and can be used from the viewpoint of operation. The emergence of sexually anaerobic microorganisms is awaited.

The present invention has been developed to meet such a demand.
An object of the present invention is to provide a novel microorganism having the above characteristics.

[0011]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, have found that microorganisms capable of growing under anaerobic conditions under conditions using a medium containing a high concentration of hydrocarbons. Specifically, it is resistant to one or a mixture of aliphatic hydrocarbons, alicyclic hydrocarbons, and aromatic hydrocarbons, and is capable of using these hydrocarbons under anaerobic conditions. First, in order to obtain microorganisms having the ability to undergo chemical degradation, the microorganisms were first searched extensively, and microorganisms having such characteristics were found. Next, the present inventors selected strains having facultative anaerobic properties from these microorganisms by further confirming growth under aerobic conditions, and furthermore, the microorganisms fixed carbon dioxide. Have the ability to produce aliphatic hydrocarbons (alkanes), and have completed the present invention.

That is, the present invention relates to Kleb
siella), a microorganism that degrades alkanes under anaerobic conditions or produces alkanes from carbon dioxide.

[0013] The invention further relates to a novel microorganism identified as Klebsiella anaerooleophila.

[0014] The present invention further provides a Klebsiella anaerooleophila FERM.
P-16920 strain.

Further, the present invention relates to a method for decomposing petroleum, characterized in that petroleum is decomposed under anaerobic conditions using the microorganism.

Further, in the above method, the petroleum is an alkane.

Further, the method of the present invention is characterized in that petroleum is produced by fixing carbon dioxide in the atmosphere using the microorganism.

Further, in the above method, the petroleum is an alkane.

A representative bacterium in the present invention is Klebsiella sp. TK-122 (Klebsiella sp.
22). The mycological properties of this fungus are as follows.

(A) Morphology Cell Shape Bacillus Cell Size 0.5 × 2-4 μm Sporulation None Motility None Gram stain Negative

(B) Growth state in various media Nutrient agar medium: The growth is abundant and the color of the colony is milky white. Somewhat shiny. Agar medium obtained by adding crude oil or alkanes to the composition shown in Table 1 shown in Example 1: colonies are milky white and shiny.

(C) Physiological properties Oxygen demand Facultative anaerobic Methanogenesis Not generated Oxidase negative Gelatin liquefaction Positive H ₂ S production Negative urease Positive / negative (weak) Indole production positive VP test negative Sugar assimilation glucose Positive sucrose positive D-mannitol positive inositol positive D-sorbitol positive L-rhamnose positive D-melibiose positive D-amygdalin positive L-arabinose positive organic assimilable citrate positive pyruvate negative amino acid decarboxylation lysine positive ornithine Growth temperature of positive bacteria 30 ℃ grows 37 ℃ grows 48 ℃ does not grow

(D) Hydrocarbon assimilation under aerobic conditions Decane Positive Dodecane Positive Tetradecane Positive Hexadecane Positive

(E) Hydrocarbon assimilation under anaerobic conditions Decane positive Dodecane positive Tetradecane positive Hexadecane positive

(F) Resistance to hydrocarbons Crude oil (10 v / v%) resistant Tetradecane (10 v / v%) resistant

According to morphological observation using a transmission electron microscope of cells of the microorganism of the present invention grown under anaerobic conditions in the above-mentioned medium containing tetradecane, the amount of cells in the cells ranges from 10 to 20% per volume. Oil droplets of low electron density bubbles are observed in almost all cells. Furthermore, many black aggregates are observed inside the cell membrane (see FIG. 1).

Further, the microorganism of the present invention is not a sulfate-reducing bacterium because it anaerobically assimilates tetradecane even in a medium containing no sulfate, it is not a hydrogen bacterium because it is a facultative anaerobic bacterium, and it is not a methane. It does not produce methane, indicating that it is not a methanogen.

Based on the above mycological properties, Bergey's Manual of Determinative Bacterio
rogy) Searched using the 8th edition (1975) and Api 20E system (manufactured by Nippon BioMerieux Biotech Co., Ltd.), and excluding the point having no motility, it was identified as Serratia odorifera and 99%. A match was found with probability.

However, when the nucleotide sequence of the 16S rRNA gene was compared, it was only 95% identical to the Serratia genus, and Klebsiella oxytoca was isolated.
oxytoca) is 99% identical to Klebsiella
The identity with planticola (Klebsiellaplanticola) was 98%. In recent years, identification of strains (especially genera) has become the mainstream based on comparison of 16S rRNA genes. As a result of the above examinations, the microorganism of the present invention was finally recognized as a new species of bacteria of the genus Klebsiella, and Klebsiella anaerooleophila (Klebsiella anaerooleophila)
a). The fact that Klebsiella bacteria generally do not have motility also coincided with the characteristics of this strain. The differences from the existing species of Klebsiella bacteria are that they can degrade alkanes under anaerobic conditions, that the VP test is negative, and that the ornithine decarboxylation reaction is positive. One strain belonging to this species, namely TK122, has been deposited as FERMP-16920 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology before the filing date of the present invention (July 29, 1998).

The medium used for culturing the microorganism of the present invention includes:
Any culture medium of any composition may be used as long as this strain can grow well, and two different culture media may be used for cell growth and for decomposition of hydrocarbons under anaerobic conditions. good. The medium used at this time may contain an appropriate carbon source, nitrogen source, inorganic ions and the like as medium components.

As the above-mentioned carbon source, carbon dioxide can be used as the carbon source in the case of autotrophic growth. In the case of a medium for decomposing hydrocarbons under anaerobic conditions, the presence of carbon dioxide and hydrogen is not essential, and the hydrocarbons used as raw materials may be used alone or in combination. An arbitrary carbon source that can be used by the microorganism of the present invention may be added to hydrocarbons used as raw materials. Those that can be used as additional carbon sources include sugars such as glucose and sucrose, and organic acids such as citric acid. In addition, in the case of a culture medium for growing cells, the above-mentioned carbon source which can utilize the microorganism of the present invention can be used.

Specific examples of the hydrocarbons used as raw materials in the case of the above-mentioned decomposition by the microorganism of the present invention include aliphatic hydrocarbons such as decane, dodecane, tetradecane and hexadecane, and crude oils which are mixtures thereof. Petroleum fractions and the like.

As the nitrogen source, inorganic nitrogen compounds such as ammonium sulfate and ammonium nitrate, and organic nitrogen sources such as peptone and yeast extract can be used. When an organic nitrogen compound is used, since the organic nitrogen compound also contains carbon, another carbon source is not necessarily required in the case of a growth medium.

The above-mentioned inorganic ions include phosphate ions, magnesium ions, calcium ions, potassium ions, iron ions, copper ions, manganese ions, and the like. These inorganic ions can be added to a medium in the form of inorganic salts or the like. You may make it contain.

As a method for culturing the microorganism of the present invention, a culture method using a liquid medium such as a shaking culture method, a culture method using a solid medium such as an agar medium, and the like can be used. The culture temperature is, for example, 15 to 42 ° C., preferably 30 to 40 ° C., the pH of the medium is near neutral, and the number of culture days is determined by the amount of bacterial cells and the decomposition of hydrocarbons (ie, the type and amount of hydrocarbons). , Degree of decomposition, etc.)
0 days, more preferably 6 days to 30 days is appropriate. Examples of the present invention will be described below, but the present invention is not limited by these examples.

[0036]

EXAMPLES Example l BM media components shown in Table 1 below, after dissolved in distilled water 1l, pH of the medium was adjusted to 7.0 with sodium hydroxide. Next, 12 g of purified agarose was added to this medium, and the mixture was sterilized by heating at 121 ° C. for 15 minutes, and then subjected to bubbling with sterile nitrogen gas (hereinafter, the bubbling treatment after the heat sterilization is referred to as anoxic / sterile treatment). ). Table 1
Was placed in an anaerobic box replaced with a mixed gas of nitrogen, carbon dioxide and hydrogen having the composition shown in Table 2 and dispensed into a petri dish, which was used as a solid medium for separating microorganisms. On the other hand, in an anaerobic box, 10 ml of distilled water subjected to anoxic and aseptic treatment in the same manner as the above medium was added to
1 g of the collected oil mud was suspended.

Crude oil 3 which has been anoxically and aseptically treated in the same manner as described above.
0 μl and 0.1 ml of the above suspension water were added to the surface of the above-mentioned petri dish, and cultured at 37 ° C. for 20 days. Liquid BM medium for which growth was observed (medium components shown in Table 1)
And cultured again at 37 ° C. for 10 days. The obtained cells were purified by a dilution culture method. All the above operations were performed in the anaerobic box.

Purification by the above-mentioned dilution method was performed as follows. The cells were appropriately diluted with sterile water, applied to an L agar medium prepared by adding 15 g of agar to the L medium shown in Table 3, and cultured at 37 ° C. for 1 day. The colonies that grew at an appropriate dilution ratio were picked up, the obtained cells were appropriately diluted again with sterile water, and spread on the above medium,
The cells were cultured at 37 ° C. for one day.

The strain obtained under anaerobic conditions is subcultured on a medium (medium having the composition shown in Table 1) supplemented with 1% of crude oil or tetradecane, cultured under aerobic conditions at 37 ° C. for 1 day, and grown. The desired microorganism was obtained by isolating the strain.

[0040]

[Table 1] Buffer: phosphate buffer 67 mM Na ₂ HPO ₄ , 6
7 mM KH ₂ PO ₄ vitamin solution (per liter);
12 g pantothein calcium, 0.59 mg bition 0.59 mg folic acid, 59 mg inositol,
0.12 g niacin, 59 mg p-aminobenzoic acid, 0.12 g pyridoxine, 59 mg ribofura
Bottle, 0.12 g thiamine hydrochloride trace elements (per _{1l); 31mg H 3 BO 3} , 57m
g CuSO ₄ , 34 mg MnSO ₄ , 14 mg Na
₂ MoO ₄ , 58 mg ZnSO ₄ , 86 mg FeS
O ₄ , 2 mg CoCl ₂ , 4 g EDTA, 8 g M
gSO ₄ , ₄ g KCl, 2 g CaCl ₂

[0041]

[Table 2]

[0042]

[Table 3] pH adjusted to 7.2 with sodium hydroxide solution

The properties, physiological properties, and the like of the strain obtained as described above were consistent with the above-mentioned description of Klebsiella anaerooleophila.

Example 2 50 ml of the medium shown in Table 1 was placed in a 0.3-liter sealed flask.
Then, 50 μl of crude oil was added thereto, and the mixture was sterilized by heating at 121 ° C. for 15 minutes, and then left overnight in an anaerobic box filled with a mixed gas shown in Table 2 to obtain a completely anaerobic state. Then, TK-122 was inoculated, sealed, and shaken at 37 ° C for 4 days.

As a result, about 20 mg / l of Klebsiella anaerooleophila TK-122 was obtained, and no contamination or growth of other microorganisms was observed. Crude oil was extracted by adding an equal amount of hexane to the culture solution, and 1 μl of the crude oil was supplied to a gas chromatograph. Crude oil components were quantified using FID as a detector. The column is HP-1 manufactured by Hewlett-Packard.
(0.25 mm X 30 m), helium was flowed as a carrier gas at a flow rate of 25 ml / min, and a linear temperature gradient of 80 to 300 ° C was applied to the column oven for rapid elution of the target component. As a result, it was confirmed that the microorganisms significantly degrade linear hydrocarbons from octane (C ₈ ) to hexadecane (C ₁₆ ). In particular, dodecane (C ₁₂ ) was decomposed best, with a decomposition rate of 50% or more (see FIGS. 2 and 3).

[0046] Comparative Example Klebsiella sp. TK-122 without using a strain hydrocarbons are known to assimilate degraded in aerobic conditions, Alice Arthrobacter globiformis (Arthrobacter gilobi
formis) IFO12137, Alice Lacta Biscosus (Art
hrobacter viscosus) IFO13497, Pseudomonas fluorescens (Paeudomonas fluorescens) IFO392, Pseudomonas putida (Paeudomonas putida) IFO12996,
The same experiment as in Example 2 was performed except that ATCC17484, ATCC12633, Pseudomonas sp. (ATCC17483) and Rhodococcus sp. (Rhodococcus sp.) ATCC19070 were used. As a result, these strains
No growth was observed in any of them, and hydrocarbons could not be assimilated under anaerobic conditions. Further, Japanese Patent Application Laid-Open No. 5-276
The HD-1 strain described in No. 933 assimilated hydrocarbons under anaerobic conditions, but the amount was 1% or less, in comparison with which TK-122 was far superior in its ability (FIG. 2, see FIG. 3).

Example 3 In order to prove the assimilation of hydrocarbons in the absence of oxygen, that is, under anaerobic conditions, it is necessary to clarify what functions as an electron acceptor. Therefore, the influence on the alkane decomposition yield by examining the components capable of functioning as electron acceptors from the medium shown in Table 1 was examined (FIG. 4 (a)). The experiment was performed in the same manner as in Example 2. First, the medium from the medium components shown in Table 1 to remove ^{_{SO 4 2- (BM: SO 4}} 2- free), the electron acceptor because alkanes comparable to BM was degraded is not SO ₄ ^2-a It has been found. Next, an experiment was conducted by sequentially removing trace elements, and it was found that when molybdate ions were removed (BM: MoO ₄ ^2- free), no alkanes were decomposed at all. That is, it was revealed that the microorganism assimilates alkanes using molybdate ions instead of oxygen under anaerobic conditions. Subsequently, the molybdate ion concentration was changed in the range of 0 to 5 mM, and the influence on the alkane decomposition yield was examined together with the growth of the bacterial cells (FIG. 4 (b)). As a result, it was found that the cells did not grow at 0 mM, and that molybdate ions were essential for the growth of the cells themselves under anaerobic conditions. Further, when the molybdate ion concentration was 0.4 mM, the decomposition yield of alkanes was the highest, and about 75% of octane to hexadecane was decomposed. On the other hand, the optimal concentration of molybdate ions for the growth of the bacteria was 0.2 mM.

Example 4 Next, the initial crude oil concentration in the medium was changed from 0 to 1%, and the gas phase in the sealed flask was subjected to gas analysis by gas chromatography. In this case, anaerobic conditions were prepared using nitrogen gas instead of the mixed gas shown in Table 2. Otherwise, Example 2 was followed. Four days after the decomposition test, 0.1 ml of the gas phase of each flask was subjected to CarboPlot (CarboPlo).
t) The sample was supplied to a column (0.53 mm × 25 m) and maintained at 40 ° C., and helium was flowed as a carrier gas at a flow rate of 10 ml / min. The detector used TCD to measure the type and amount of gas in the sample gas phase. As a result, generation of carbon dioxide was recognized only when crude oil was contained. The amount is
If the crude oil concentration is 0.1% or more, it is almost 0.07 to 0.1
It was constant between 09 mM. This proved that the microorganism assimilated crude oil under anaerobic conditions, and that the final product was carbon dioxide (FIG. 5).

Example 5 Production of alkanes from carbon dioxide using TK-122 was carried out as follows. Medium (BM) shown in Table 1
50 ml was placed in a 0.3 l sealed flask,
, And then left overnight in an anaerobic box filled with a mixed gas containing carbon dioxide as shown in Table 2. This was inoculated with TK-122 and shaken at 37 ° C. for 4 days. As a result, 13 m of TK-122 cells
g / l was obtained, and no contamination or growth of other microorganisms was observed. As described above, it was confirmed that TK-122 fixed carbon dioxide. Next, 10 to the cell volume
The same amount of hexane was added and the bacteria were crushed with a homogenizer, and at the same time, a hydrophobic component was extracted. This was analyzed by gas chromatography according to the method described in Example 2.
As a result, it was found that 0.13 mg and 0.02 mg of alkanes and alkenes were contained per 1 g of the cells (FIG. 6, (c)). Further, the same experiment was carried out using the medium (MBM) shown in Table 4 to find that about 80 mg
/ L of cells were obtained, and no contamination or growth of other microorganisms was observed. Alkanes and alkenes recovered from the cells by hexane extraction were about 0.28 m in total.
g / g of cells. In a control experiment containing no carbon dioxide as a gas component, the cells were cultured in a medium (MBM) shown in Table 3 using nitrogen.
/ L of cells were obtained. However, the total amount of alkanes and alkenes contained per gram of cells was only 0.06 mg. That is, TK-122
It is evident that the production of alkanes and alkenes in is dependent on carbon dioxide.

[0050]

[Table 4]

In FIG. 6, (a) shows culture conditions in which the MBM medium (Table 4) was filled with nitrogen gas.
(B) is a culture condition in which a mixed gas containing carbon dioxide (Table 2) is filled in the MBM medium (Table 4), and (C) is
Mixed gas containing carbon dioxide in BM medium (Table 1) (Table 2)
Culture conditions filled with:
In (b) and (c), YAlkane / x indicates the alkane production yield per cell weight, and YAlkene / x indicates
The alkene production yield per cell weight is shown, and Yp /
x is the total production yield of alkanes and alkenes per cell weight.

[0052]

As described above, the microorganism of the present invention
It is resistant to hydrocarbons and can assimilate alkanes and alkenes under anaerobic or aerobic conditions or produce alkanes and alkenes from carbon dioxide. Therefore, the present invention has an effect that it can be widely used in fields such as petroleum refining processes, bioreactors, wastewater treatment, and protein engineering.

[Brief description of the drawings]

FIG. 1 is a microscopic photograph (magnification: 20,000 times) of the microorganism of the present invention obtained in Example 1 using tetradecane as a carbon source.

FIG. 2 is a graph comparing the assimilation resolution of crude oil with time under anaerobic conditions using TK-122 (after 0, 2, 4, and 6 days). (A) shows the results showing the assimilative cracking ability of crude oil after 0 days, (B) after 2 days, (C) after 4 days, and (D) after 6 days.

FIG. 3 is a graph showing a temporal change when crude oil is kept under anaerobic conditions without using microorganisms.

FIG. 4 (a) shows a culture medium (BM: SO ₄ ^2- free and B
M: Mo ₄ ^2-free) and the yield of the decomposed alkanes with, (b) is an exploded yield and relative strains of alkanes, varying MoO ₄ ^{2-concentration} in the medium It is a graph which each shows a growth ratio.

FIG. 5 is a graph showing the relationship between initial crude oil (% (v / v)) and the concentration of carbon dioxide in the gas phase (mM).

FIG. 6 is a graph showing a comparison of the production yield of alkanes and alkenes in each medium.

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission Date] June 25, 1999 (1999.6.2
5)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction contents]

[Document Name] Statement

[Title of the Invention] New microorganism

[Claims]

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to novel microorganisms, and more particularly, the present invention relates to Klebsiell
a) a microorganism belonging to the genus, which decomposes alkanes under anaerobic conditions or produces alkanes from carbon dioxide, and decomposes petroleum under anaerobic conditions using said microorganisms, or decomposes petroleum using said microorganisms; It relates to a method of manufacturing.

[0002]

2. Description of the Related Art With respect to the action of microorganisms on hydrocarbons, many studies have been made on the decomposition of these compounds from the viewpoint of preventing environmental pollution. In addition, many studies on fermentation using hydrocarbons as a raw material using microbial cultivation methods or studies on microbial conversion reactions have been reported, and these studies are also very useful as techniques for producing useful substances using microorganisms. It is considered important ("Petroleum Fermentation" edited by Petroleum Fermentation Research Society, published by Koshobo 1970).

[0003] Specific examples include environmental measures such as microbial decomposition of coastal contaminated crude oil, activated sludge treatment of hydrocarbon-containing wastewater, and microbiological methods of dibasic acid production using n-paraffin as a raw material. Manufacturing technologies and the like have been put to practical use.

Further, there are reports on application to petroleum refining processes and research on the production of aromatic carboxylic acids by microbial oxidation of aromatic hydrocarbons. (1) "Conversion of organic compounds by microorganisms" (G.K. L.A.
M. Gorobreaver (Academic Publishing Center)) and (2) R.G. M. Atlas, Microbial Degradation of Petr
oleum Hydrocarbons. Microbiological Reviews, 45, 1
80-209 (1981)]. In these studies, the genus Arthrobacter, the genus Brevibacterium, the genus Corynebacterium, the genus Micrococcus, the genus Mycobacterium, the genus Nocardia. , Pseudomonas (Pseu
domonas, Rhodococcus
cus), Candida, Bacillus, Acinetobacter (Ac)
It has been clarified that microorganisms such as the genus inetobacter have hydrocarbon-assimilating properties.

[0005]

However, most of these hydrocarbon-assimilating bacteria are aerobic bacteria, which require nutrient replenishment, large amounts of water, and continuous ventilation. There are drawbacks such as the need for

[0006] For this reason, the use of aerobic bacteria in the microbiological application of hydrocarbons has many physical limitations, such as under conditions that permit ventilation, or economic limitations, such as the provision of large ventilating equipment. In addition, there is a problem that there is a danger of fire, explosion, and the like, because the condition is such that a dangerous substance such as hydrocarbons and oxygen coexist.

Further, hydrocarbons generally show strong toxicity to microorganisms, and therefore, when a microbial reaction using hydrocarbons as a substrate is carried out, these compounds are in gaseous form so as not to come into direct contact with the microorganisms. It is common practice to supply at low concentration or contact at low concentration (0.1% or less).
Therefore, the decomposition rate and productivity are often low, and there is a problem that the concentration of hydrocarbons as substrates must be controlled to a low concentration.

[0008] In recent years, in order to overcome these drawbacks, researches for broadly searching for microorganisms resistant to organic solvents have been actively conducted, and novel microorganisms resistant to hydrocarbons have been identified ( JP-A-63-216473,
JP-A-63-216474, JP-A-5-2770
No. 33). Many of these resistant microorganisms are also aerobic bacteria, and some of them have the ability to assimilate hydrocarbons. However, at present, there are various restrictions such as the need for molecular oxygen for assimilation. The microorganism HD- described in JP-A-5-276933.
1 has the ability to assimilate under anaerobic conditions, but its ability is very weak. There are also reports of assimilative decomposition of hydrocarbons using anaerobic bacteria under anaerobic conditions (eg, Nature, 372, 455-458 (1994)), but the microorganisms do not tolerate oxygen. Therefore, the operation is very difficult at present.

Under the above circumstances, hydrocarbons can be assimilated under anoxic conditions (anaerobic conditions), have a resistance to hydrocarbons, and can be used from the viewpoint of operation. The emergence of sexually anaerobic microorganisms is awaited.

The present invention has been developed to meet such a demand.
An object of the present invention is to provide a novel microorganism having the above characteristics.

[0011]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, have found that microorganisms capable of growing under anaerobic conditions under conditions using a medium containing a high concentration of hydrocarbons. Specifically, it is resistant to one or a mixture of aliphatic hydrocarbons, alicyclic hydrocarbons, and aromatic hydrocarbons, and is capable of using these hydrocarbons under anaerobic conditions. First, in order to obtain microorganisms having the ability to undergo chemical degradation, the microorganisms were first searched extensively, and microorganisms having such characteristics were found. Next, the present inventors selected strains having facultative anaerobic properties from these microorganisms by further confirming growth under aerobic conditions, and furthermore, the microorganisms fixed carbon dioxide. Have the ability to produce aliphatic hydrocarbons (alkanes), and have completed the present invention.

That is, the present invention decomposes alkanes under anaerobic conditions or produces alkanes from carbon dioxide and is deposited as FERM P-16920, Klebsiella species TK122 (Klebsiel).
la sp. TK122).

Further, the present invention relates to a method for decomposing alkanes contained in petroleum under anaerobic conditions by using the microorganism.

Further, the present invention relates to a method for producing alkanes and alkenes by fixing carbon dioxide in the atmosphere using the microorganism.

The microbiological properties of the fungus of the present invention, Klebsiella sp. TK-122, are as follows.

(A) Morphology Cell Shape Bacillus Cell Size 0.5 × 2-4 μm Sporulation None Motility None Gram staining Negative

(B) Growth state in various media Nutrient agar medium: The growth is abundant and the color of the colony is milky white. Somewhat shiny. Agar medium obtained by adding crude oil or alkanes to the composition shown in Table 1 shown in Example 1: colonies are milky white and shiny.

(C) Physiological properties Oxygen demand Facultative anaerobic Methanogenesis Not generated Oxidase negative Gelatin liquefaction Positive H ₂ S production Negative urease Positive / negative (weak) Indole production positive VP test negative Sugar assimilation glucose Positive sucrose positive D-mannitol positive inositol positive D-sorbitol positive L-rhamnose positive D-melibiose positive D-amygdalin positive L-arabinose positive organic assimilable citrate positive pyruvate negative amino acid decarboxylation lysine positive ornithine Growth temperature of positive bacteria 30 ℃ grows 37 ℃ grows 48 ℃ does not grow

(D) Hydrocarbon assimilation under aerobic conditions Decane Positive Dodecane Positive Tetradecane Positive Hexadecane Positive

(E) Hydrocarbon assimilation under anaerobic conditions Decane-positive Dodecane-positive Tetradecane-positive Hexadecane-positive

(F) Resistance to hydrocarbons Crude oil (10 v / v%) resistant Tetradecane (10 v / v%) resistant

According to morphological observation using a transmission electron microscope of cells of the microorganism of the present invention grown under anaerobic conditions in the above-mentioned medium containing tetradecane, the amount of cells in the cells ranges from 10 to 20% per volume. Oil droplets of low electron density bubbles are observed in almost all cells. Furthermore, many black aggregates are observed inside the cell membrane (see FIG. 1).

Further, the microorganism of the present invention is not a sulfate-reducing bacterium because it anaerobically assimilates tetradecane even in a medium containing no sulfate, it is not a hydrogen bacterium because it is a facultative anaerobic bacterium, and it is not a methane. It does not produce methane, indicating that it is not a methanogen.

Based on the mycological properties described above, Bergey's Manual of Determinative Bacterio
rogy) Searched using the 8th edition (1975) and Api 20E system (manufactured by Nippon BioMerieux Biotech Co., Ltd.), and excluding the point having no motility, it was identified as Serratia odorifera and 99%. A match was found with probability.

However, when the nucleotide sequence of the 16S rRNA gene was compared, it had only 95% identity with the genus Serratia, indicating that Klebsiella oxytoca was present.
oxytoca) is 99% identical to Klebsiella
The identity with planticola (Klebsiellaplanticola) was 98%. In recent years, identification of strains (especially genera) has become the mainstream based on comparison of 16S rRNA genes. As a result of the above examinations, the microorganism of the present invention was finally recognized as a new species of bacteria of the genus Klebsiella, and Klebsiella anaerooleophila (Klebsiella anaerooleophila)
a). The fact that Klebsiella bacteria generally do not have motility also coincided with the characteristics of this strain. The differences from the existing species of Klebsiella bacteria are that they can degrade alkanes under anaerobic conditions, that the VP test is negative, and that the ornithine decarboxylation reaction is positive. One strain belonging to this species, namely TK122, has been deposited as FERMP-16920 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology before the filing date of the present invention (July 29, 1998).

The medium used for culturing the microorganism of the present invention is as follows:
Any culture medium of any composition may be used as long as this strain can grow well, and two different culture media may be used for cell growth and for decomposition of hydrocarbons under anaerobic conditions. good. The medium used at this time may contain an appropriate carbon source, nitrogen source, inorganic ions and the like as medium components.

In the case of autotrophic growth, carbon dioxide can be used as the above-mentioned carbon source. In the case of a medium for decomposing hydrocarbons under anaerobic conditions, the presence of carbon dioxide and hydrogen is not essential, and the hydrocarbons used as raw materials may be used alone or in combination. An arbitrary carbon source that can be used by the microorganism of the present invention may be added to hydrocarbons used as raw materials. Those that can be used as additional carbon sources include sugars such as glucose and sucrose, and organic acids such as citric acid. In addition, in the case of a culture medium for growing cells, the above-mentioned carbon source which can utilize the microorganism of the present invention can be used.

Specific examples of hydrocarbons used as raw materials in the case of decomposition by the microorganism of the present invention include aliphatic hydrocarbons such as decane, dodecane, tetradecane and hexadecane, and crude oil such as a mixture thereof. Petroleum fractions and the like.

As the nitrogen source, inorganic nitrogen compounds such as ammonium sulfate and ammonium nitrate, and organic nitrogen sources such as peptone and yeast extract can be used. When an organic nitrogen compound is used, since the organic nitrogen compound also contains carbon, another carbon source is not necessarily required in the case of a growth medium.

Further, the above-mentioned inorganic ions include phosphate ions, magnesium ions, calcium ions, potassium ions, iron ions, copper ions, manganese ions, and the like. These inorganic ions are added to the medium in the form of inorganic salts and the like. You may make it contain.

As a method for culturing the microorganism of the present invention, a culture method using a liquid medium such as a shaking culture method, and a culture method using a solid medium such as an agar medium can be used. The culture temperature is, for example, 15 to 42 ° C., preferably 30 to 40 ° C., the pH of the medium is near neutral, and the number of culture days is determined by the amount of bacterial cells and the decomposition of hydrocarbons (ie, the type and amount of hydrocarbons). , Degree of decomposition, etc.)
0 days, more preferably 6 days to 30 days is appropriate. Examples of the present invention will be described below, but the present invention is not limited by these examples.

[0032]

EXAMPLES Example l BM media components shown in Table 1 below, after dissolved in distilled water 1l, pH of the medium was adjusted to 7.0 with sodium hydroxide. Next, 12 g of purified agarose was added to this medium, and the mixture was sterilized by heating at 121 ° C. for 15 minutes, and then subjected to bubbling with sterile nitrogen gas (hereinafter, the bubbling treatment after the heat sterilization is referred to as anoxic / sterile treatment). ). Table 1
Was placed in an anaerobic box replaced with a mixed gas of nitrogen, carbon dioxide and hydrogen having the composition shown in Table 2 and dispensed into a petri dish, which was used as a solid medium for separating microorganisms. On the other hand, in an anaerobic box, 10 ml of distilled water subjected to anoxic and aseptic treatment in the same manner as the above medium was added to
1 g of the collected oil mud was suspended.

Crude oil 3 which has been anoxically and aseptically treated as described above.
0 μl and 0.1 ml of the above suspension water were added to the surface of the above-mentioned petri dish, and cultured at 37 ° C. for 20 days. Liquid BM medium for which growth was observed (medium components shown in Table 1)
And cultured again at 37 ° C. for 10 days. The obtained cells were purified by a dilution culture method. All the above operations were performed in the anaerobic box.

Purification by the above-mentioned dilution method was carried out as follows. The cells were appropriately diluted with sterile water, applied to an L agar medium prepared by adding 15 g of agar to the L medium shown in Table 3, and cultured at 37 ° C. for 1 day. The colonies that grew at an appropriate dilution ratio were picked up, the obtained cells were appropriately diluted again with sterile water, and spread on the above medium,
The cells were cultured at 37 ° C. for one day.

The strain obtained under anaerobic conditions is subcultured on a medium (medium having the composition shown in Table 1) supplemented with 1% of crude oil or tetradecane, cultured under aerobic conditions at 37 ° C. for 1 day, and grown. The desired microorganism was obtained by isolating the strain.

[0036]

[Table 1] Buffer: phosphate buffer 67 mM Na ₂ HPO ₄ , 6
7 mM KH ₂ PO ₄ vitamin solution (per liter);
12 g pantothein calcium, 0.59 mg bition 0.59 mg folic acid, 59 mg inositol,
0.12 g niacin, 59 mg p-aminobenzoic acid, 0.12 g pyridoxine, 59 mg ribofura
Bottle, 0.12 g thiamine hydrochloride trace elements (per _{1l); 31mg H 3 BO 3} , 57m
g CuSO ₄ , 34 mg MnSO ₄ , 14 mg Na
₂ MoO ₄ , 58 mg ZnSO ₄ , 86 mg FeS
O ₄ , 2 mg CoCl ₂ , 4 g EDTA, 8 g M
gSO ₄ , ₄ g KCl, 2 g CaCl ₂

[0037]

[Table 2]

[0038]

[Table 3] pH adjusted to 7.2 with sodium hydroxide solution

The properties, physiological properties, and the like of the strain obtained as described above corresponded to the above-mentioned description of Klebsiella anaerooleophila.

Example 2 50 ml of the medium shown in Table 1 was placed in a 0.3 l sealed flask.
Then, 50 μl of crude oil was added thereto, and the mixture was sterilized by heating at 121 ° C. for 15 minutes, and then left overnight in an anaerobic box filled with a mixed gas shown in Table 2 to obtain a completely anaerobic state. Then, TK-122 was inoculated, sealed, and shaken at 37 ° C for 4 days.

As a result, about 20 mg / l of Klebsiella anaerooleophila TK-122 cells were obtained, and no contamination or growth of other microorganisms was observed. Crude oil was extracted by adding an equal amount of hexane to the culture solution, and 1 μl of the crude oil was supplied to a gas chromatograph. Crude oil components were quantified using FID as a detector. The column is HP-1 manufactured by Hewlett-Packard.
(0.25 mm X 30 m), helium was flowed as a carrier gas at a flow rate of 25 ml / min, and a linear temperature gradient of 80 to 300 ° C was applied to the column oven for rapid elution of the target component. As a result, it was confirmed that the microorganisms significantly degrade linear hydrocarbons from octane (C ₈ ) to hexadecane (C ₁₆ ). In particular, dodecane (C ₁₂ ) was decomposed best, with a decomposition rate of 50% or more (see FIGS. 2 and 3).

[0042] Comparative Example Klebsiella sp. TK-122 without using a strain hydrocarbons are known to assimilate degraded in aerobic conditions, Alice Arthrobacter globiformis (Arthrobacter gilobi
formis) IFO12137, Alice Lacta Biscosus (Art
hrobacter viscosus) IFO13497, Pseudomonas fluorescens (Paeudomonas fluorescens) IFO392, Pseudomonas putida (Paeudomonas putida) IFO12996,
The same experiment as in Example 2 was performed except that ATCC17484, ATCC12633, Pseudomonas sp. (ATCC17483) and Rhodococcus sp. (Rhodococcus sp.) ATCC19070 were used. As a result, these strains
No growth was observed in any of them, and hydrocarbons could not be assimilated under anaerobic conditions. Further, Japanese Patent Application Laid-Open No. 5-276
The HD-1 strain described in No. 933 assimilated hydrocarbons under anaerobic conditions, but the amount was 1% or less, in comparison with which TK-122 was far superior in its ability (FIG. 2, see FIG. 3).

Example 3 In order to prove the assimilation of hydrocarbons in the absence of oxygen, that is, under anaerobic conditions, it is necessary to clarify what functions as an electron acceptor. Therefore, the influence on the alkane decomposition yield by examining the components capable of functioning as electron acceptors from the medium shown in Table 1 was examined (FIG. 4 (a)). The experiment was performed in the same manner as in Example 2. First, the medium from the medium components shown in Table 1 to remove ^{_{SO 4 2- (BM: SO 4}} 2- free), the electron acceptor because alkanes comparable to BM was degraded is not SO ₄ ^2-a It has been found. Next, an experiment was conducted by sequentially removing trace elements, and it was found that when molybdate ions were removed (BM: MoO ₄ ^2- free), no alkanes were decomposed at all. That is, it was revealed that the microorganism assimilates alkanes using molybdate ions instead of oxygen under anaerobic conditions. Subsequently, the molybdate ion concentration was changed in the range of 0 to 5 mM, and the influence on the alkane decomposition yield was examined together with the growth of the bacterial cells (FIG. 4 (b)). As a result, it was found that the cells did not grow at 0 mM, and that molybdate ions were essential for the growth of the cells themselves under anaerobic conditions. Further, when the molybdate ion concentration was 0.4 mM, the decomposition yield of alkanes was the highest, and about 75% of octane to hexadecane was decomposed. On the other hand, the optimal concentration of molybdate ions for the growth of the bacteria was 0.2 mM.

Example 4 Next, the initial crude oil concentration in the medium was changed from 0 to 1%, and the gas phase in the sealed flask was subjected to gas analysis by gas chromatography. In this case, anaerobic conditions were prepared using nitrogen gas instead of the mixed gas shown in Table 2. Otherwise, Example 2 was followed. Four days after the decomposition test, 0.1 ml of the gas phase of each flask was subjected to CarboPlot (CarboPlo).
t) The sample was supplied to a column (0.53 mm × 25 m) and maintained at 40 ° C., and helium was flowed as a carrier gas at a flow rate of 10 ml / min. The detector used TCD to measure the type and amount of gas in the sample gas phase. As a result, generation of carbon dioxide was recognized only when crude oil was contained. The amount is
If the crude oil concentration is 0.1% or more, it is approximately 0.07 to 0.1.
It was constant between 09 mM. This proved that the microorganism assimilated crude oil under anaerobic conditions, and that the final product was carbon dioxide (FIG. 5).

Example 5 Production of alkanes from carbon dioxide using TK-122 was carried out as follows. Medium (BM) shown in Table 1
50 ml was placed in a 0.3 l sealed flask,
, And then left overnight in an anaerobic box filled with a mixed gas containing carbon dioxide as shown in Table 2. This was inoculated with TK-122 and shaken at 37 ° C. for 4 days. As a result, 13 m of TK-122 cells
g / l was obtained, and no contamination or growth of other microorganisms was observed. As described above, it was confirmed that TK-122 fixed carbon dioxide. Next, 10 to the cell volume
The same amount of hexane was added and the bacteria were crushed with a homogenizer, and at the same time, a hydrophobic component was extracted. This was analyzed by gas chromatography according to the method described in Example 2.
As a result, it was found that 0.13 mg and 0.02 mg of alkanes and alkenes were contained per 1 g of the cells (FIG. 6, (c)). Further, the same experiment was carried out using the medium (MBM) shown in Table 4 to find that about 80 mg
/ L of cells were obtained, and no contamination or growth of other microorganisms was observed. Alkanes and alkenes recovered from the cells by hexane extraction were about 0.28 m in total.
g / g of cells. In a control experiment containing no carbon dioxide as a gas component, the cells were cultured in a medium (MBM) shown in Table 3 using nitrogen.
/ L of cells were obtained. However, the total amount of alkanes and alkenes contained per gram of cells was only 0.06 mg. That is, TK-122
It is evident that the production of alkanes and alkenes in is dependent on carbon dioxide.

[0046]

[Table 4]

In FIG. 6, (a) shows the culture conditions in which the MBM medium (Table 4) was filled with nitrogen gas.
(B) is a culture condition in which a mixed gas containing carbon dioxide (Table 2) is filled in the MBM medium (Table 4), and (C) is
Mixed gas containing carbon dioxide in BM medium (Table 1) (Table 2)
Culture conditions filled with:
In (b) and (c), YAlkane / x indicates the alkane production yield per cell weight, and YAlkene / x indicates
The alkene production yield per cell weight is shown, and Yp /
x is the total production yield of alkanes and alkenes per cell weight.

[0048]

As described above, the microorganism of the present invention
It is resistant to hydrocarbons and can assimilate alkanes and alkenes under anaerobic or aerobic conditions or produce alkanes and alkenes from carbon dioxide. Therefore, the present invention has an effect that it can be widely used in fields such as petroleum refining processes, bioreactors, wastewater treatment, and protein engineering.

[Brief description of the drawings]

FIG. 1 is a microscopic photograph (magnification: 20,000 times) of the microorganism of the present invention obtained in Example 1 using tetradecane as a carbon source.

FIG. 2 is a graph comparing the assimilation resolution of crude oil with time under anaerobic conditions using TK-122 (after 0, 2, 4, and 6 days). (A) shows the results showing the assimilative cracking ability of crude oil after 0 days, (B) after 2 days, (C) after 4 days, and (D) after 6 days.

FIG. 3 is a graph showing a temporal change when crude oil is kept under anaerobic conditions without using microorganisms.

FIG. 4 (a) shows a culture medium (BM: SO ₄ ^2- free and B
M: Mo ₄ ^2-free) and the yield of the decomposed alkanes with, (b) is an exploded yield and relative strains of alkanes, varying MoO ₄ ^{2-concentration} in the medium It is a graph which each shows a growth ratio.

FIG. 5 is a graph showing the relationship between initial crude oil (% (v / v)) and the concentration of carbon dioxide in the gas phase (mM).

FIG. 6 is a graph showing a comparison of the production yield of alkanes and alkenes in each medium.

Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court II (Reference) C12R 1:22) (C12P 5/02 C12R 1:22)

Claims (7)

[Claims] 1. The genus of the genus Klebsiella,
A microorganism that degrades alkanes under anaerobic conditions or produces alkanes from carbon dioxide. 2. The microorganism according to claim 1, wherein the microorganism is Klebsiella anaerooleophila. 3. The method according to claim 1, wherein the microorganism is a Klebsiella species TK122 (Kleb Deposited as FERM P-16920).
The microorganism according to claim 1, which is siella sp. TK122). 4. A method for cracking petroleum, comprising the steps of:
A method for decomposing petroleum under anaerobic conditions using the microorganism according to any one of the above. 5. The method according to claim 4, wherein the petroleum is an alkane. 6. A method for producing petroleum, comprising the steps of:
A method for producing petroleum by fixing carbon dioxide in the atmosphere using the microorganism according to any one of the above. 7. The oil according to claim 6, wherein the petroleum is an alkane.
The method described in.