JP4877900B2 - Hydrocarbon treatment method and hydrocarbon treatment system - Google Patents

Description

  The present invention relates to a hydrocarbon treatment method and a hydrocarbon treatment system, and more particularly, to an efficient hydrocarbon treatment method and a hydrocarbon treatment system using a kind of bacteria belonging to the genus Rhodococcus.

Rhodococcus (genus Rhodococcus) is a kind of gram-positive bacteria commonly found in soil and the ocean, and a coryneform bacterium with a high G + C content. Rhodococcus bacteria have the ability to decompose and assimilate a number of persistent compounds including petroleum hydrocarbons and polychlorinated biphenyls (PCB), as well as acrylamide and useful enzymes. It is known that there are many producing bacteria such as functional biopolymers including group or extracellular polysaccharides. Therefore, it is positioned as an industrially important fungus group, and is expected to be applied to environmental purification and substance production by a bioprocess capable of reducing energy and reducing environmental burden (Non-patent Document 1). In particular, when considering a bioprocess, microorganisms that are expected to be applied are required to have properties such as good growth and active metabolic activity in a special environment containing an organic solvent. In addition, in order to show good growth while decomposing these microorganisms in the presence of high-concentration non-volatile compounds, microorganisms necessary for purification of the oil-contaminated environment due to an oil spill accident, etc., not only have the ability to resolve them but also coexist. Therefore, it is required to have high resistance to toxicity of the hardly volatile compound.

  In order to analyze the above-described properties, first, the organic solvent resistance of the microorganism is required as a starting point, and particularly in a bioprocess, growth in the presence of a high concentration organic solvent is required. In the research on organic solvent resistance of microorganisms, genetic biochemical research has been conducted mainly on model microorganisms such as Escherichia coli and Pseudomonas bacteria, which are Gram-negative bacteria. A resistance mechanism such as vesicle formation has been proposed (Non-patent Document 2). On the other hand, in gram-positive bacteria, genetic chemical research on hydrocarbon degrading genes has progressed, but there are not many studies on organic solvent resistance. This is presumably due to the fact that Gram-positive bacteria are generally considered to have a lower level of organic solvent resistance than negative bacteria. However, when considering a bioprocess, information on resistance to organic solvents under conditions close to the actual use environment is required for microorganisms at a very close to application stage. As described above, since Rhodococcus bacteria are expected to be applied to bioprocesses, it is necessary to accumulate knowledge about the organic solvent resistance of the bacteria.

  Iwabuchi et al. Discovered that Rhodococcus rhodochrous S-2 strain is a high-concentration oil-resistant oil-degrading bacterium, and as a result of investigating its oil resistance, an extracellular polysaccharide produced by the bacterium (hereinafter, It was revealed that "EPS" is deeply involved in the resistance of hardly volatile organic solvents such as long-chain alkanes. Furthermore, when the colony morphology and solvent resistance of Rhodococcus genus bacteria were examined, rough type bacteria with a low EPS production amount have high affinity for the solvent and as a result are solvent-sensitive. On the other hand, the EPS production amount is large. Since mucoid bacteria showed resistance, it was clarified that there is a high correlation between colony morphology and organic solvent resistance in bacteria belonging to the same genus. EPS has also been shown to impart solvent resistance to solvent-sensitive rough bacteria, and from these, the formation of mucoid colonies is one index for considering the solvent resistance of the genus bacteria. (Non-Patent Document 3).

Rhodococcus erythropolis PR-4 strain is a strain isolated as a pristane (2,6,10,14-tetramethyl-pentadecane) degrading bacterium which is a kind of branched alkane (Non-patent Document 4), It is a strain that changes its colony form based on EPS production from the rough type to the mucoid type to the rough type as the culture progresses. Since this strain is known to exhibit resistance to a hardly volatile organic solvent, it has been selected as a genome analysis strain and has also begun to develop a host-vector system. Therefore, the Rhodococcus erythropolis PR-4 strain is expected to become a strain with a developed genetic manipulation system among the bacteria of the genus Rhodococcus in the near future.
Finnerty, WR et al. (1992) Annual Review of Microbiology, p193-218. Ramos, JL et al. (2002) Annual Review of Microbiology, p743-768. Iwabuchi, N. et al. (2000) Applied Environmental Microbiology, 66: 5073-5077. Komukai-Nakamura, S. et al. (1996) Journal of Fermentation and Bioengineering, 82: p570-574

  In general, when an organic solvent is treated with bacteria, it is carried out in a two-layer culture system of an organic solvent and an aqueous solvent containing medium components. However, since bacteria added in an aqueous solvent can only react at the interface between an organic solvent and an aqueous solvent, it is necessary to improve the lipophilicity of the cells and apply hydrocarbons to the environment purification and material production by bioprocesses. There is a need to improve processing efficiency.

  Accordingly, an object of the present invention is to provide an efficient hydrocarbon processing method and hydrocarbon processing system in which the lipophilicity of Rhodococcus erythropolis PR-4 strain is improved.

  The present inventors cultured Rhodococcus erythropolis PR-4 strain under medium conditions of a two-layer culture system consisting of an organic layer containing a hardly volatile organic solvent and an aqueous layer containing a liquid medium, and Rhodococcus erythropolis PR. The solvent resistance mechanism of strain -4 was examined. At that time, Rhodococcus erythropolis PR-4 strain was hardly observed in the aqueous layer, and it was observed that it migrated into the organic solvent particles and grew in the organic solvent particles.

  The present invention has been made on the basis of the above findings, and a step of adding Rhodococcus erythropolis PR-4 strain to an aqueous solvent containing a medium component and an organic solvent containing a hydrocarbon having 14 or more carbon atoms are added. The present invention provides a method for treating hydrocarbons, comprising the steps of: transferring the Rhodococcus erythropolis PR-4 strain into the organic solvent and metabolizing the hydrocarbon having 14 or more carbon atoms.

  With such a configuration, since a hydrocarbon having 14 or more carbon atoms can be metabolized in a state where the Rhodococcus erythropolis PR-4 strain is transferred into an organic solvent, the surface area of the Rhodococcus erythropolis PR-4 strain is reduced. It becomes the contact area with the hydrocarbon as it is. Therefore, as a result of the increase in the contact area between the bacteria and the hydrocarbon, the treatment efficiency can be significantly improved as compared with the case where the hydrocarbon is treated at the conventional organic solvent-aqueous solvent interface.

The present invention also includes a step of adding Rhodococcus erythropolis PR-4 strain to a first aqueous solvent containing a medium component, a step of adding a first organic solvent containing a hydrocarbon having 14 or more carbon atoms, Transferring the Rhodococcus erythropolis PR-4 strain into the first organic solvent and metabolizing the hydrocarbon having 14 or more carbon atoms; and a second aqueous solvent containing a medium component, A step of adding Rhodococcus erythropolis PR-4 strain metabolized with the above hydrocarbon, a step of adding a second organic solvent containing a hydrocarbon having 13 or less carbon atoms, and the Rhodococcus erythropolis PR-4 And transferring the strain into the second organic solvent to metabolize the hydrocarbon having 13 or less carbon atoms.

  With such a configuration, the Rhodococcus erythropolis PR-4 strain can be cultured in a state of being transferred to a hydrocarbon having 13 or less carbon atoms, similarly to the case where the hydrocarbon having 14 or more carbon atoms is treated. Therefore, the surface area of Rhodococcus erythropolis PR-4 becomes the contact area with the hydrocarbon as it is. Therefore, as a result of the increase in the contact area between the bacteria and the hydrocarbon, the treatment efficiency can be significantly improved as compared with the case of treating the hydrocarbon at the conventional organic solvent-aqueous solvent interface.

  The present invention also provides an organic solvent supply means for supplying an organic solvent containing a hydrocarbon having 14 or more carbon atoms, an aqueous solvent supply means for supplying an aqueous solvent containing a medium component, and Rhodococcus erythropolis PR-4 strain. Cell addition means to be added, treatment means for transferring the Rhodococcus erythropolis PR-4 strain into the organic solvent, and treating the hydrocarbon having 14 or more carbon atoms, and production generated in the aqueous solvent The present invention provides a hydrocarbon treatment system comprising product separation means for separating products.

  With such a structure, the Rhodococcus erythropolis PR-4 strain that has migrated into the organic solvent can be treated with hydrocarbons in the organic solvent, and water-soluble usefulness produced by the reaction that occurs during the treatment. The material can be eluted in an aqueous solvent. And the process of hydrocarbon and production of a useful substance can be performed simultaneously by collect | recovering the eluted water-soluble useful substance.

  According to the hydrocarbon treatment method of the present invention, Rhodococcus erythropolis PR-4 strain is transferred from an aqueous solvent to an organic solvent, and the hydrocarbon is treated by using the property of growing in the organic solvent. Therefore, the surface area of Rhodococcus erythropolis PR-4 becomes the hydrocarbon contact area as it is. Therefore, the treatment efficiency is significantly improved as compared with the case of treating hydrocarbons at the conventional organic solvent-aqueous solvent interface.

  Moreover, according to the hydrocarbon processing system which concerns on this invention, the process of a hydrocarbon and the production of a useful substance can be performed simultaneously. Therefore, it is possible to apply to environmental purification and material production by a bioprocess that can reduce energy consumption and reduce environmental load.

(Embodiment 1)
Next, embodiments of the present invention will be described in more detail. As described above, the hydrocarbon treatment method according to the present invention includes the step of adding Rhodococcus erythropolis PR-4 strain to an aqueous solvent containing a medium component, and the addition of an organic solvent containing a hydrocarbon having 14 or more carbon atoms. And a step of transferring the Rhodococcus erythropolis PR-4 strain into the organic solvent and metabolizing the hydrocarbon having 14 or more carbon atoms.

  In the present embodiment, “hydrocarbon” includes chain hydrocarbons and cyclic hydrocarbons. “Treatment of hydrocarbon” means that the Rhodococcus erythropolis PR-4 is metabolized with a hydrocarbon having 14 or more carbon atoms. In addition, “moving into an organic solvent” means that the Rhodococcus erythropolis PR-4 strain existing in an aqueous solvent is moved from an aqueous solvent into a hydrocarbon having 14 or more carbon atoms contained in the organic solvent. To do.

  The present invention uses Rhodococcus erythropolis PR-4 strain. The same strain can take either rough or mucoid form as its colony grows, but it can transfer Rhodococcus erythropolis PR-4 into an organic solvent in any form. . Moreover, you may use the mutant strain which maintains the rough type or mucoid type form of the same strain. Furthermore, transformants of the same strain can also be used.

  As an aqueous solvent containing a culture medium component, a general bacterial culture medium can be used. Moreover, other culture medium components may be included in the culture medium for general bacteria. Examples of the medium for general bacteria include IB liquid medium, YG liquid medium, LB medium, marine broth, nutritive broth, tryptic soy broth and the like. Among general bacterial media, it is particularly preferable to use an IB liquid medium.

  When the IB liquid medium is used, the yeast extract is an important medium component when the Rhodococcus erythropolis PR-4 strain is transferred from an aqueous solvent to an organic solvent. Even when Rhodococcus erythropolis PR-4 strain is added to an aqueous solvent in which no yeast extract is present, transfer into an organic solvent is unlikely to occur.

  The addition amount of the yeast extract in the aqueous solvent is preferably 0.05% (w / w) or more, based on the total amount of the aqueous solvent (100%), 0.5 to 5% (W / w) is more preferable, and about 1% (w / w) is even more preferable.

  The organic solvent containing hydrocarbons having 14 or more carbon atoms is preferably 100% occupied by hydrocarbons having 14 or more carbon atoms, but contains hydrocarbons having 13 or less carbon atoms or other hydrophobic substances. Also good. In the case of containing hydrocarbons having 13 or less carbon atoms, hydrocarbons having 14 or more carbon atoms are preferably contained in an amount of 20% (v / v) or more based on the total amount of the organic solvent, and 40% (v / v) or more. More preferably, it is more preferably 60% (v / v) or more.

In order to transfer Rhodococcus erythropolis PR-4 into hydrocarbon, it is necessary to be a hydrocarbon having 14 or more carbon atoms. Rhodococcus erythropolis PR-4 does not migrate into hydrocarbons with hydrocarbons having less than 14 carbon atoms. Moreover, there is no restriction | limiting in particular in the upper limit of carbon number. For example, even if it is solid at room temperature, it can be used as long as it can be made into a liquid by dissolving solid hydrocarbons in the presence of other hydrocarbons.

  Specific examples of the hydrocarbon having 14 or more carbon atoms include, for example, n-tetradecane (C14), n-pentadecane (C15), n-hexadecane (C16), n-heptadecane (C17), n-octadecane (C18), Examples include pristane (C19) and squalane (C30). These hydrocarbons can be used alone or in combination of two or more.

  After the Rhodococcus erythropolis PR-4 strain is added to the aqueous solvent, when an organic solvent containing a hydrocarbon having 14 or more carbon atoms is added, the Rhodococcus erythropolis PR-4 strain is included in the organic solvent from the aqueous solvent. It moves into hydrocarbons having 14 or more carbon atoms. Then, the hydrocarbon having 14 or more carbon atoms contained therein is metabolized in the organic solvent. In addition, various metabolites are generated depending on the type of hydrocarbon.

  The mechanism by which Rhodococcus erythropolis PR-4 is transferred into organic solvents is often unknown, but Rhodococcus erythropolis PR-4 is transferred into organic solvents by changing its cell surface properties. Is estimated to be possible.

  FIG. 1 is a diagram conceptually showing a series of processes from the start of hydrocarbon processing to the production of substances. FIG. 1 (a) is a view showing a state immediately after Rhodococcus erythropolis PR-4 strain 12... Is added to the aqueous solvent 10. Immediately after the addition of Rhodococcus erythropolis PR-4 strain 12..., Rhodococcus erythropolis PR-4 strain 12... Is dispersed around the organic solvent 14, that is, in the aqueous solvent 10.

  Thereafter, as shown in FIG. 1 (b), Rhodococcus erythropolis PR-4 strain 12... Moves into the organic solvent 14 and agglomerates in the organic solvent 14. At this point, the aqueous solvent 10 is almost free of Rhodococcus erythropolis PR-4 strain 12.

  As shown in FIG. 1 (c), Rhodococcus erythropolis PR-4 strain 12... Metabolizes and grows hydrocarbons in organic solvent 12 and produces water-soluble product P. Since the organic solvent 14 is hydrophobic, the water-soluble product P moves from the organic solvent 14 into the aqueous solvent 10 and is dispersed in the aqueous solvent 10.

  The hydrocarbon treatment may or may not be performed, but the viewpoint of bringing more Rhodococcus erythropolis PR-4 strain 12 into contact with the organic solvent 14 and the viewpoint of securing the dissolved oxygen amount in the medium. Is preferably treated with stirring. Stirring can be performed by using, for example, a stirring device or a shaking device.

(Embodiment 2)
The hydrocarbon treatment method according to the second embodiment is different from the first embodiment in that it includes a step of treating a hydrocarbon having 13 or less carbon atoms after treating a hydrocarbon having 14 or more carbon atoms. That is, this embodiment includes a step of adding Rhodococcus erythropolis PR-4 strain to a first aqueous solvent containing a medium component, and a step of adding a first organic solvent containing a hydrocarbon having 14 or more carbon atoms. , Transferring the Rhodococcus erythropolis PR-4 strain into the first organic solvent to metabolize the hydrocarbon having 14 or more carbon atoms, and a second aqueous solvent containing a medium component, Adding a Rhodococcus erythropolis PR-4 strain metabolized with 14 or more hydrocarbons, adding a second organic solvent containing a hydrocarbon having 13 or less carbon atoms, and the Rhodococcus erythropolis PR- Transferring 4 strains into the second organic solvent, and metabolizing the hydrocarbon having 13 or less carbon atoms.

  Usually, as in Embodiment 1, even if the hydrocarbon having 14 or more carbon atoms is simply changed to a hydrocarbon having 13 or less carbon atoms and metabolized to Rhodococcus erythropolis PR-4 strain, Rhodococcus erythropolis PR-4 Strains cannot migrate into hydrocarbons with 13 or fewer carbon atoms. However, as in this embodiment, the cells obtained by culturing Rhodococcus erythropolis PR-4 strain in an organic solvent containing a hydrocarbon having 14 or more carbon atoms are recovered, and the organic solvent containing a hydrocarbon having 13 or less carbon atoms When suspended in, Rhodococcus erythropolis PR-4 can be transferred into an organic solvent containing hydrocarbons having 13 or less carbon atoms.

  Therefore, in the present embodiment, “hydrocarbon treatment” means metabolizing hydrocarbons having 14 or more carbon atoms to Rhodococcus erythropolis PR-4 strain and 13 or less carbon atoms to Rhodococcus erythropolis PR-4 strain. Means to metabolize the hydrocarbons. In addition, “moving into an organic solvent” means that the Rhodococcus erythropolis PR-4 strain existing in an aqueous solvent is moved from an aqueous solvent into a hydrocarbon having 14 or more carbon atoms contained in the organic solvent. And Rhodococcus erythropolis PR-4 move from an aqueous solvent into a hydrocarbon having 13 or less carbon atoms contained in an organic solvent.

  Examples of the hydrocarbon having 13 or less carbon atoms include n-hexane, n-heptane, n-octane, n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane and the like. These hydrocarbons can be used alone or in combination of two or more.

  In the present embodiment, since the types of hydrocarbons to be metabolized are different, a plurality of types of hydrocarbons can be processed. Also, a plurality of types of metabolites (products) can be obtained.

  The definition of hydrocarbons, specific examples of hydrocarbons having 14 or more carbon atoms, the medium composition of the first and second aqueous solvents, and other culture conditions are the same as in Embodiment 1, and thus the description thereof is omitted here. To do.

(Embodiment 3)
Embodiment 3 relates to a hydrocarbon treatment system. FIG. 2 is a conceptual diagram of the hydrocarbon treatment system 2 of the present embodiment. As shown in FIG. 2, the hydrocarbon treatment system 2 includes an organic solvent supply unit 20, an aqueous solvent supply unit 22, a fungus body addition unit 24, a treatment unit 26, and a product separation unit 28. Yes.

  The organic solvent supply means 20 supplies an organic solvent containing a hydrocarbon having 14 or more carbon atoms to the processing means 26 described later. Specific examples of the hydrocarbon having 14 or more carbon atoms include, for example, n-tetradecane (C14), n-pentadecane (C15), n-hexadecane (C16), n-heptadecane (C17), n-octadecane (C18), Examples include pristane (C19) and squalane (C30). These hydrocarbons can be used alone or in combination of two or more.

  The aqueous solvent supply means 22 supplies an aqueous solvent containing a medium component to the processing means 26 described later. As an aqueous solvent containing a culture medium component, a general bacterial culture medium can be used. Moreover, other culture medium components may be included in the culture medium for general bacteria. Examples of the medium for general bacteria include IB liquid medium, YG liquid medium, LB medium, marine broth, nutritive broth, tryptic soy broth and the like. Among general bacterial media, it is particularly preferable to use an IB liquid medium.

  The fungus body addition means 24 adds Rhodococcus erythropolis PR-4 strain to the processing means 26 described later. The Rhodococcus erythropolis PR-4 strain can take either a rough type or a mucoid type according to the progress of the culture, but any form can be used. Moreover, you may use the mutant strain which maintains the rough type or mucoid type form of the same strain. The cell addition means 24 may be provided with a pre-culture means having a function of pre-culturing Rhodococcus erythropolis PR-4 strain in advance. Pre-culture can be performed, for example, by shaking culture of Rhodococcus erythropolis PR-4 at 28-30 ° C. using the IB medium.

  The treatment means 26 treats hydrocarbons having 14 or more carbon atoms contained in the organic solvent by Rhodococcus erythropolis PR-4 strain in a medium composed of an organic solvent and an aqueous solvent. The organic solvent is supplied from the organic solvent supply means 20, and the aqueous solvent is supplied from the aqueous solvent supply means 22. As a result, an organic solvent-aqueous solvent bilayer culture system is formed. Then, Rhodococcus erythropolis PR-4 strain is supplied from the cell supply means 24 into the aqueous solvent. Thereafter, the organic solvent and the aqueous solvent are stirred by a stirrer (not shown), and Rhodococcus erythropolis PR-4 strain starts metabolism and production of hydrocarbons having 14 or more carbon atoms. Hydrocarbon treatment conditions are performed, for example, at a temperature of 28 to 30 ° C. for several days to two weeks while stirring the medium with a stirring device (not shown). In addition, process conditions can be suitably set according to the kind of hydrocarbon to process.

  Since a series of processes from the start of hydrocarbon treatment to the production of substances is as described in FIGS. 1A to 1C, description thereof is omitted here.

  The product separation means 18 separates the product P, the organic solvent 12 and the aqueous solvent 10 produced in the aqueous solvent. As the separation method, a conventionally known separation / purification method (various chromatography, various electrophoresis or the like) can be used. In addition, using the property of the organic solvent-aqueous solvent two-layer culture system, the medium is allowed to stand for a predetermined time, and after the organic solvent and the aqueous solvent are separated into two layers, only the aqueous solvent is separated. The product P can also be purified. In addition, since Rhodococcus erythropolis PR-4 strain | stump | stock exists in an organic solvent, isolation | separation of an aqueous solvent and a microbe is also easy.

  In addition, although the above demonstrated the case where a C14 or more hydrocarbon is processed, as demonstrated in Embodiment 2, it can also apply to the process of a C13 or less hydrocarbon. Specifically, first, hydrocarbons having 14 or more carbon atoms are treated with Rhodococcus erythropolis PR-4 strain in the treatment means 26, the cells are collected by the product separation means 28, and then returned to the treatment means 26 again. .

  Next, an organic solvent containing hydrocarbons having 13 or less carbon atoms is introduced into the treatment means 26 by the organic solvent supply means 20, and the culture medium is agitated by a stirrer (not shown). As a result, Rhodococcus erythropolis PR-4 is transferred into hydrocarbons having 13 or less carbon atoms, and hydrocarbons having 13 or less carbon atoms can be treated. Note that a hydrocarbon having 14 or more carbon atoms may be contained in the organic solvent containing a hydrocarbon having 13 or less carbon atoms. After the treatment, the product separation means 18 separates and purifies the organic solvent 12, the aqueous solvent 10 and the product P.

[Test Example 1] Treatment of various hydrocarbons (1) Preparation of IB liquid medium An IB liquid medium was prepared in the following manner. That is, 1 L of Milli-Q was prepared, 10 g of glucose (Wako Pure Chemical Industries), 10 g of yeast extract (DIFCO LABORATOREIS), 0.2 g of MgCl ₂ .7H ₂ O (Wako Pure Chemical Industries), CaCl ₂・ 0.1 g of 2H ₂ O (Wako Pure Chemical Industries), 0.1 g of NaCl (Wako Pure Chemical Industries), 0.02 g of FeCl ₂ .6H ₂ O (Wako Pure Chemical Industries), (NH ₄ ) ₂ SO ₄ (Wako Pure Chemical Industries, Ltd.) 0.5 g was added, adjusted to pH 7.2 with NaOH solution, and then autoclaved at 121 ° C. for 15 minutes.

(2) Various hydrocarbons As n-alkanes, n-hexane (C6), n-octane (C8), n-nonane (C9), n-decane (C10), n-undecane (C11), n-dodecane ( C12), n-tridecane (C13), n-tetradecane (C14), n-pentadecane (C15), n-hexadecane (C16), n-heptadecane (C17), and n-octadecane (C18) were used. Pristane (C19) and squalane (C30) were used as branched alkanes.

(3) Test strain As a test strain, Rhodococcus erythropolis PR-4 strain and Rhodococcus rhodochrous S-2 strain were used.

(4) Treatment method of hydrocarbon One platinum loop of the test strain shown in (3) above was inoculated into the IB liquid medium prepared in (1) above, and cultured with shaking at 28 ° C. for 3 days. 1 ml of the preculture was collected and centrifuged at 15,000 rpm, 4 ° C. for 10 minutes. The obtained precipitate was suspended by adding 1 ml of physiological saline, and centrifuged again. Thereafter, this washing operation was repeated twice, and the resulting precipitate was suspended in 1 ml of physiological saline, which was used as a stock solution.

The stock solution was appropriately diluted so that the initial concentration of the test strain was 10 ⁴ cfu / ml, and added to fresh IB medium contained in a φ24 test tube (IWAKI GLASS). Subsequently, the various hydrocarbons shown in (2) above were added to a final concentration of 5% (v / v), followed by shaking culture at 28 ° C. and 110 rpm. Then, on the third day from the start of culture, the growth status of the test strain and the location where the test strain was localized were observed. Here, when it was present in the organic solvent, it was “internal”, and when it was present on the surface of the organic solvent, it was “superficial”. The results are shown in Table 1.

  When Rhodococcus erythropolis PR-4 strain (referred to as “PR-4 strain” in the table) was used, the state of the culture solution was compared. As a result, n-alkane having 14 or more carbon atoms and branched alkane were added. Under the conditions, an increase in turbidity between the organic solvent and the aqueous solvent was confirmed. Further, when microscopic observation was performed, it was confirmed that Rhodococcus erythropolis PR-4 strain was transferred and present in hydrocarbon particles having 14 or more carbon atoms.

  Under the conditions in which n-alkane having 10 to 12 carbon atoms was added, an increase in turbidity was observed only at the interface between the organic solvent and the aqueous solvent. When these were observed with a microscope, it was confirmed that Rhodococcus erythropolis PR-4 strain was adsorbed on the surface of the hydrocarbon particles.

  Under the condition using n-alkane having 8 or less carbon atoms, almost no turbidity was observed. Further, when microscopic observation was performed, since Rhodococcus erythropolis PR-4 strain was not observed in the aqueous solvent and the organic solvent, the number of Rhodococcus erythropolis PR-4 strain was remarkably small compared with other conditions. It was expected.

  On the other hand, the same examination was performed for Rhodococcus rhodochrous S-2 strain (in the table, referred to as “S-2 strain”). As a result, the formation of an emulsion was observed in the whole culture solution under the conditions in which an n-alkane having 14 or more carbon atoms and a branched alkane were added. Further, when microscopic observation was performed, it was observed that Rhodococcus rhodochrous S-2 was present in an aqueous solvent under these conditions, and that Rhodococcus rhodochrous S-2 was adsorbed on the surface of the hydrocarbon particles. It was.

  Under the condition where n-alkane having 12 or less carbon atoms was added, formation of an emulsion was not observed, and Rhodococcus rhodochrous S-2 strain could not be confirmed in the culture solution.

  From this, it was found that Rhodococcus erythropolis PR-4 strain and Rhodococcus rhodochrous S-2 strain have different interactions with hydrocarbons. In addition, it was suggested that Rhodococcus erythropolis PR-4 recognizes hydrocarbons having different carbon numbers and performs an interaction adapted to the carbon number.

[Test Example 2] Examination of the influence of the mixing ratio of different hydrocarbons on the processing of hydrocarbons Select two types of hydrocarbons with different interactions, change their proportions, add them to the IB liquid medium, The effect of erythropolis PR-4 strain on the treatment of hydrocarbons was examined.

  Rhodococcus erythropolis PR-4 strain (referred to as “PR-4 strain” in the table) was inoculated into IB liquid medium by one platinum loop, and cultured with shaking at 28 ° C. for 3 days. 1 ml of the preculture was collected and centrifuged at 15,000 rpm, 4 ° C. for 10 minutes. The obtained precipitate was suspended by adding 1 ml of physiological saline, and centrifuged again. Thereafter, this washing operation was repeated twice, and the resulting precipitate was suspended in 1 ml of physiological saline, which was used as a stock solution.

The stock solution was appropriately diluted so that the initial concentration of Rhodococcus erythropolis PR-4 was 10 ⁴ cfu / ml, and Rhodococcus erythropolis PR-4 was added to the separately prepared IB medium. Subsequently, different hydrocarbons were added, respectively, and cultured with shaking at 28 ° C. and 110 rpm.

  As hydrocarbons, Rhodococcus erythropolis PR-4 strain, pristane (C19), which is a hydrocarbon that can migrate and metabolize, and Rhodococcus erythropolis PR-4 strain are adsorbed on the surface of organic solvents and metabolized. A mixture of dodecane (C12), which is a hydrocarbon to be used, was used. And each mixing ratio was changed so that the final concentration of the mixed hydrocarbon might be 5% (v / v), and it added to the culture solution, and processed the hydrocarbon.

  Also, hydrocarbons were treated in the same manner as described above using pristane and octane (C8), which is a hydrocarbon that Rhodococcus erythropolis PR-4 strain cannot migrate and grow. The results are shown in Table 2.

  As shown in Table 2, when dodecane and pristane were mixed, the transfer of Rhodococcus erythropolis PR-4 to an organic solvent was confirmed when the mixing ratio of dodecane and pristane was 8: 2 to 1: 9. It was.

  On the other hand, when pristane and octane were mixed, the transfer of Rhodococcus erythropolis PR-4 to an organic solvent was confirmed when the mixing ratio of octane and pristane was 6: 4 to 1: 9.

[Test Example 3] Examination of the adaptation of Rhodococcus erythropolis PR-4 strain Rhodococcus erythropolis PR-4 strain (referred to as "PR-4 strain" in the table) was inoculated into a IB liquid medium by one platinum loop, The culture was shaken at 28 ° C. for 3 days. 1 ml of the preculture was collected and centrifuged at 15,000 rpm, 4 ° C. for 10 minutes. The obtained precipitate was suspended by adding 1 ml of physiological saline, and centrifuged again. Thereafter, this washing operation was repeated twice, and the resulting precipitate was suspended in 1 ml of physiological saline, which was used as a stock solution.

The stock solution was appropriately diluted so that the initial concentration of Rhodococcus erythropolis PR-4 was 10 ⁴ cfu / ml and added to a fresh IB liquid medium contained in a φ24 test tube (IWAKI GLASS). Subsequently, the final concentration of pristane (C19) was added to 5% (v / v), followed by shaking culture at 28 ° C. and 110 rpm for 3 days.

After 3 days from the start of culture, Rhodococcus erythropolis PR-4 cultured in IB liquid medium supplemented with pristane (C19) was recovered. Then, the recovered Rhodococcus erythropolis PR-4 strain was added to a separately prepared IB liquid medium so that the initial concentration was 10 ⁴ cfu / ml. This time, the final concentration of dodecane (C12) was added to 5% (v / v), followed by shaking culture at 28 ° C. and 110 rpm.

  As a control, Rhodococcus erythropolis PR-4 strain was cultured in an IB liquid medium supplemented with dodecane (C12), and then Rhodococcus erythropolis PR-4 strain was cultured in an IB liquid medium supplemented with pristane (C19). did.

  On the third day from the start of the culture, the growth status of Rhodococcus erythropolis PR-4 strain and the location where Rhodococcus erythropolis PR-4 strain was localized were observed. Here, when the Rhodococcus erythropolis PR-4 strain was present in the hydrocarbon, it was “internal”, and when it was present on the surface of the hydrocarbon, it was “superficial”. The results are shown in Table 3.

  As shown in Table 3, it was found that the Rhodococcus erythropolis PR-4 strain was transferred into dodecane (C12) in the same manner as when pristane (C19) was treated alone. Conversely, Rhodococcus erythropolis PR-4 strain cultured in a medium supplemented with dodecane (C12) was collected and cultured in a medium supplemented with pristane (C19). Rhodococcus erythropolis PR-4 strain was found to be pristane (C19). ) Was not transferred to the surface, and was found to be present on the surface of pristane (C19). From this, during the first stage treatment, the cell wall and / or cell membrane of Rhodococcus erythropolis PR-4 strain adapts to the type of hydrocarbon (causes adaptation), and the nature of the second stage It was inferred that even if the hydrocarbon used for eye treatment changed, it was maintained.

  Since the present invention can metabolize hydrocarbons by transferring Rhodococcus erythropolis PR-4 strain into an organic solvent, it is possible to reduce the energy and reduce the environmental burden by environmental purification.・ Applicable to material production.

It is the figure which showed notionally the series of processes from the start of a hydrocarbon process to a substance production. 1 is a conceptual diagram of a hydrocarbon treatment system 2. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 2 ... Hydrocarbon processing system, 10 ... Aqueous solvent, 12 ... Rhodococcus erythropolis PR-4 strain, 14 ... Organic solvent, 20 ... Organic solvent supply means, 22 ... Aqueous solvent supply means, 24 ... Bacterial cell addition means, Processing Means ... 26, product separation means ... 28, P ... product

Claims (6)

Adding Rhodococcus erythropolis PR-4 strain to an aqueous solvent containing medium components;
Adding an organic solvent containing a hydrocarbon having 14 or more carbon atoms;
Transferring the Rhodococcus erythropolis PR-4 strain into the organic solvent to metabolize the hydrocarbon having 14 or more carbon atoms;
A method for treating hydrocarbons.   The method for treating hydrocarbons according to claim 1, wherein the aqueous solvent contains a medium for general bacteria.   The hydrocarbon treatment method according to claim 1 or 2, wherein the hydrocarbon having 14 or more carbon atoms is at least one hydrocarbon selected from the group consisting of tetradecane, pentadecane, hexadecane, pristane, and squalane.   The hydrocarbon treatment method according to claim 1, wherein the hydrocarbon treatment is performed while stirring. Adding Rhodococcus erythropolis PR-4 strain to a first aqueous solvent containing media components;
Adding a first organic solvent containing a hydrocarbon having 14 or more carbon atoms;
Transferring the Rhodococcus erythropolis PR-4 strain into the first organic solvent to metabolize the hydrocarbon having 14 or more carbon atoms;
Adding Rhodococcus erythropolis PR-4 strain metabolized with the hydrocarbon having 14 or more carbon atoms to a second aqueous solvent containing a medium component;
Adding a second organic solvent containing a hydrocarbon having 13 or less carbon atoms;
Transferring the Rhodococcus erythropolis PR-4 strain into the second organic solvent to metabolize the hydrocarbon having 13 or less carbon atoms;
A method for treating hydrocarbons. An organic solvent supply means for supplying an organic solvent containing a hydrocarbon having 14 or more carbon atoms;
An aqueous solvent supply means for supplying an aqueous solvent containing a medium component;
A cell addition means for adding Rhodococcus erythropolis PR-4 strain,
A treatment means for transferring the Rhodococcus erythropolis PR-4 strain into the organic solvent and treating the hydrocarbon having 14 or more carbon atoms;
Product separation means for separating the product produced in the aqueous solvent;
Hydrocarbon treatment system with

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