JP5747371B2 - Lipophilic solvent treatment method using Rhodococcus bacteria - 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 Rhodococcus bacteria.

  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. Found 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 PR4 strain is a strain isolated as a degrading bacterium of pristane (2,6,10,14-tetramethyl-pentadecane) which is a kind of branched alkane (Non-patent Document 4). It is a strain that changes its colony form based on the production of EPS over time from rough type to mucoid type to rough type. 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. Accordingly, the Rhodococcus erythropolis PR4 strain (hereinafter referred to as “Rh-PR4” or “PR4 strain”) has developed a genetic manipulation system in the near future in the genus Rhodococcus. It is expected to become a stock.

  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 the bacteria added in the aqueous solvent can only react at the interface between the organic solvent and the aqueous solvent, it is necessary to improve the lipophilicity of the microbial cells for application to environmental purification and substance production by bioprocess. It is necessary to improve the treatment efficiency by transferring to hydrocarbons.

  R. erythropolis strain PR4 is a two-layer culture system of culture medium / alkane, and it has two different stations, “adsorption type” that adsorbs to the surface of alkane particles and “transfer type” that transfers into alkane particles, depending on the number of carbons of the added alkane. It is a strain exhibiting sexuality and is expected as a host for bioremediation and bioprocesses.

  Iwabuchi et al. Revealed that Rh-PR4 can be transferred to tetradecane, pentadecane, hexadecane, pristane, squalane and the like having 14 or more carbon atoms to metabolize and decompose these hydrocarbons (Patent Document 1).

JP 2006-325433 A

Finnerty, W. R. et al. (1992) Annual Review of Microbiology, p193-218. Ramos, J. L. 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

  As mentioned above, Rhodococcus microorganisms are useful for hydrocarbon decomposition and metabolism, but there are restrictions on hydrocarbons that can be decomposed and metabolized, such as being unable to transfer to hydrocarbons with a certain number of carbon atoms or less. was there.

  Therefore, the present invention is capable of efficiently decomposing a new oily solvent by controlling the affinity of the microorganism of the genus Rhodococcus to the lipophilic solvent, that is, by changing from the state of being adsorbed to the lipophilic solvent to the state of transfer to this. It is intended to provide a method that can be used.

  To achieve the above object, the present invention provides a method for treating an object using Rhodococcus bacteria, which is added to the medium by limiting the concentration of magnesium salt added to the medium of the bacteria. The localization of the bacterium with respect to the metabolized new oily compound is changed from a form adsorbed to the compound to a form transferred to the compound.

  Furthermore, the present invention includes a step of adding Rhodococcus bacteria to a water-soluble medium substantially free of magnesium salt or containing magnesium salt at a concentration not more than a limited concentration; Adding an object and metabolizing the object to be metabolized by the bacterium.

  The Rhodococcus bacterium is Rhodococcus erythropolis, preferably its PR4 strain. Rhodococcus erythropolis genus bacteria include transformants that maintain and improve the properties of decomposing and metabolizing hydrocarbons, in addition to naturally occurring strains.

  There are no particular restrictions on the hydrocarbons in which Rhodococcus erythropolis bacteria can be transferred to and decomposed into hydrocarbons. Preferred are n-alkanes and branched alkanes that are semi-solid and have 16 or less carbon atoms, preferably 13 or less carbon atoms.

  As described in Patent Document 1, conventionally, Rhodococcus erythropolis genus bacteria only adsorb to hydrocarbons having a low carbon number, for example, hydrocarbons having 13 or less carbon atoms. However, it was not possible to metabolize and decompose it, but according to the present invention, by limiting the concentration of inorganic salts added to the bacterial culture medium, Can be transferred to the hydrocarbon.

  The concentration of magnesium is 88.5 nM or less, preferably 8.9 nM or less, and more preferably 0.9 nM or less. The magnesium salt is in particular magnesium chloride.

  As described above, according to the present invention, by controlling the affinity of the Rhodococcus microorganism to the metabolic target component such as hydrocarbons, that is, by changing from the state adsorbed to the component to the state transferred to this, Can be efficiently decomposed.

The examination result of the yeast extract (YE) density | concentration with respect to a culture medium is shown, The microscope picture and the characteristic table | surface which show the localization with respect to the hydrocarbon (C12, C19) of PR4 strain | stump | stock. The examination result of the glucose (Glu) density | concentration with respect to a culture medium is shown, It is a characteristic table | surface which shows the micrograph and the localization with respect to the hydrocarbon (C12, C19) of PR4 strain | stump | stock. The examination result of the yeast extract (YE) density | concentration at the time of glucose non-addition to a culture medium is shown, The microscope picture and the characteristic table | surface which show the localization with respect to hydrocarbon (C12, C19) of PR4 strain | stump | stock. The measurement examination result of PR4 strain | stump | stock when an inorganic salt is added to a culture medium is shown, It is a characteristic table | surface which shows the micrograph and the localization with respect to hydrocarbon (C12, C19) of PR4 strain | stump | stock. This shows the measurement study results when a component that inhibits the PR4 strain from being transferred to hydrocarbons is added to the medium, and a micrograph and a characteristic table showing the localization of the PR4 strain to hydrocarbons (C12, C19) It is. The measurement examination result of PR4 strain | stump | stock when magnesium chloride is added to a culture medium is shown, It is a characteristic table | surface which shows the micrograph and the localization with respect to hydrocarbon (C12, C19) of PR4 strain | stump | stock.

  1. A metabolic method using Rhodococcus erythropolis PR4 strain Rhodococcus genus bacteria can be obtained from the Biogenetic Resource Division, Biotechnology Division, National Institute of Technology and Evaluation.

2. Preparation of culture solution
A medium and solution of Rh-PR4 were prepared as follows.

IB liquid medium: about 700 ml Milli-Q water (Synthesis A10, Millipore), 10 g glucose (Wako Pure Chemical Industries), 10 g yeast extract (Becton, Dickison and company), 0.2 g MgCl ₂ · 6H ₂ O (Wako Pure Chemical Industries), 0.10 g CaCl ₂・ 2H ₂ O (Wako Pure Chemical Industries), 0.10 g NaCl (Wako Pure Chemical Industries), 0.02 g FeCl ₂・ 4H ₂ O (Wako Pure Chemical Industries) ), 0.50 g of (NH ₄ ) ₂ SO ₄ (Wako Pure Chemical Industries) was added, the pH was adjusted to 7.2 using a pH meter, the volume was increased to 1 L, and autoclaved at 121 ° C. for 15 minutes.

NP solution: 0.5 g of (NH ₄ ) ₂ SO ₄ and 0.5 g of K ₂ HPO ₄ were added to an appropriate amount of milli-Q water to make up to 1 L, and autoclaved at 121 ° C. for 15 minutes.

Total mineral solution: 0.18 g MgCl ₂ · 6H ₂ O, 0.10 g CaCl ₂ · 2H ₂ O, 0.10 g NaCl, 0.02 g FeCl ₂ · 4H ₂ O, 0.50 g ( NH ₄ ) ₂ SO ₄ , 0.50 g of K ₂ HPO ₄ was added to make up to 1 L, and autoclaved at 121 ° C. for 15 minutes.

Make a solution that combines the components contained in the total mineral solution individually. Magnesium acetate, magnesium lactate, magnesium nitrate, the magnesium sulfate was adjusted such that the magnesium chloride and the concentration, as _{appropriate, (CH 3 COO) 2 ·} 4H 2 O ( Wako Pure Chemical Industries), [CH ₃ CH ( OH) COO] ₂ Mg · 3H ₂ O (Wako Pure Chemical Industries), Mg (NO ₃ ) ₂ · 6H ₂ O (Wako Pure Chemical Industries), MgSO ₄ (Wako Pure Chemical Industries) were prepared and added to the medium.

3. Preparation of culture system First, one platinum loop of PR4 strain was taken in 5 ml of IB liquid medium and cultured at 28 ° C. and 110 rpm for 2 to 3 days. This preculture was transferred to a 15 ml-dissolved centrifuge tube and centrifuged at 4 ° C. and 10,000 rpm for 10 minutes. Thereafter, the supernatant was discarded, 5 ml of sterilized milli-Q water was added, the cells were well suspended, and then sufficiently stirred by vortexing.

This suspension was centrifuged again at 4 ° C. and 10,000 rpm for 10 minutes, and the supernatant was removed. This operation was performed a total of 6 times to wash the cells. Next, the washed cells were diluted and ingested into 10 ml of various media so that the initial concentration of the cells was 10 ⁶ cfu / ml. Finally, alkanes were ingested to a final concentration of 5-10% (v / v) and cultured at 28 ° C. and 110 rpm until the stationary phase was reached.

4.Microscopic observation After the organic layer and the aqueous layer of the obtained culture sample are completely separated, the aqueous layer and the organic layer are separated separately or sufficiently suspended, and the mixed solutions are collected and prepared in a preparation (IWAKI). It was dropped and a cover glass (IWAKI) was placed, and each was prepared as an aqueous layer, an organic layer and a mixed solution.

  A sample was placed on the stage of a system biological microscope (OLYMPUS BX50), a drop of immersion oil was dropped on the slide glass, and the objective lens was magnified 100 times for observation. For each sample, five typical fields of view were selected, photographed with a connected microscope camera (OLYMPAS DP72), and analyzed with image processing software (OLYMPAS DP2-BSW ver 2.1).

5. Results and Discussion Using n-dodecane (C12) as a representative alkane adsorbed by cells and pristane (C19) as a representative for metastasis, cells can be combined with various medium components of these two-layer culture systems individually or in combination. Attempts were made to search for conditions that change the localization of the. The main components of IB medium were roughly divided into yeast extract, glucose, and inorganic salts, and each was examined individually.

5-1. Examination of yeast extract concentration The concentration of yeast extract was examined. Prepare the medium so that the final concentration of yeast extract in IB liquid medium is 1.0, 0.1, 0.08, 0.06, 0.04, 0.03, 0.02, 0.01%, 0.001% (w / v). A bilayer culture system added at 5% (v / v) was prepared to examine whether or not the growth of PR4 strain and cell localization changed (FIG. 1).

  The IB medium containing C12 or C19 grown as a control showed the same results as before. When the yeast extract was between 1% and 0.03%, white turbidity was observed in the culture solution, but no white turbidity was observed at a concentration of 0.02% or less.

  As a result of observing the localization of the cells with a phase contrast microscope, it was confirmed that the Rh-PR4 cells were adsorbed on the surface of the oil droplets under the condition of 0.1% to 0.02% yeast extract solution with dodecane added. In addition, it was confirmed that Rh-PR4 cells were transferred to the oil droplets when pristane was added. The above results were similar to those in the control, and in both cases, when the condition was 0.02% or more, cell localization, that is, whether the cells adsorbed to the surface of the hydrocarbon oil droplets or transferred to it. It was judged that there was no effect on the distinction.

  On the other hand, microbial cells could not be observed under the condition of 0.02% or less. From this, it was considered that the limit value of yeast extract concentration necessary for growth of PR4 strain was 0.02%.

5-2. Examination of glucose concentration Subsequently, glucose was examined. Adjust the yeast extract so that the final concentration is 1%, the final concentration of glucose is 1.0, 0.1, 0.08, 0.06, 0.04, 0.02, 0.01, 0.001, 0%, and add a C12 or C19 added bilayer culture system. It was prepared and examined whether changes in the growth and cell localization of the PR4 strain occurred (FIG. 2). As a result, the same tendency as the control was confirmed both in the culture state and the microscopic observation.

  Next, the conditions in which the yeast extract was not added were set, and glucose was added to the conditions mentioned above and examined. As a result, cell proliferation could not be confirmed under all conditions. This suggests that yeast extract is essential for growth, although glucose does not affect the growth in the two-layer culture environment tested by the same bacterium.

5-3. Examination of the concentration of yeast extract without addition of glucose As described above, it was found that glucose has no effect on the growth of PR4 strain, so the lower limit of yeast extract concentration under the condition of no addition of glucose was examined. (Fig. 3). As a result, as in the case where glucose was added, the growth lower limit concentration was 0.02%, and growth could not be confirmed under these conditions.

  As a result, it was revealed that the component necessary for growth of PR4 strain was yeast extract, and its minimum concentration was 0.02%. At this time, under all conditions up to 1.0, 0.1, 0.08, 0.06, 0.04, and 0.02%, the PR4 strain grew while showing a transfer type when C19 was added and an adsorbed type when C12 was added. Therefore, it was determined that yeast extract and glucose do not affect the localization of cells of the same fungus.

5-4. Examination of inorganic salts Inorganic salts were examined. A medium prepared only with all the inorganic salts added to the IB medium was prepared, and C12 or C19 was added thereto, and cultured and observed under the same conditions as before (FIG. 4).

Conditions As a result, in all the conditions that do not contain K ₂ HPO ₄ and (NH _{4) 2} SO ₄ containing although growth of PR4 strain can not be confirmed, K ₂ HPO ₄ and (NH ₄₎ only ₂ SO ₄ In C19 and C12, growth was confirmed.
When C19 was added, it was confirmed that the cells had been transferred as before, but interestingly, when C12 was added, the cells were transferred and existed in the oil droplets. Was confirmed.

  On the other hand, when all inorganic salts are added, the PR4 strain is known to be adsorbed and not transferred to C12. We anticipated that there was an inhibitory component, and then examined that component.

Various mineral salts were added to the medium based on C12 as alkane and K ₂ HPO ₄ and (NH ₄ ) ₂ SO ₄ as inorganic salts at the same concentration as IB medium, respectively. When the presence was confirmed, it was confirmed that when MgCl ₂ was added, the PR4 strain was only adsorbed and not transferred to the oil droplets (FIG. 5). From the above, it was found that the localization of the bacterium to alkane can be controlled by adding MgCl ₂ .

5-5.Concentration of magnesium salt
Since MgCl ₂ was found to be involved in changing the localization of PR4 strains to hydrocarbons, the effect of MgCl ₂ concentration on bacterial localization was further investigated. Since the MgCl ₂ concentration of the IB medium is 885 mM, the medium in which the concentration of magnesium salt diluted stepwise is sequentially reduced to a medium in which the concentrations of K ₂ HPO ₄ and (NH ₄ ) ₂ SO ₄ are constant. And the localization of the cells of the same bacteria to hydrocarbons was examined (FIG. 6).

  Since the cell localization could not be clearly distinguished as either metastatic or adsorbed, the micrographs were divided into four patterns: adsorption, partial adsorption, partial adsorption, and transition. The ratio of the four patterns was examined according to the transition according to the magnesium salt concentration.

As a result, cells that had partially transferred even under the same concentration conditions as the control IB medium were confirmed, but as the MgCl ₂ concentration decreased, the proportion of adsorbed cells decreased, and all photographs were transferred. The concentration of magnesium chloride was 88.5 nM.

On the other hand, although some cells were adsorbed even when MgCl _{2 was} not added, it was confirmed that many cells were transferred into C12.

As a result, by decreasing the MgCl ₂ concentration, the localization tends to change from the transition type to the adsorption type, and the ratio of the transition type to the adsorption type is reversed when the concentration is around 8.9 mM. It was suggested that the concentration of metastatic cells increased at lower concentrations.

In addition, in order to examine whether the phenomenon described above is a phenomenon peculiar to magnesium chloride, the above-mentioned medium containing only K ₂ HPO ₄ and (NH ₄ ) ₂ SO ₄ is added with magnesium sulfate, magnesium nitrate, magnesium acetate, lactic acid. Magnesium was added to the same concentration as magnesium chloride in the IB medium, and localization was examined in the presence of C12. As a result, even when magnesium sulfate and magnesium nitrate were added, the cells were adsorbed on the surface of the C12 particles like magnesium chloride.

  Therefore, it was found that the localization of Rhodococcus bacteria to hydrocarbons can be adjusted by limiting the concentration of magnesium salts other than magnesium chloride such as magnesium sulfate and magnesium nitrate.

Claims (5)

A form in which Rhodococcus bacteria are added to a medium containing a lipophilic solvent, the concentration of the inorganic salt in the medium is adjusted for the localization of Rhodococcus bacteria in the medium, and the Rhodococcus bacteria are adsorbed to the lipophilic solvent And the ratio of the form transferred to the lipophilic solvent, metabolizing Rhodococcus bacteria and decomposing the lipophilic solvent ,
The lipophilic solvent is a hydrocarbon having 13 or less carbon atoms,
As the medium, an NP solution containing (NH ₄ ) ₂ SO ₄ and K ₂ HPO ₄ is used,
A lipophilic solvent treatment method in which a magnesium salt is not added as the inorganic salt or is adjusted to 88.5 nM or less . The lipophilic solvent is a hydrocarbon liquids at normal temperature under normal pressure, lipophilic solvent treatment method according to claim 1. The Rhodococcus bacterium is Rhodococcus erythropolis (Rhodococcus erythropolis), lipophilic solvent treatment method according to claim 1 or claim 2. 4. The lipophilic solvent treatment method according to claim 3 , wherein the Rhodococcus bacterium is Rhodococcus erythropolis PR4 strain. 5. The lipophilic solvent treatment method according to any one of claims 1 to 4, wherein the magnesium salt is magnesium chloride.

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