JPH099981A - Separation of oil and water in microbial reaction of oily and aqueous two-phase system - Google Patents

Abstract

PURPOSE: To efficiently recover a metabolite or a reactional product from an aqueous phase by filtering a microbial reactional solution comprising a microorganism, an oil and water through a ceramic filter module and separating a microbial cell/oil from water. CONSTITUTION: A microbial reactional solution of an oily and aqueous two-phase system in production, etc. of 2,6-naphthalenedicarboxylic acid by culturing Sphingomonas pauclmobilis AK2M16 (FERM P-13996) is filtered through a ceramic filter module. Thereby, the aqueous phase can selectively be separated without containing a microbial or an oil in the separated aqueous phase. The reactional product can readily be recovered in high yield.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for selectively and continuously separating oil / water from a reaction solution in an oil / water two-phase microbial reaction.

[0002]

2. Description of the Related Art The present petroleum refining process is recognized as one of the most efficient and most industrially efficient processes, but on the other hand, it is supported by high temperature and high pressure energy loads. There is a background. And it is also a fact that it triggers global environmental problems.
In the future, it is considered that petroleum refining processes that apply biotechnology and the like with even less environmental impact will be required or expected.

The conventional microbial reaction is carried out exclusively in a water system, and it has been considered to be extremely disadvantageous for a reaction with a water-insoluble substance using petroleum or various oils as a substrate.
On the other hand, in recent years, organic solvent-resistant microorganisms have been found, and studies on practical examples of microbial reactions in an oil-water two-phase system in which an oil such as an organic solvent intervenes have been started. In addition, microorganisms that can be converted by such a reaction system have also been reported. However, these studies have only just begun and are related to the reactivity of microorganisms in the organic solvent two-phase system, and there are still few studies and reports on specific processes after the microbial reaction step. The oil-water separation process in the oil-water two-phase system reaction has many problems due to lack of rational technology.
Of course, examples of successful continuous processing, industrialization, etc. have not yet been announced.

An advantage of the microbial reaction in the oil-water two-phase system is a microbial reaction of a water-insoluble raw material (substrate). In this case, a solid substrate can also be dissolved in an organic solvent as a raw material, facilitate mixing of the substrate and the medium, and make an efficient reaction system. That is, since it is possible to supply a water-insoluble raw material like the liquid raw material, it is possible to expand the applicable raw material, improve the reaction rate, prevent microbial contamination, control foaming, and continuously supply the raw material. It is an extremely effective method as compared with the method of supplying the raw material as a solid, and is expected as a highly useful microbial reaction process for petroleum-based substances.

However, in order to process the microbial reaction in the oil-water two-phase system efficiently and in a large amount, it is necessary to recover the product, supply the substrate / medium components, and remove the reaction inhibiting substances such as metabolic intermediates. A targeted efficient oil-water separation technology is essential.

[0006] Usually, the oil-water separation method used is a static separation method, and a more effective method is a centrifugation method or a method of filtering with a hydrophobic filter paper. However, these methods are not suitable for industrial treatment, as described below.

[0007]

In general, the reaction solution of oil-water two-phase system is remarkably emulsified by the action of the surfactant produced by the bacterial cells. A container is required, and it takes a long time to separate oily water, and it is completely unsuitable for separating bacterial cells.
Of course, regardless of the degree of emulsification, separation of oily water is accompanied by great difficulty.

The method of filtering with a hydrophobic filter paper has many problems in terms of efficiency such as low durability of the filter material and deterioration of separation performance due to clogging of the filter paper, which is also unsuitable.

Although oily water can be separated by the centrifugal separation method, it is difficult to completely separate the microbial cells because a surfactant is deposited on the interface and the microbial cells are also present therein. In addition, when only the aqueous phase is extracted from the reaction solution during or after the reaction and a new nutrient source is supplied, that is, a fresh energy source is supplied while removing reaction products and reaction inhibitors, continuous In the conventional reaction process, the centrifugation process makes the reaction process intermittent, and the operation is complicated and resuspension work of the bacterial cells is required, which requires a great deal of labor and time, which simplifies the process and improves the efficiency. There are still issues to be solved. In addition, it is not efficient in terms of loss associated with the recovery operation of active cells, and is not suitable for continuous large-scale processing.

In view of the above problems, the present inventors have developed an oil-water separation technique in an oil-water two-phase microbial reaction of petroleum, in which emulsification of a microbial reaction liquid proceeds, and therefore, in view of the above problems, an efficient continuous reaction In establishing the process, the establishment of the technique that can be suitably used is newly set as the object of the present invention.

That is, it is extremely difficult to separate oil moisture from a microbial reaction liquid consisting of microorganisms, oil, and water. In the present invention, as described above, in the ternary reaction liquid containing microorganisms, Microorganisms and oil,
Further, the purpose of the present invention is to separate into water and industrially efficiently, but such a technical problem is novel since it does not exist in itself. Needless to say, when these three components are emulsified to form an emulsion, it becomes more difficult to separate them. The present invention is also an object of the present invention to industrially separate bacterial cells / oil and water in such a reaction solution.

[0012] The present inventors not only newly set a technical problem that is extremely difficult to realize to separate cells / oil and water from such a three-component reaction solution, and further , The reaction product is recovered from the separated water, the substrate and the culture component are additionally supplied, and / or the reaction inhibitor is removed, and then returned to the culture tank (reaction tank), On the other hand, it was also set as an object to establish a technique of circulating culture or circulation reaction by returning microorganisms and oil to the culture tank (reaction tank) again, and culturing or reacting there again. According to this technology,
Not only can valuable microorganisms and oil components be reused, the use of expensive culture media can be reduced, which is excellent not only in terms of efficiency but also in economic terms. There are new technical issues that have not been known so far.

[0013]

Means for Solving the Problems The inventors of the present invention newly set the above-mentioned new technical problems which have not been heretofore known and which are highly effective in terms of technical significance. As a result of earnest research to solve all at once, it is appropriate to adopt a filtration method as a method for continuously separating oil / water without interrupting the reaction process from the oil / water two-phase microbial reaction solution. As a result of further investigations on the filtering material, which is one of the problems in the filtration method, it was found for the first time that the ceramic filter has an effective function for the oil-water two-phase microbial reaction liquid. It was That is, the ceramic filter has a function of selectively filtering the water phase even in the emulsified oil-water two-phase microbial reaction liquid, and by interposing the ceramic filter module, more efficient and continuous oil-water separation can be achieved. I discovered that is possible.

Further, by using the ceramic filter, not only the bacterial cells / oil phase can be efficiently separated from the aqueous phase, but also the separated bacterial cells have not been deteriorated or changed in function, so that they can be continuously cultured and reacted. Also confirmed that it can be used for. Further, it was further confirmed that it is possible to add necessary medium components and substrates to the separated aqueous phase and return them to the culture tank or the reaction tank, and continuous culture is also possible. The present invention was finally completed as a result of discovering these useful new findings and further examining them.

In the oil-water two-phase microbial reaction, there has never been a petroleum bioprocess involving such a continuous oil-water separation technique. Furthermore, it is possible to apply this technology to the treatment of spilled oil and waste oil, and to apply it to environmental purification and pretreatment of oil alternative fuels such as oil shale and tar sand. It can be a technology that makes a great contribution to

The present invention will be described in detail below. The oil-water separation method of the present invention efficiently filters cells / oil phase and water phase from the reaction solution by filtering through a ceramic filter module in an oil-water two-phase microbial reaction even in an emulsion state. Can be separated.

In a petroleum bioprocess, a complex oil-water two-phase reaction liquid containing an organic solvent and containing extremely delicate microorganisms is separated through a ceramic filter module into an oil / bacteria phase and an aqueous phase. However, there has not been reported a method for selectively and continuously separating each phase without damaging the bacterial cells. Furthermore, there are no reports of separation of each phase in the emulsified reaction solution, and there are no reports of efficient production of substances by continuous culture using this continuous separation treatment. I can't.

When selectively separating the aqueous phase from the oil-water two-phase microbial reaction solution, it is difficult to scale up by using the centrifugal separation method, but in the method using the ceramic filter module, a plurality of filters are used. By combining them, it becomes possible to easily scale up. Further, in terms of strength, it is stronger and more durable than general filter media, and is superior in terms of chemical resistance and heat resistance, and is superior in terms of industry.

In the separation method of the present invention, the microorganism to be subjected to the oil-water two-phase reaction may be any microorganism having a substrate converting ability and capable of growing or reacting in the two-phase system.

The oil-water two-phase system referred to in the present invention is as follows.
It is a reaction system in which an aqueous phase composed of a medium and / or a buffer solution and an organic solvent containing a substrate are contained at a constant ratio.

Regarding the aqueous phase component used in the oil-water two-phase system,
In the microbial cell reaction method, which is not particularly limited, components composed of inorganic salts and organic substances that are usually used for culturing microorganisms can be used, and in the resting microbial cell reaction method,
A buffer solution is used, and a phosphate buffer solution is preferably used. or,
Regarding the oil phase, depending on the microorganism to be subjected to the reaction, it is possible to form a two-phase with the aqueous phase, and the type is not particularly limited as long as the microorganism can grow or react, and aliphatic hydrocarbons, alicyclic It is also possible to use a mixture of one or more of hydrocarbons, aromatic hydrocarbons, ethers, ketones, and derivatives thereof, and various petroleum-based mixtures including crude oil.

In the oil-water two-phase microbial reaction liquid, the oil-water two-phase microbial reaction liquid is obtained by reacting with the microorganism in the oil-water two-phase system, extracting the reaction liquid with a pump or the like, and then filtering through a ceramic filter module. The aqueous phase can be selectively separated to recover the water-soluble reaction product. The separated aqueous phase is in a clean state because the bacterial cells have been completely removed.

By circulating the reaction solution, filtration is performed while continuously reacting to remove water-soluble inhibitors such as metabolic intermediates and to continuously supply a new energy source or insufficient energy source. be able to. Further, even when the filtration capacity is lowered due to clogging, the filtration capacity can be easily recovered by backwashing.

The ceramic filter module used in the present invention has a structure in which a fine porous thin film is laminated on an inorganic porous support, does not permeate the microorganisms and oil phase used, and selects only the aqueous phase. It is possible to preferably use a material that has a pore size that allows transparent permeation and that is resistant to sterilization by steam and chemical cleaning, such as alumina ceramics and zirconia ceramics. Commercially available products can be used well.

Therefore, according to the present invention, in an oil-water two-phase microbial reaction, by filtering the reaction solution through a ceramic filter module, even if the reaction solution is emulsified, the aqueous phase is removed from them. Since it can be selectively separated, it can be utilized in various oil-water two-phase microbial reaction processes and enables an efficient continuous reaction process. Therefore, not only the separation of oil and water,
The present invention can be advantageously used for continuous culture.

For example, to an organic solvent such as decalin 2,6-
Sphingomonas spp (for example, Sphingomonas spp.
gomonas paucimobilis FERM
P-13632, 13996, etc.)
Culturing is carried out at 300 ° C. for a predetermined period (for example, 3 to 5 days). After culturing, the culture solution is treated with a ceramic filter module. As a result, the bacterial cell / oil phase and the aqueous phase are rapidly and sharply separated. Since the separated aqueous phase is free from bacterial cells and oil, the reaction product 2,6-naphthalenedicarboxylic acid can be recovered very easily in a high yield.

The cell / oil phase is returned to the original culture tank, and the separated aqueous phase is added to and supplemented with medium components, if necessary, and then returned to the culture tank. In this way, continuous culture can be carried out efficiently and economically.

The medium components for culturing the bacterium include:
Except for containing 2,6-dimethylnaphthalene as a carbon source and a conversion substrate, other medium components are not particularly limited as long as the bacterium grows well. That is, as a growth substrate, in a nitrogen source, for example, ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium carbonate, ammonium acetate, ammonium nitrate, sodium nitrate, urea, etc., and as inorganic salts, for example, potassium, sodium, iron, magnesium, Salts such as manganese, copper, calcium, and cobalt can be used. In addition to 2,6-dimethylnaphthalene, salicylic acid, 2-methylnaphthalene, anthracene, glucose, fructose, starch and the like can be added as a carbon source as an auxiliary substrate.

As the solvent for dissolving 2,6-dimethylnaphthalene, for example, heptane, n-octane, isooctane, cyclooctane, nonane, decane, dodecane,
Tetralin, decalin, hexyl ether, etc. can be used, but they do not decrease in volatilization in a few days of culture, and their microbial toxicity is neither too strong nor too weak in order to suppress the growth of various bacteria, and 2 Solvents other than the above can also be used as long as they do not assimilate and dissolve 6,6-dimethylnaphthalene. And any one of these,
Alternatively, two or more kinds may be arbitrarily mixed and used.

[0030]

[Examples] Examples will be shown below using an oil-water two-phase assimilating microorganism as an example, but the present invention is not limited to the following examples.

[0031]

Example 1 Strain Sphingomonas pauc
immobilis AK2M16 (FERM P-13
996) was proliferated and cultured in the KF medium shown in Table 1 below, and when the culture solution reached an optical density (OD660) of about 20, 1 liter of this culture solution and 1 liter of decalin were added to a mini jar of 3 liter capacity. Introduced to mentor, ceramic filter module (housing: S
US304, Element: Alumina (0.2 μm outer membrane type, 22φ × 300 mm), Inner column: Teflon, Seal part: Silicon O-ring and Teflon seal tape) were used to perform filtration by a cross flow method. A peristaltic pump was used for extraction, and filtration conditions were: membrane surface flow rate: 0.5 to 1.0 m / sec (1,000 ml / min),
The temperature was 30 ° C., the aeration rate and stirring speed of the mini jar fermenter were 1.6 l / min and 400 rpm, and filtration was carried out at a rate of 120 ml after 1.8 hours and 4.5 hours after the start of filtration. And the ceramic filter module was backwashed with 200 ml of water.

[0032]

[Table 1]

The obtained results are shown in Table 2 below. The filtrate was a clean aqueous phase, and the organic solvent decalin and the bacterial cells were completely removed. Also, as shown in Table 2, by backwashing the ceramic filter module, the lowered filtration flow rate could be recovered.

[0034]

[Table 2]

[0035]

INDUSTRIAL APPLICABILITY According to the present invention, an oil-water two-phase microbial reaction solution is filtered through a ceramic filter module to selectively separate the aqueous phase from the reaction solution, thereby producing a metabolite or a reaction product. Items can be collected efficiently. Further, it is possible to remove the inhibitory substances in the reaction solution and continuously supply the medium components, so that an efficient continuous reaction process in an oil-water two-phase microbial reaction such as an organic solvent-mediated microbial reaction can be constructed.

Further, according to the present invention, since valuable microorganisms and organic solvents can be repeatedly used and the medium components can be reused without discarding them, it is economically superior and the present invention is an industrial treatment. Is outstanding.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location C12R 1:01) (C12N 1/20 C12R 1:01)

Claims (6)

[Claims] 1. In an oil-water two-phase microbial reaction, the reaction solution is filtered through a ceramic filter module,
A method for separating oil and water, comprising selectively separating an aqueous phase containing a metabolite or a reaction product from the reaction solution. 2. The method according to claim 1, wherein the separation treatment is continuously performed. 3. The method according to claim 1, wherein a metabolite or a reaction product is recovered after separating the aqueous phase. 4. The method according to claim 1, wherein necessary components are added to the separated aqueous phase, supplemented, and / or unnecessary components are removed, and then this is recycled to the reaction. Three
The method according to any one of claims 1 to 4. 5. The oil / bacterial body phase after separating the aqueous phase is reused for the reaction as it is or after adding and supplementing necessary components. The method according to claim 4. 6. The method according to any one of claims 1 to 5, wherein the reaction is an emulsified oil-water two-phase microbial reaction.