US20060263870A1

US20060263870A1 - Hydrocarbon processing method and hydrocarbon processing system

Info

Publication number: US20060263870A1
Application number: US11/280,824
Authority: US
Inventors: Mutsuyasu Nakajima; Michio Sunairi; Noriyuki Iwabuchi; Emi Kishi
Original assignee: Nihon University
Current assignee: Nihon University
Priority date: 2005-05-23
Filing date: 2005-11-17
Publication date: 2006-11-23
Also published as: JP2006325433A; JP4877900B2

Abstract

A hydrocarbon treatment process of the present invention comprises the steps of: adding Rhodococcus erythropolis PR-4 to an aqueous medium containing culture medium ingredients; adding an organic solvent containing a hydrocarbon with 14 or more carbon atoms; and enabling the Rhodococcus erythropolis PR-4 to migrate into the organic solvent and metabolize the hydrocarbon with 14 or more carbon atoms.

Description

BACKGROUND

The present invention relates to a hydrocarbon treatment process and hydrocarbon treatment system, and more particularly to a highly efficient hydrocarbon treatment process and hydrocarbon treatment system utilizing a species of bacteria belonging to the genus Rhodococcus.
Rhodococcus is a type of gram-positive, coryneform bacteria with a high G+C content, and these bacteria are abundant in very ordinary environments such as the soil and the ocean. Many species of Rhodococcus bacteria can not only decompose and utilize recalcitrant substances such as petroleum-based hydrocarbons, polychlorinated biphenyl (PCB) compounds, or the like, but can also produce acrylamide and useful enzymes, as well as functional biopolymers such as extracellular polysaccharides. For that reason, Rhodococcus bacteria are very important industrially, and they are expected to find applications in environmental cleanup and the production of substances through bioprocesses that can reduce energy consumption and alleviate the burden on the environment [Finnerty, W. R. et al. (1992) Annual Review of Microbiology, p193-218]. Especially when considering bioprocesses, characteristics of excellent growth, vigorous metabolic activity, or the like, under special environments that contain organic solvents are required of microorganisms from which these applications are expected. Moreover, in order for the microorganisms that are needed for cleaning up an oil-polluted environment caused by an accidental oil spill, or the like, to show excellent growth while performing degradation in the presence of high concentrations of refractory compounds, these microorganisms must not only be able to degrade these compounds, but they must also have a high tolerance of the toxicity of these coexisting refractory compounds.
To analyze these aforementioned characteristics, first of all, the microorganisms must have organic solvent tolerance and especially, growth under a high concentration of organic solvent is necessary for the bioprocess. In research on the organic solvent tolerance of microorganisms, genetic and biochemical research has been performed centering on model microorganisms such as the gram-negative bacteria E. coli and Pseudomonas, and tolerance mechanisms such as changes in the cell surface structure, efflux pumps, and vesicle formation have been proposed [Ramos, J. L. et al. (2002) Annual Review of Microbiology, p 743-768]. On the other hand, although genetic and biochemical research concerning the hydrocarbon decomposition genes, or the like, has progressed in the gram-positive bacteria, not much research on the organic solvent tolerance has been performed. This situation is attributable to the fact that in general, the organic solvent tolerance level in gram-positive bacteria is considered to be low compared with gram-negative bacteria. However, when thinking about bioprocesses, data on organic solvent tolerance in conditions approaching the actual use environment is needed for microorganism at a stage very close to their application. As noted above, because application of Rhodococcus bacteria to bioprocesses is expected, it is necessary to accumulate information concerning the organic solvent tolerance of these bacteria.
Iwabuchi et al. discovered that the S-2 strain of Rhodococcus rhodochrous will tolerate high concentrations of petroleum and also degrade petroleum, and as a result of additional investigations into the petroleum tolerance thereof, they found that extracellular polysaccharides (hereinafter, EPS) produced by these bacteria strongly contribute to the tolerance of recalcitrant organic solvents such as long-chain alkanes, or the like. Furthermore, when they investigated the relationship between colony morphology and solvent tolerance in Rhodococcus, they found that the rough morphotype, which produces little EPS, has a high affinity for the solvent and consequently is susceptible to the solvent, but the mucoid morphotype, which produces large quantities of EPS, is tolerant, thereby demonstrating a strong correlation between colony morphology and organic solvent tolerance in bacteria of the genus Rhodococcus. Finally, they demonstrated that EPS can provide solvent tolerance to the susceptible rough morphotype, and thus discovered that mucoid colony formation is one indicator of solvent tolerance in Rhodococcus [Iwabuchi, N. et al. (2000) Applied Environmental Microbiology, 66: 5073-5077].
Strain PR-4 of Rhodococcus erythropolis was isolated as an organism that degrades the branched alkane pristane (2,6,10,14-tetramethyl-pentadecane) [Komukai-Nakamura, S. et al. (1996) Journal of Fermentation and Bioengineering, 82: p570-574], and as culturing time progresses, PR-4 changes its own colony morphotype from rough to mucoid and vice versa based on the production of EPS. Because PR-4 is known to exhibit tolerance to refractory organic solvents, it has been selected for genome analysis, and development of a host-vector system is also underway. As a result, strain PR-4 of Rhodococcus erythropolis is expected to become the strain among the Rhodococcus bacteria in which a genetic manipulation system will be developed in the near future.

SUMMARY

Generally, when treating organic solvents with bacteria, treatment is performed in a bilayer culture system consisting of an organic solvent and an aqueous medium containing culture medium ingredients. However, because bacteria added to the aqueous medium can only react at the organic solvent-aqueous medium interface, it is necessary to improve the lipophilicity of the bacterial cells and enhance their hydrocarbon treatment efficiency for their application in environmental cleanup and production of substances through bioprocesses.
Therefore, an object of the present invention is to provide an efficient hydrocarbon treatment process and hydrocarbon treatment system whereby the lipophilicity of Rhodococcus erythropolis strain PR-4 has been improved.
The inventors cultured Rhodococcus erythropolis strain PR-4 under culturing conditions using a bilayer culture system comprising an organic layer containing a recalcitrant organic solvent and an aqueous layer containing liquid culture medium, and they investigated the solvent tolerance mechanism of strain PR-4. At that time, they found that almost no PR-4 could be observed in the aqueous layer, and it appeared that they had migrated to the interior of the organic solvent particles and were growing inside those organic solvent particles.
The present invention was created based on that knowledge, and it provides a hydrocarbon treatment process comprising the steps of: adding Rhodococcus erythropolis PR-4 to an aqueous medium containing culture medium ingredients; adding an organic solvent containing a hydrocarbon with 14 or more carbon atoms; and enabling the Rhodococcus erythropolis PR-4 to migrate into the organic solvent and metabolize the hydrocarbon with 14 or more carbon atoms.
By adopting this constitution it is possible to metabolize a hydrocarbon with 14 or more carbon atoms by enabling the PR-4 to migrate into the organic solvent, and thereby the surface area of the PR-4 becomes the contact area with the hydrocarbon. As a result, the contact area between the bacteria and the hydrocarbon increases, and the treatment efficiency can be greatly improved over prior art treatment of hydrocarbons at the organic solvent-aqueous medium interface.
The present invention also provides a hydrocarbon treatment process comprising the steps of: adding Rhodococcus erythropolis PR-4 to a first aqueous medium containing culture medium ingredients; adding a first organic solvent containing a hydrocarbon with 14 or more carbon atoms; enabling the Rhodococcus erythropolis PR-4 to migrate into the first organic solvent and metabolize the hydrocarbon with 14 or more carbon atoms; adding the Rhodococcus erythropolis PR-4 that have metabolized the hydrocarbon with 14 or more carbon atoms to a second aqueous medium containing culture medium ingredients; adding a second organic solvent containing a hydrocarbon with 13 or fewer carbon atoms; and enabling the Rhodococcus erythropolis PR-4 to migrate into the second organic solvent and metabolize the hydrocarbon with 13 or fewer carbon atoms.
By adopting this constitution it is possible to metabolize a hydrocarbon with 13 or fewer carbon atoms by enabling the PR-4 to migrate into the organic solvent in the same manner as treating a hydrocarbon with 14 or more carbon atoms, and thereby the surface area of the PR-4 becomes the contact area with the hydrocarbon. As a result, the contact area between the bacteria and the hydrocarbon increases, and the treatment efficiency can be greatly improved over prior art treatment of hydrocarbons at the organic solvent-aqueous medium interface.
Furthermore, the present invention provides a hydrocarbon treatment system comprising: means for supplying an organic solvent containing a hydrocarbon with 14 or more carbon atoms; means for supplying an aqueous medium containing culture medium ingredients; means for adding Rhodococcus erythropolis PR-4; means for enabling the Rhodococcus erythropolis PR-4 to migrate into the organic solvent and treat the hydrocarbon with 14 or more carbon atoms; and means for separating the product produced in the aqueous medium.
By adopting this constitution it is possible to treat hydrocarbons in an organic solvent with Rhodococcus erythropolis PR-4 that have migrated into the organic solvent, and it is possible for a water-soluble useful substance produced by a reaction during treatment to be eluted into the aqueous medium. Moreover, by recovering the eluted water-soluble useful substance, it is possible to treat a hydrocarbon and produce a useful substance simultaneously.
In accordance with the hydrocarbon treatment process in the present invention, the hydrocarbon is treated by utilizing the trait whereby the Rhodococcus erythropolis PR-4 migrate from the aqueous medium into the organic solvent and grow within the organic solvent. As a result, the treatment efficiency can be greatly improved over prior art treatment of hydrocarbons at the organic solvent-aqueous medium interface. Furthermore, in accordance with the hydrocarbon treatment system of the present invention, it is possible to treat a hydrocarbon and produce a useful substance simultaneously. As a result it is possible to apply this system to environmental cleanup and the production of substances through bioprocesses that can reduce energy consumption and alleviate the burden on the environment.

DESCRIPTION OF DRAWINGS

FIG. 1A-1C are conceptual figures that shows the continuous process from the start of hydrocarbon treatment until the production of a substance; and
FIG. 2 is a conceptual figure of the hydrocarbon treatment system 2.

DETAILED DESCRIPTION

Embodiment 1

A further, more detailed explanation of the present invention is provided through embodiments below. As previously described, the hydrocarbon treatment process of the present invention comprises the following steps: adding Rhodococcus erythropolis PR-4 to an aqueous medium containing culture medium ingredients; adding an organic solvent containing a hydrocarbon with 14 or more carbon atoms; and enabling the PR-4 to migrate into the organic solvent and metabolize the hydrocarbon with 14 or more carbon atoms.
In these embodiments, the term “hydrocarbon” includes straight-chain hydrocarbons and cyclic hydrocarbons. The term “hydrocarbon treatment” means metabolism of a hydrocarbon with 14 or more carbon atoms by Rhodococcus erythropolis PR-4. In addition, the term “migrate into the organic solvent” means that the Rhodococcus erythropolis PR-4 migrate from the aqueous medium into the hydrocarbon with 14 or more carbon atoms contained in the organic solvent.
The present invention utilizes Rhodococcus erythropolis PR-4. As culturing time progresses, strain PR-4 can spontaneously assume either a rough or mucoid colony morphotype, and either morphotype of PR-4 can migrate into the organic solvent. A PR-4 mutant that maintains either the rough or mucoid morphotype can also be used. Furthermore, a PR-4 transformant can also be used.
A common bacteria culture medium can be used as the aqueous medium containing culture medium ingredients. Other culture medium ingredients can also be included in the common bacteria culture medium. Common bacteria culture media include the following examples: IB liquid medium, YG liquid medium, LB medium, marine broth, nutrient broth, trypto-soy broth, or the like. Among the common bacteria culture media, the use of IB liquid medium is especially preferred.
When the above IB liquid medium is used, yeast extract is an important medium ingredient when the Rhodococcus erythropolis PR-4 migrate from the aqueous medium to the organic solvent. Migration into the organic solvent does not easily occur if Rhodococcus erythropolis PR-4 are added to aqueous medium that does not contain yeast extract.
The amount of the aforementioned yeast extract to be added to the aforementioned aqueous medium when the total volume of the aqueous medium is used as a reference standard (100%) is preferably 0.05% (w/w) or more, and more preferably 0.5 to 5% (w/w), with about 1% (w/w) being most preferable.
The organic solvent containing a hydrocarbon with 14 or more carbon atoms is preferably one in which the hydrocarbon with 14 or more carbon atoms comprises 100%, and it may also contain a hydrocarbon with 13 or fewer carbon atoms or another hydrophobic substance. If the organic solvent contains a hydrocarbon with 13 or fewer carbon atoms, when the total volume is used as a reference standard, the amount of hydrocarbon with 14 or more carbon atoms contained therein is preferably 20% (v/v) or more, more preferably 40% (v/v) or more, and most preferably 60% (v/v) or more.
For the Rhodococcus erythropolis PR-4 to migrate into the hydrocarbon, it must be a hydrocarbon with 14 or more carbon atoms. With a hydrocarbon with less than 14 carbon atoms, the Rhodococcus erythropolis PR-4 do not migrate into the hydrocarbon. Moreover, the present invention places no restriction on the upper limit of the number of carbon atoms. For example, even if a hydrocarbon is a solid at ambient room temperature, it can be used if the solid hydrocarbon dissolves in the presence of another hydrocarbon to become a liquid.
Concrete examples of the hydrocarbon with 14 or more carbon atoms include, for example, n-tetradecane (C14), n-pentadecane (C15), n-hexadecane (C16), n-heptadecane (C17), n-octadecane (C18), pristane (C19), squalane (C30), or the like. These hydrocarbons can either be used alone or as a mixture of two or more.
When the organic solvent containing a hydrocarbon with 14 or more carbon atoms is added after the Rhodococcus erythropolis PR-4 are added to the aqueous medium, the Rhodococcus erythropolis PR-4 migrate from the aqueous medium into the hydrocarbon with 14 or more carbon atoms contained in the organic solvent. Then the bacteria metabolize the hydrocarbon with 14 or more carbon atoms contained in the organic solvent. Furthermore, the bacteria produce various metabolites depending on the species of hydrocarbon.
The mechanism whereby the Rhodococcus erythropolis PR-4 migrate into the organic solvent is still not entirely understood, but it is presumed that the bacteria are able to migrate into the organic solvent by altering their cell surface properties.
FIG. 1 is a conceptual figure that shows the continuous process from the start of hydrocarbon treatment until the production of a substance. FIG. 1A is a figure showing the appearance immediately after Rhodococcus erythropolis PR-4 12 has been added to the aqueous medium 10. Immediately thereafter, the bacteria 12 are present dispersed surrounding the organic solvent 14, i.e., in the aqueous medium 10.
Subsequently, as shown in FIG. 1B, the Rhodococcus erythropolis PR-4 12 migrate into the organic solvent 14 and aggregate within the organic solvent 14. At this time, a state is reached wherein almost no bacteria 12 are present in the aqueous medium 10.
Finally, as shown in FIG. 1C, the Rhodococcus erythropolis PR-4 12 metabolize the hydrocarbon and grown in the organic solvent 14, and concurrently they produce a water-soluble product P. Because the organic solvent 14 is hydrophobic, the water-soluble product P migrates from the organic solvent 14 into the aqueous medium 10, and is dispersed in the aqueous medium 10.
The hydrocarbon treatment may be performed without agitation, but from the standpoint of bringing the Rhodococcus erythropolis PR-4 12 into contact with the organic solvent 14 more greatly and from the stand point of attaining the amount of dissolved oxygen in the medium, it is preferable to perform the treatment with agitation. The agitation can be performed with the use of a stirring device and shaking device, or the like.

Embodiment 2

The hydrocarbon treatment process of Embodiment 2 differs from that of Embodiment 1 because it includes a step wherein a hydrocarbon with 13 or fewer carbon atoms is treated after the hydrocarbon with 14 or more carbon atoms is treated. In other words, this embodiment comprises the steps of: adding Rhodococcus erythropolis PR-4 to a first aqueous medium containing culture medium ingredients; adding a first organic solvent containing a hydrocarbon with 14 or more carbon atoms; enabling the Rhodococcus erythropolis PR-4 to migrate into the first organic solvent and metabolize the hydrocarbon with 14 or more carbon atoms; adding the Rhodococcus erythropolis PR-4 that have metabolized the hydrocarbon with 14 or more carbon atoms to a second aqueous medium containing culture medium ingredients; adding a second organic solvent containing a hydrocarbon with 13 or fewer carbon atoms; and enabling the Rhodococcus erythropolis PR-4 to migrate into the second organic solvent and metabolize the hydrocarbon with 13 or fewer carbon atoms.
Normally, if the hydrocarbon with 14 or more carbon atoms is simply replaced by a hydrocarbon with 13 or fewer carbon atoms and metabolism by the Rhodococcus erythropolis PR-4 is attempted as in Embodiment 1, the bacteria cannot migrate into the hydrocarbon with 13 or fewer atoms. However, if a bacteria wherein Rhodococcus erythropolis PR-4 have been cultured in an organic solvent containing a hydrocarbon with 14 or more carbon atoms is collected and suspended in an organic solvent containing a hydrocarbon with 13 or fewer carbon atoms in accordance with this embodiment, it is possible for the bacteria to migrate into the organic solvent containing the hydrocarbon with 13 or fewer carbon atoms.
Therefore, in this embodiment the term “hydrocarbon treatment” means the metabolism of a hydrocarbon with 14 or more carbon atoms by the Rhodococcus erythropolis PR-4 and the metabolism of a hydrocarbon with 13 or fewer carbon atoms by the Rhodococcus erythropolis PR-4. Moreover, term “migrate into the organic solvent” means that the Rhodococcus erythropolis PR-4 migrate from the aqueous medium into the hydrocarbon with 14 or more carbon atoms contained in the organic solvent and the term “migrate into the organic solvent” means that the Rhodococcus erythropolis PR-4 migrate from the aqueous medium into the hydrocarbon with 13 or fewer carbon atoms contained in the organic solvent.
Concrete examples of the hydrocarbon with 13 or fewer carbon atoms include, for example, n-hexane, n-heptane, n-octane, n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane, or the like. These hydrocarbons can either be used alone or as a mixture of two or more.
Because the type of hydrocarbons metabolized in this embodiment differ, it is possible treatment of a plurality of types of hydrocarbons is possible. It is also possible to obtain a plurality of types of metabolites (products).
Because the definition of hydrocarbon, specific examples of hydrocarbons having 14 or more carbon atoms, the medium composition of the first and second aqueous mediums, and the other culturing conditions are the same as in Embodiment 1, the explanation thereof is omitted here.

Embodiment 3

Embodiment 3 relates to a hydrocarbon treatment system. FIG. 2 is a conceptual figure of the hydrocarbon treatment system 2 of this embodiment. As shown in FIG. 2, the hydrocarbon treatment system 2 comprises means for supplying organic solvent 20, means for supplying aqueous medium 22, means for adding bacteria 24, treatment means 26, and product separation means 28.
The means for supplying organic solvent 20 is one that supplies organic solvent containing a hydrocarbon with 14 or more carbon atoms to the treatment means 26 described below. Concrete examples of the hydrocarbon with 14 or more carbon atoms include, for example, n-tetradecane (C14), n-pentadecane (C15), n-hexadecane (C16), n-heptadecane (C17), n-octadecane (C18), pristane (C19), squalane (C30), or the like. These hydrocarbons can either be used alone or as a mixture of two or more.
The means for supplying aqueous medium 22 is one that supplies aqueous medium containing culture medium ingredients to the treatment means 26 described below. A common bacteria culture medium can be used as the aqueous medium containing culture medium ingredients. Other culture medium ingredients can also be included in the common bacteria culture medium. Common bacteria culture media include the following examples: IB liquid medium, YG liquid medium, LB medium, marine broth, nutrient broth, trypto-soy broth, or the like. Among the common bacteria culture media, the use of IB liquid medium is especially preferred.
The means for adding bacteria 24 is one that adds the Rhodococcus erythropolis PR-4 to the treatment means 26 described below. As culturing time progresses, the Rhodococcus erythropolis PR-4 can spontaneously assume either a rough or mucoid colony morphotype, and either morphotype can be used. A PR-4 mutant that maintains either the rough or mucoid morphotype can also be used. The means for adding bacteria 24 is provided with preculturing means having a function wherein the Rhodococcus erythropolis PR-4 are precultured prior to addition. Preculturing can be performed by culturing the Rhodococcus erythropolis PR-4 with agitation at 28° C. to 30° C. using, for example, the aforementioned IB medium.
The treatment means 26 is one wherein the Rhodococcus erythropolis PR-4 treat a hydrocarbon with 14 or more carbon atoms contained in the organic solvent in a culture medium comprising the organic solvent and the aqueous medium. The organic solvent is supplied by the means for supplying organic solvent 20, and the aqueous medium is supplied by the means for supplying aqueous medium 22. An organic solvent-aqueous medium bilayer culture system is formed thereby. Then the Rhodococcus erythropolis PR-4 in organic solvent are supplied by the bacteria supply means 24. Thereafter, the organic solvent and aqueous medium are stirred by a stirrer not shown in the figure, and the Rhodococcus erythropolis PR-4 begin to metabolize the hydrocarbon with 14 or more carbon atoms and produce a substance. The hydrocarbon treatment is performed, for example under conditions wherein the medium is stirred by the stirring device not shown in the figure for about several days to two weeks at 28° C. to 30° C. The treatment conditions can be established as needed in accordance with they type of hydrocarbon to be treated.
The continuous process from the start of hydrocarbon treatment until the production of a substance is explained in FIGS. 1A-1C, and therefore the explanation thereof is omitted here.
The process separation means 18 is one that separates the product P produced in the aqueous medium, the organic solvent 12, and the aqueous medium 10. The separation process can utilize publicly known prior art separation/purification means (various types of chromatography or electrophoresis, or the like.). It is possible to separate out only the aqueous medium and purify the product P thereby through utilizing the properties of the organic solvent-aqueous medium bilayer culture system after allowing the medium to stand for a specified amount of time until it has reached a state wherein the organic solvent and the aqueous medium have separated into two layers. Because the Rhodococcus erythropolis PR-4 are present in the organic solvent, separation of the bacteria from the aqueous medium is easily performed.
An explanation was provided above concerning the treatment of a hydrocarbon with 14 or more carbon atoms, but as explained in Embodiment 2, this treatment process can be applied to the treatment of hydrocarbons of 13 or fewer carbon atoms. More specifically a hydrocarbon with 14 or more carbon atoms is treated by Rhodococcus erythropolis PR-4 in the treatment means 26, and after the bacteria is recovered by the product separation means 28, it is returned once more to the treatment means 26.
Next an organic solvent containing a hydrocarbon with 13 or fewer carbon atoms is supplied into the treatment means 26 by the means for supplying organic solvent 20, and the medium is stirred by a stirrer not shown in the figure. The Rhodococcus erythropolis PR-4 migrates into the hydrocarbon with 13 or fewer carbon atoms thereby, and it is possible to treat the hydrocarbon with 13 or fewer carbon atoms. A hydrocarbon with 14 or more carbon atoms may also be contained in the organic solvent containing the hydrocarbon with 13 or fewer carbon atoms. After treatment, the organic solvent 12, the aqueous medium 10, and the product P are separated/purified by the product separation means 28.

EXPERIMENTAL EXAMPLE 1

Treatment of Various Hydrocarbons

(1) Preparation of IB Liquid Medium
IB liquid medium was prepared as shown below. More specifically, 1 L of Milli-Q ultrapure water was prepared; 10 g of glucose (Wako Pure Chemical Industries), 10 g of yeast extract (DIFCO LABORATORIES), 0.2 g of MgCl₂.7H₂O (Wako Pure Chemical Industries), 0.1 g of CaCl₂.2H₂O (Wako Pure Chemical Industries), 0.1 g of NaCl (Wako Pure Chemical Industries), 0.02 g of FeCl₂.6H₂O (Wako Pure Chemical Industries), and 0.5 g of (NH₄)₂SO₄(Wako Pure Chemical Industries) were added; the pH was adjusted to 7.2 with NaOH solution; and the solution was sterilized by autoclave for 15 minutes at 121° C.
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. As branched alkanes, pristane (C19), and squalane (C30) were used.
Test Strains
As test strains, Rhodococcus erythropolis PR-4 and Rhodococcus rhodochrous S-2 were used.
Hydrocarbon Treatment
The test strains shown in (3) above were inoculated into the IB liquid medium prepared in (1) above using a single platinum loop, and cultured with agitation for 3 days at 28° C. Centrifugal separation was then performed at 15,000 rpm and 4° C. for 10 minutes on 1 mL of this precultured liquid. The precipitate obtained thereby was suspended in 1 mL of physiological saline, and centrifugal separation was performed once more. Thereafter, this rinse procedure was repeated two times, and the precipitate obtained thereby was suspended in 1 mL of physiological saline and this used as the stock solution.

The stock solution was suitably diluted so the initial concentration of the test bacteria was 10⁴cfu/mL, and the bacteria were added to fresh IB medium in a 24 mm diameter test tube (Iwaki Glass). Next, each of the various hydrocarbons shown in (2) above was added so that the final concentration of each would be 5% (v/v), and culturing with agitation was performed at 28° C. and 110 rpm. On the third day after the start of culturing, the growth status of the test bacteria and sites where the test bacteria had localized were observed. When the test bacteria were present in the organic solvent, they were labeled “inside” and when they were present on the surface of the organic solvent, they were labeled “surface.” The results are shown in Table 1.

	TABLE 1


	PR-4	S-2

hydrocarbon	growth	localization	growth	localization

n-hexane	−	—	−	—
(C6)
n-octane	−		−
(C8)
n-decane	±	surface	−
(C10)
n-dodecane	±		−
(C12)
n-	+	inside	±	surface
tetradecane
(C14)
n-	+		±
hexadecane
(C16)
n-pristane	+		±
(C19)
n-squalane	+		±
(C30)

−: growth is not shown or markedly few bacteria grow
±: growth is shown at interface of organic solvent and aqueous medium
+: growth is shown in organic solvent

When the Rhodococcus erythropolis PR-4 (labeled PR-4 in the table) were used and the appearance of the culture liquid was compared, an increase in turbidity in the organic solvent and the aqueous medium was confirmed in the tubes to which the n-alkanes and branched alkanes of 14 or more carbon atoms had been added. It was also confirmed by microscopy that the Rhodococcus erythropolis PR-4 had migrated and were present in the hydrocarbons of 14 or more carbon atoms.
In the tubes to which n-alkanes of 10-12 carbon atoms had been added, an increase in turbidity was confirmed only at the interface of the organic solvent and the aqueous medium. It was also confirmed by microscopy that the Rhodococcus erythropolis PR-4 had adsorbed and were present on the surface of the hydrocarbon.
Almost no turbidity was seen under conditions using n-alkanes of 8 or fewer carbon atoms. In addition, in microscopy no Rhodococcus erythropolis PR-4 were seen in both the aqueous medium and organic solvent, and therefore it was presumed that the bacterial count was markedly less than under other conditions.
A similar investigation was performed using the S-2 strain of Rhodococcus rhodochrous (labeled S-2 in the table). As a result, the formation of an emulsion throughout the liquid culture medium was seen under conditions wherein an n-alkane or branched alkane of 14 or more carbon atoms was added. Microscopic examination revealed that under these conditions the S-2 were present in the aqueous medium and appeared to be adsorbed onto the surface of the hydrocarbon particles.
No emulsion formation was observed when an n-alkane of 12 carbon atoms or fewer was added, and no S-2 could be seen in the liquid culture medium.
From these results it became clear that the interaction with a hydrocarbon differs between Rhodococcus erythropolis PR-4 and Rhodococcus rhodochrous S-2. These findings indicate, therefore, that PR-4 recognize hydrocarbons with different numbers of carbon atoms and interact in accordance with the number of carbon atoms.

EXPERIMENTAL EXAMPLE 2

Investigation of the Effect of the Mix Ratio of Different Hydrocarbons on Hydrocarbon Treatment

Two species of hydrocarbons having different interactions with PR-4 were selected, they were prepared in a series of different ratios and added to the IB liquid medium, and the effect thereof on the hydrocarbon treatment by PR-4 was investigated.
A single platinum loop of Rhodococcus erythropolis PR-4 (labeled PR-4 in the table) was used to inoculate IB liquid culture medium, and the bacteria were cultured with agitation for 3 days at 28° C. Centrifugal separation was then performed at 15,000 rpm and 4° C. for 10 minutes on 1 mL of this precultured liquid. The precipitate obtained thereby was suspended in 1 mL of physiological saline, and centrifugal separation was performed once more. Thereafter, this rinse procedure was repeated two times, and the precipitate obtained thereby was suspended in 1 mL of physiological saline and was used as the stock solution.
The stock solution was suitably diluted so the initial concentration of the PR-4 was 10⁴cfu/mL, and the bacteria were added to separately prepared IB medium. Then each of the different hydrocarbons was added, and culturing with agitation was performed at 28° C. and 110 rpm.
For the hydrocarbons, a mixture of pristane (C19), a hydrocarbon that PR-4 metabolize by migrating into, and dodecane (C12), a hydrocarbon that PR-4 metabolize by adsorbing onto the surface of the organic solvent, was used. The hydrocarbons were added to the liquid culture medium by altering the mix ratios so the final concentration of mixed hydrocarbons would be 5% (v/v), and the hydrocarbon treatment was performed.

In addition, a hydrocarbon treatment was performed using the same procedure as above using pristane and octane (C8), a hydrocarbon that PR-4 that cannot migrate into and grow. The results are shown in Table 2.

	TABLE 2


	growth of PR-4

mix ratio of	dodecane:pristane	dodecane:pristane
hydrocarbon	(C12:C19)	(C8:C19)

9:1	−	−
8:2	+	−
7:3	+	−
6:4	+	+
5:5	++	++
4:6	++	++
3:7	++	++
2:8	++	++
1:9	++	++

−: growth is not shown or markedly few bacteria grow
+: growth is shown in pristane
++: a number of bacteria grow in pristane

As shown in Table 2, when dodecane and pristane are mixed, it was confirmed that the PR-4 migrated into the organic solvent when the mix ratio of dodecane to pristane ranged from 8:2 to 1:9.
On the other hand, when pristane and octane were mixed, it was confirmed that the PR-4 migrated into the organic solvent when the mix ratio of octane to pristane ranged from 6:4 to 1:9.

EXPERIMENTAL EXAMPLE 3

Investigation of Rhodococcus erythropolis PR-4 Adaptation

A single platinum loop of Rhodococcus erythropolis PR-4 (labeled PR-4 in the table) was used to inoculate IB liquid culture medium, and the bacteria were cultured with agitation for 3 days at 28° C. Centrifugal separation was then performed at 15,000 rpm and 4° C. for 10 minutes on 1 mL of this precultured liquid. The precipitate obtained thereby was suspended in 1 mL of physiological saline, and centrifugal separation was performed once more. Thereafter, this rinse procedure was repeated two times, and the precipitate obtained thereby was suspended in 1 mL of physiological saline and was used as the stock solution.
The stock solution was suitably diluted so the initial concentration of the PR-4 was 10⁴cfu/mL, and the bacteria were added to fresh IB medium in a 24 mm diameter test tube (Iwaki Glass). Next, pristane (C19) was added to a final concentration of 5% (v/v), and the bacteria were cultured with agitation for 3 days at 28° C. and 110 rpm.
After 3 days had elapsed after the start of culture, the PR-4 cultured in the IB liquid culture medium to which pristane (C19) had been added were collected. The collected PR-4 were added to separately prepared IB liquid culture medium so the initial concentration would be 10⁴cfu/mL. Then dodecane (C12) was added to a final concentration of 5% (v/v), and the bacteria were cultured with agitation at 28° C. and 110 rpm.
As a control, after PR-4 had been cultured in IB liquid culture medium to which dodecane (C12) had been added, they were cultured in IB liquid culture medium to which pristane (C19) was added.

On the third day after the start of culture, the growth status of the PR-4 and their site of localization were examined. When the PR-4 were present in the hydrocarbon, they were labeled “inside” and when they were present on the surface of the hydrocarbon, they were labeled “surface.” The results are shown in Table 3.

	TABLE 3


	growth	localization

pristane (C19) into	+	inside
dodecane (C12)
dodecane (C12) into	±	surface
pristane (C19)

±: growth is shown at interface of hydrocarbon and IB liquid culture medium
+: growth is shown in hydrocarbon

As shown in Table 3, it is clear that the PR-4 migrated into the dodecane (C12) in the same manner as when treatment was performed on pristane (C19) alone. Conversely, when the PR-4 that had been cultured in a medium to which dodecane (C12) was added were then cultured in medium to which pristane (C19) was added, the PR-4 did not migrate into the pristane (C19), and were present on the surface of the pristane (C19). From this investigation it is presumed that during the first stage of treatment the cell wall and/or cell membrane of the PR-4 changes in response to the species of hydrocarbon (adaptation), and that trait is retained even if the hydrocarbon used in the second stage of treatment is different.
The present invention enables Rhodococcus erythropolis PR-4 to migrate into the organic solvent and metabolize the hydrocarbon. As a result, it the present invention can find applications in environmental cleanup and the production of substances through bioprocesses that can reduce energy consumption and alleviate the burden on the environment.

Claims

1. A hydrocarbon treatment process comprising the steps of:

adding Rhodococcus erythropolis PR-4 to an aqueous medium containing culture medium ingredients,

adding an organic solvent containing a hydrocarbon with 14 or more carbon atoms; and

enabling the Rhodococcus erythropolis PR-4 to migrate into the organic solvent and metabolize the hydrocarbon with 14 or more carbon atoms.

2. The hydrocarbon treatment process according to claim 1 wherein the aqueous medium comprises a common bacteria culture medium.

3. The hydrocarbon treatment process according to claim 1 wherein the hydrocarbon with 14 or more carbon atoms is at least one or more hydrocarbons selected from a group consisting of tetradecane, pentadecane, hexadecane, pristane and squalane.

4. The hydrocarbon treatment process according to any of claim 1 wherein the hydrocarbon treatment process is performed with stirring.

5. A hydrocarbon treatment process comprising the steps of:

adding Rhodococcus erythropolis PR-4 to a first aqueous medium containing culture medium ingredients;

adding a first organic solvent containing a hydrocarbon with 14 or more carbon atoms;

enabling the Rhodococcus erythropolis PR-4 to migrate into the first organic solvent and metabolize the hydrocarbon with 14 or more carbon atoms;

adding the Rhodococcus erythropolis PR-4 that have metabolized the hydrocarbon with 14 or more carbon atoms to a second aqueous medium containing culture medium ingredients;

adding a second organic solvent containing a hydrocarbon with 13 or fewer carbon atoms; and

enabling the Rhodococcus erythropolis PR-4 to migrate into the second organic solvent and metabolize the hydrocarbon with 13 or fewer carbon atoms.

6. A hydrocarbon treatment system comprising:

means for supplying an organic solvent containing a hydrocarbon with 14 or more carbon atoms;

means for supplying an aqueous medium containing culture medium ingredients;

means for adding Rhodococcus erythropolis PR-4;

means for enabling the Rhodococcus erythropolis PR-4 to migrate into the organic solvent and treat the hydrocarbon with 14 or more carbon atoms; and

means for separating the product produced in the aqueous medium.