JP2011078911A - Microcapsule encapsulating denitrifying bacteria and production method thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microcapsule encapsulating denitrifying bacteria, exceedingly physically chemically stable, large in immobilized volume of denitrifying bacteria, excellent in diffusibility of nitrate nitrogen inside and activity of the denitrifying bacteria, and obtaining a high treatment efficiency as a microcapsule immobilized with autotrophic denitrifying bacteria. <P>SOLUTION: The microcapsule encapsulating denitrifying bacteria is provided in which a protective agent aqueous polymer solution containing the autotrophic denitrifying bacteria is internally capsuled in the lumen part of a mononuclear structure having a porous capsule wall containing a hydrophobic polymer and polyalkylene glycol as a principal component. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a microcapsule containing autotrophic denitrifying bacteria suitable for removing nitrate nitrogen as a contaminant in a water treatment system and the like, and a method for producing the same.

  In recent years, contamination of groundwater by nitrate nitrogen has become a serious problem worldwide. This nitrate nitrogen is generated when nitrogen compounds contained in fertilizers, livestock manure, domestic wastewater, etc. are decomposed or chemically oxidized by nitrite and nitrate bacteria, and penetrate into the underground to cause extensive groundwater contamination. However, since it is a water pollutant that cannot be completely treated at current water treatment plants, it is inevitable that it will be ingested in drinking water. When nitrate nitrogen is ingested by the body, it is reduced to nitrite nitrogen, causing methemoglobinemia to cause cyanosis (choking symptoms), and involved in the production of nitrosamines that are carcinogens. The establishment of effective removal means is an urgent issue.

  In general, nitrate nitrogen is removed by physicochemical denitrification using ion exchange resin or reverse osmosis membrane and by denitrification by microorganisms to decompose and remove nitrate nitrogen into nitrogen gas. It is roughly divided into biological denitrification methods. However, in the former physicochemical denitrification method, nitrate water in the water is separated to produce purified water, but the separated nitrate nitrogen is transferred to the regenerated wastewater and concentrated wastewater that is generated at the same time, and does not disappear. The treatment of wastewater containing high concentrations of nitrate nitrogen becomes a new problem. On the other hand, the latter biological denitrification methods include a method using heterotrophic denitrification bacteria that reduce nitrate nitrogen when ingesting an organic carbon source as nutrients and performing nitrate respiration, and hydrogen gas or the like as an electron donor. There is a method using an autotrophic denitrifying bacterium that performs denitrification using an inorganic substance such as a sulfur compound, but the organic carbon source ingested by the heterotrophic denitrifying bacterium also causes deterioration of water quality. A method using autotrophic denitrifying bacteria that does not require a source is preferred.

The denitrification action by autotrophic denitrifying bacteria is expressed by the following reaction formulas (I) and (II) when hydrogen gas is used as an electron donor, for example.
2NO ₃ ⁻ + 2H ₂ → 2NO ₂ ⁻ + 2H ₂ O (I)
2NO ₂ ⁻ + 3H ₂ → N ₂ + 2H ₂ O + 2OH ⁻ (II)
When both reaction formulas (I) and (II) are put together, the following formula (III) is obtained.
2NO ₃ ⁻ + 5H ₂ → N ₂ + 4H ₂ O + 2OH ⁻ (III)

  However, autotrophic denitrifying bacteria are difficult to handle by themselves and may cause secondary environmental contamination, so it is desirable to immobilize them in microcapsules. Conventionally, as a general method for immobilizing useful microorganisms in microcapsules, water-soluble synthetic polysaccharides such as alginate, κ-carrageenan, chitosan and polyvinyl alcohol (hereinafter abbreviated as PVA) are used. There is a method of supporting microorganisms on porous gel particles having high permeability. As a specific means for obtaining polysaccharide-based porous gel particles, a method of producing gel particles by dropping a polysaccharide aqueous solution into a calcium chloride aqueous solution is generally used. Further, as a specific means for obtaining PVA-based porous gel particles, a method of forming spherical gel particles by dropping an aqueous solution in which PVA and sodium alginate are dissolved into an aqueous calcium chloride solution (Patent Document 1), a microorganism is used. A method in which an aqueous solution of sodium alginate is dropped into an aqueous calcium chloride solution to form gel particles, and the gel particles are immersed in a high-concentration solution of soluble solids to contract (Patent Document 2). After impregnating the aqueous calcium chloride solution, the gel layer is formed on the outside of the core by immersing in an aqueous solution in which the PVA polymer and sodium alginate are dissolved, and then the core is immersed in a cross-linking agent-containing liquid. A method of dissolving and removing calcium alginate gel by crosslinking a polymer and further immersing in an aqueous sodium hydroxide solution (Patent Document 3) is known. That.

  However, when microcapsules with immobilized autotrophic denitrifying bacteria are used to remove nitrate nitrogen on an industrial scale such as a water treatment system, the immobilized denitrifying bacteria are less likely to dissipate and nitrate nitrogen is introduced into the interior. In addition to being easy to diffuse, the microcapsules themselves are excellent in physical and chemical strength and are not easily disintegrated. In addition, the denitrifying bacteria have a large immobilization volume and increase the diffusibility of nitrate nitrogen into the interior in order to increase the processing efficiency. It is necessary to be good, and it is also required to be excellent in mass productivity and economy. However, the conventional porous gel particles are insufficient in physical and chemical strength and easily disintegrated, have a small fixed volume of denitrifying bacteria and are inferior in processing efficiency, and an aqueous solution of the gel-forming polymer is an aqueous calcium chloride solution. In terms of production method, it is not suitable for use on an industrial scale such as a water treatment system in terms of supply capacity and cost.

  On the other hand, the present inventors have previously proposed several methods for producing microcapsules containing microorganisms. First, polymer beads such as sodium alginate containing microorganisms are emulsified and dispersed in an organic solvent in which the outer wall material polymer is dissolved, and this S / O emulsion is transferred into an aqueous solution to gradually remove the organic solvent. This is a method (Patent Document 4) for obtaining microcapsules in which the core substance of microorganisms is covered with an outer wall material coating. The second is a microorganism and a protective agent in an oil phase comprising an organic solvent A in which a wall material polymer is dissolved as a good solvent and an organic solvent B having a higher boiling point and a poor solvent for the wall material polymer. An aqueous solution containing a polymer is added and emulsified to prepare a W / O emulsion in which the aqueous solution is dispersed as water droplets in the organic phase, and this is added to the aqueous phase and emulsified to obtain W / O. This is a method of crystallizing the wall material polymer by sequentially evaporating and removing the good solvent and the poor solvent by heating / heating / depressurizing the / W emulsion (Patent Document 5).

JP-A-8-116974 JP-A-2005-224160 JP 2005-42037 A JP 2003-88747 A JP 2004-329159 A

  According to the method of the present inventors' proposal, it has been demonstrated that a useful microbe can be encapsulated and a physically and chemically stable microcapsule can be produced. In particular, the microcapsules obtained by the second method have a so-called core-shell type hollow structure with a large immobilized volume of microorganisms, and can be produced by sequentially mixing liquid agents, so that the production efficiency is good. There was an advantage. However, in the course of the subsequent studies by the present inventors, even the microcapsules with immobilized autotrophic denitrifying bacteria prepared based on these proposed methods are further improved in terms of processing efficiency, that is, nitrate nitrate removal ability. It turns out that there is room for.

  In view of the above circumstances, the present invention is a microcapsule in which autotrophic denitrifying bacteria are immobilized, which is physically and chemically very stable, has a large fixed volume of denitrifying bacteria, and has nitrate nitrogen inside. It is an object of the present invention to provide a denitrifying bacteria-encapsulated microcapsule that is excellent in diffusibility and activity of denitrifying bacteria and that can achieve high processing efficiency, and a production method that can reliably mass-produce this.

  In order to achieve the above object, a denitrifying bacteria-encapsulating microcapsule according to the invention of claim 1 is provided in a lumen of a core-shell type structure having a porous capsule wall mainly composed of a hydrophobic polymer and polyalkylene glycol. In addition, a protective polymer aqueous solution containing autotrophic denitrifying bacteria is included.

  Further, in the denitrifying bacteria-encapsulated microcapsule of claim 1, in the invention of claim 2, the capsule wall has a hydrophobic polymer: polyalkylene glycol mass ratio in the range of 1: 0.5 to 5, In the invention, the hydrophobic polymer of the capsule wall is polymethyl methacrylate, and the polyalkylene glycol is polyethylene glycol having an average molecular weight of 500 to 500,000. In the invention of claim 4, the average particle diameter is 50 to 1,500 μm, A configuration in which the relative specific surface area S2 / S1, which is the ratio of the calculated specific surface area S1 and the measured specific surface area S2 based on the average lumen diameter and the average particle diameter, is 40 or more is a preferable aspect.

  On the other hand, the method for producing the microcapsules containing denitrifying bacteria according to the invention of claim 5 is independent of the organic phase in which the wall material polymer mainly composed of a hydrophobic polymer and polyalkylene glycol is dissolved in a low-boiling organic solvent. A W / O emulsion in which droplets of the protective polymer aqueous solution containing autotrophic denitrifying bacteria are dispersed in the organic phase as an inner aqueous phase by adding and mixing the protective polymer aqueous solution containing the nutritional denitrifying bacteria. After the preparation, the W / O emulsion is added and mixed in the aqueous phase and stirred for a predetermined time, whereby the W / O / W emulsion in which the droplets of the W / O emulsion are dispersed in the outer aqueous phase. As prepared, the dispersed droplets are converted into droplets with a structure in which the inner single aqueous phase is covered with the outer organic phase by coalescence of the inner aqueous phase droplets, and then the organic solvent in the organic phase is added. Temperature or / and By gelling the wall material polymer was removed liquid drying by vacuum, it is characterized in that to generate any of the denitrifying bacteria containing microcapsules of the claims 1-4.

  Furthermore, in the method for producing microcapsules containing denitrifying bacteria according to claim 5, the invention according to claim 6 is a constitution in which the organic solvent having a low boiling point is mainly composed of dichloromethane, and the invention according to claim 7 is a method having a low boiling point of the organic phase. The organic solvent comprises a good solvent for the hydrophobic polymer, and a poor solvent having a higher boiling point than the good solvent and a low solubility for the hydrophobic polymer is blended in the organic phase in a range of 4% by mass or less in the solvent component. In the invention of claim 8, the protective polymer aqueous solution added and mixed in the organic phase is an aqueous solution of sodium alginate having a concentration of 0.5 to 10% by mass, and the invention of claim 9 is an aqueous phase as an outer aqueous phase. A configuration containing tricalcium phosphate as a dispersion stabilizer is a preferred embodiment.

  Since the denitrifying bacteria-encapsulating microcapsules according to the invention of claim 1 have a core-shell structure, the volume of the lumen, ie, the autotrophic denitrifying bacteria, is large for the particle size, and the denitrifying bacteria are contained inside the capsule. It is possible to secure a wide reaction field to move actively and decompose nitrate nitrogen. In addition, because the capsule wall is composed mainly of a hydrophobic polymer and polyalkylene glycol, it exhibits excellent physical and chemical strength based on the gel structure of the hydrophobic polymer and is difficult to disintegrate. But sufficient durability is obtained. Moreover, since the polyalkylene glycol, which is one component of the capsule wall, has both hydrophilicity and lipophilicity, the capsule wall has pores that are generated due to solvent volatilization during gelation of the hydrophobic polymer, A part of the polyalkylene glycol dissolves in the aqueous phase from the entangled state of the polymer of the hydrophobic polymer and the polyalkylene glycol, so that an infinite number of appropriate microporous channels leading to the inside and outside are formed. Thus, a large diffusibility of contaminants inside the capsule can be obtained. Therefore, by using this denitrifying microcapsule containing microcapsules in purification treatment of water and sewage containing nitrate nitrogen as a contaminant, nitrate nitrogen can be removed with high treatment efficiency over a long period of time.

  According to the invention of claim 2, since the hydrophobic polymer and the polyalkylene glycol constituting the capsule wall are in a specific ratio as the denitrifying bacteria-encapsulating microcapsule, the physical / chemical strength and the inside of the nitrate nitrogen are Both are excellent in diffusibility to the surface.

  According to the invention of claim 3, as the denitrifying bacteria-encapsulating microcapsules, the hydrophobic polymer and polyalkylene glycol constituting the capsule wall are specific species, so that the physical / chemical strength and the inside of nitrate nitrogen are A superior diffusivity is provided.

  According to invention of Claim 4, as said denitrifying microbe inclusion microcapsule, it is a specific average particle diameter, and calculated specific surface area S1 based on an average lumen | bore diameter and an average particle diameter and measured specific surface area S2 are a specific ratio. Therefore, there is provided an autotrophic denitrifying bacterium having a very large immobilization volume and a capsule wall having a very good porous structure, which can exhibit higher processing efficiency.

  According to the invention of claim 5, as a method for producing a denitrifying microcapsule-containing microcapsule, there is provided means capable of easily and reliably producing a microcapsule having the above-mentioned excellent characteristics with high production efficiency.

  According to the invention of claim 6, in the above production method, since the low-boiling organic solvent in the organic phase is mainly composed of dichloromethane having a low boiling point, the gelation of the wall material polymer by in-liquid drying efficiently proceeds. At the same time, there is an advantage that the temperature and pressure conditions at the time of drying in the liquid are relaxed and energy consumption can be reduced.

  According to the invention of claim 7, the low-boiling organic solvent in the organic phase is a good solvent for the hydrophobic polymer, and the organic phase contains an appropriate amount of a poor solvent having a higher boiling point than the good solvent. The advantage is that the walls are more porous.

  According to the invention of claim 8, in the above production method, the protective polymer aqueous solution added and mixed in the organic phase is a sodium salt aqueous solution having a specific concentration. Capsules are obtained.

  According to the ninth aspect of the invention, since the specific dispersion stabilizer is included in the aqueous phase as the outer aqueous phase in the above production method, a stable W / O / W emulsion can be prepared.

It is a schematic diagram which shows the production | generation mechanism of the denitrifying bacteria inclusion microcapsule of this invention. It is a scanning electron micrograph figure which shows the cross section and the whole of the denitrifying bacteria inclusion microcapsule obtained by manufacture example ac in the Example of this invention. It is a scanning electron micrograph figure which shows the cross section of the denitrifying bacteria inclusion microcapsule obtained by manufacture example dg in the Example, and the whole. It is a correlation characteristic figure of nitrate nitrogen and nitrite nitrogen concentration-time by a batch denitrification reaction test using the denitrifying bacteria inclusion microcapsule obtained by manufacture example g in the example. It is a correlation characteristic figure of the total nitrogen concentration-time by the same batch denitrification reaction test.

  The denitrifying bacteria-encapsulating microcapsule of the present invention is a protective polymer containing an autotrophic denitrifying bacterium in a core-shell type lumen having a porous capsule wall mainly composed of a hydrophobic polymer and polyalkylene glycol. An aqueous solution is included. Next, the generation mechanism will be described with reference to FIG.

  First, by adding and mixing a protective polymer aqueous solution containing autotrophic denitrifying bacteria into an organic phase in which a wall polymer mainly composed of a hydrophobic polymer and a polyalkylene glycol is dissolved in a low-boiling organic solvent. Then, a W / O emulsion is prepared in which droplets of an aqueous protective polymer solution containing autotrophic denitrifying bacteria are dispersed in the organic phase as an inner aqueous phase. Then, by adding this W / O emulsion to the aqueous phase and stirring and mixing, as shown in FIG. 1A, W / O in which the droplets Ed of the W / O emulsion are dispersed in the outer aqueous phase. / W emulsion is prepared. When the stirring of the W / O / W emulsion is further continued, as shown in FIG. 1B, the inner aqueous phase droplets Wd in the droplets Ed of the dispersed W / O emulsion are combined. Eventually, as shown in FIG. 1 (c), the inner single aqueous phase Wp, which is the united droplet, is converted into a droplet having a structure covered with the outer organic phase Op. Next, by removing the organic solvent in the organic phase Op by heating or / and drying in liquid under reduced pressure, the capsule outer shell Cs solidified by the gelation of the wall material polymer is formed as shown in FIG. Thus, denitrifying bacteria-encapsulating microcapsules MC having a core-shell structure are produced.

  In such denitrifying bacteria-encapsulating microcapsules, since the capsule particles have a core-shell structure, the volume of the inner cavity, that is, the autotrophic denitrifying bacteria is increased for the particle size, and the denitrifying bacteria are encapsulated inside the capsule. Can move widely and can secure a wide reaction field to decompose nitrate nitrogen. In addition, since the capsule particles are composed mainly of a hydrophobic polymer and a polyalkylene glycol, the capsule particles exhibit excellent physical and chemical strength based on the gel structure of the hydrophobic polymer and are not easily disintegrated. Sufficient durability can be obtained even under severe use conditions in a water treatment system or the like. Moreover, since the polyalkylene glycol, which is one component of the capsule wall, has both hydrophilicity and lipophilicity, the capsule wall has pores that are generated due to solvent volatilization during gelation of the hydrophobic polymer, A part of the polyalkylene glycol dissolves in the aqueous phase from the entangled state of the polymer of the hydrophobic polymer and the polyalkylene glycol, so that an infinite number of appropriate microporous channels leading to the inside and outside are formed. Thus, a large diffusibility of contaminants inside the capsule can be obtained. Therefore, by using this denitrifying microcapsule-containing microcapsules in purification treatment of water and sewage containing nitrate nitrogen as a contaminant, nitrate nitrogen can be removed with a very high treatment efficiency over a long period of time. .

  As the hydrophobic polymer used for the wall material polymer in the present invention, various synthetic polymer materials known as immobilization carriers for microcapsules can be used. However, in forming a capsule wall having excellent chemical and physical strength. Polymethyl methacrylate, polystyrene, polyacrylic acid, polylactic acid, poly ε-caprolactam and the like are suitable, and polymethyl methacrylate is particularly recommended as the optimum one. The polymethyl methacrylate preferably has an average molecular weight of about 500 to 1,000,000.

  Examples of the polyalkylene glycol used for the wall material polymer include polyethylene glycol, polypropylene glycol, polyethylene / polypropylene glycol, and the like. Particularly, polyethylene glycol is preferable, and the average molecular weight is 500 from the balance of hydrophilicity and lipophilicity. A polyethylene glycol of about ~ 500,000 is recommended as the optimum.

  The mixing ratio of the hydrophobic polymer of the wall material polymer and the polyalkylene glycol is preferably in the range of 1: 0.5 to 5 in terms of the mass ratio of the former: the latter, and if the proportion of the polyalkylene glycol is too small, the contaminant However, if the proportion of the polyalkylene glycol is too large, the chemical and physical strength of the capsule wall is lowered. The wall material polymer concentration in the organic phase is preferably about 5 to 20% by mass.

  The low boiling point organic solvent for dissolving the wall material polymer to constitute the organic phase is preferably a nonpolar solvent having a boiling point of 85 ° C. or lower for drying in liquid by heating and / or reduced pressure, such as dichloromethane (boiling point). 40 ° C.), chloroform (61 ° C.), ethyl acetate (77 ° C.), 1.2-dichloroethane (83.5 ° C.) and the like. Although it is good, it is recommended to use dichloromethane alone or in combination with others as a main component. That is, since dichloromethane has a particularly low boiling point, gelation of the wall material polymer by in-liquid drying proceeds efficiently, and the temperature and pressure conditions required for in-liquid drying are eased and energy consumption can be reduced.

  Moreover, although the low boiling point organic solvents such as dichloromethane exemplified above are good solvents for the wall material polymer, the organic phase may contain a small amount of a poor solvent for the wall material polymer having a higher boiling point than the good solvent. When a small amount of such a high-boiling poor solvent is blended, the good solvent is volatilized and removed first when drying in the liquid, and then the poor solvent is volatilized and removed. As a result, the capsule wall formed becomes more porous. Examples of such poor solvents include n-hexane (boiling point 69 ° C.), isooctane (99.25 ° C.), n-octane (125.7 ° C.), n-nonane (149.5 ° C.), n- Examples include decane (174 ° C.) and n-tetradecane (252.5 ° C.). Therefore, particularly when dichloromethane is mainly used, it is recommended that n-hexane is blended as the poor solvent. In addition, the compounding quantity of a poor solvent has a good range of 4 mass% or less in a solvent component, and when there are too many compounding ratios, it will become easy to produce aggregation.

  In the organic phase in which the wall material polymer is dissolved in a low-boiling organic solvent, an appropriate emulsion stabilizer is preferably blended in order to prepare a W / O emulsion by adding the inner aqueous phase. Examples of such emulsion stabilizers include a spanning surfactant such as sorbitan monooleate, and various surfactants, water-soluble resins, water-soluble polysaccharides and the like generally used for emulsion preparation.

  The autotrophic denitrifying bacteria contained in the protective polymer aqueous solution is not particularly limited, and examples thereof include Paracoccus denitrificans, Alcaligenes eutrophas, Pseudomonas pseudoflava and the like.

  The protective polymer is not particularly limited as long as it is compatible with autotrophic denitrifying bacteria and has gel-forming properties in water. For example, water-soluble polymeric polysaccharides such as alginate, κ-carrageenan, chitosan, and polyvinyl Although alcohol is mentioned, sodium alginate is particularly preferable. The concentration of the aqueous solution in the case of using this sodium alginate is preferably about 0.5 to 10% by mass, and if it is too high, the dispersion stability of the W / O emulsion is lowered and aggregation tends to occur. In addition, polypeptone, a yeast extract, magnesium sulfate, etc. can be suitably mix | blended in the protective agent polymer aqueous solution as a nutrient source of autotrophic denitrifying bacteria.

  In order to prepare a W / O emulsion in which droplets of a protective polymer aqueous solution containing autotrophic denitrifying bacteria are dispersed as an inner aqueous phase in the organic phase, the protective polymer aqueous solution is added to the organic phase at room temperature and mixed. Just do it. In order to prepare the W / O / W emulsion using the W / O emulsion, the W / O emulsion may be added and mixed at room temperature to the aqueous phase that is also the outer aqueous phase, and the stirring is performed as it is. By continuing for about several tens of minutes, the droplets of the inner aqueous phase are coalesced in each particle of the W / O emulsion droplets which are the dispersed phase. By this droplet coalescence, the W / O emulsion droplet is converted into a droplet having a structure in which the inner single aqueous phase Wp is covered with the outer organic phase Op.

  The aqueous phase used for the outer aqueous phase of the W / O / W emulsion desirably contains a dispersion stabilizer. The dispersion stabilizer is not particularly limited, and any of those generally used for emulsion preparation can be used. However, it is recommended that tribasic calcium phosphate is included as at least one component for suppressing aggregation of droplet particles. Is done.

  After the droplets are combined, in order to volatilize and remove the low-boiling organic solvent in the organic phase by drying in the liquid, one or both of heating and decompression is performed with stirring. It is recommended to do it simultaneously. In the heating, the temperature of the emulsion may be raised stepwise or continuously over several hours, but the highest temperature may be lower than the boiling point of the low boiling organic solvent. Further, in the reduced pressure, the pressure of the atmosphere in contact with the liquid level of the emulsion may be reduced stepwise or continuously over several hours, but the maximum reduced pressure may be about a fraction of the atmospheric pressure. Thus, when the low-boiling organic solvent of the organic phase is mainly composed of dichloromethane, the maximum ultimate temperature is about 35 ° C. and the maximum pressure reduction is about 300 hPa in the liquid drying in which heating and decompression are performed simultaneously. In addition, it is good for the stirring speed in this liquid drying to be about 100-1,000 rpm.

  In the process where the low-boiling organic solvent in the organic phase is volatilized and removed by drying in the liquid, the wall material polymer in the organic phase is phase-separated from the organic solvent and crystallizes to form a solid porous capsule wall. As a result, a core-shell type microcapsule is generated. The denitrifying bacteria-encapsulated microcapsules thus produced are collected by filtration and separation from the outer aqueous phase, followed by washing. In addition, when tribasic calcium phosphate is contained in the outer aqueous phase as a dispersion stabilizer, it is preferable to wash with distilled water or the like after removing the tertiary calcium phosphate by acid washing with a dilute hydrochloric acid aqueous solution or the like.

  About the particle size of the denitrifying bacteria-encapsulated microcapsules obtained, the liquid composition and polymer concentration used as the organic phase and the inner aqueous phase, the liquid mixing ratio and the stirring speed at the time of preparing the W / O emulsion and W / O / W emulsion Although it can adjust by condition setting, the range of 50-1500 micrometers is suitable as an average particle diameter from the applicability to a water treatment system. Further, in order to ensure high diffusibility of nitrate nitrogen as a contaminant into the capsule, the ratio of the calculated specific surface area S1 and the measured specific surface area S2 based on the average lumen diameter and the average particle diameter of the capsule particles. The relative specific surface area S2 / S1 is preferably 40 or more.

  Hereinafter, as examples of the present invention, production examples a to h of denitrifying bacteria-encapsulating microcapsules under different preparation conditions are shown.

[Cultivation of autotrophic denitrifying bacteria]
Paracoccus denitrificans NBRC13301 is used as an autotrophic denitrifying bacterium and pre-cultured in 5 ml of a culture solution containing 1.0% by weight polypeptone, 0.2% by weight yeast extract and 0.1% by weight MgSO ₄ .7H ₂ O (Liquid temperature 30 ° C., stirring speed 150 rpm, 24 hours), followed by main culture (liquid temperature 30 ° C., stirring speed 150 rpm, 24 hours) in 100 ml of 4 times the concentration of the preculture, The denitrifying bacteria thus obtained were washed and recovered with 0.9% by weight physiological saline.

(Preparation of organic phase)
For 20 g of dichloromethane, 2 g of polymethyl methacrylate (average molecular weight 100,000), 4 g of polyethylene glycol (average molecular weight 20,000), n-hexane described in Table 1 below, 0.6 g of sorbitan monooleate The organic phase was prepared by addition and mixing.

(Preparation of inner aqueous phase)
4 g of an aqueous solution containing sodium alginate at a concentration shown in Table 1 below, 4% by weight polypeptone, 0.8% by weight yeast extract and 0.4% by weight MgSO ₄ .7H ₂ O was added to 4 g of autotrophic denitrifying bacteria ( Wet weight) was added and mixed to prepare an inner aqueous phase.

(Preparation of outer aqueous phase)
An outer aqueous phase was prepared by adding and mixing 250 g of tribasic calcium phosphate to 500 g of a 1 wt% aqueous polyvinyl alcohol solution.

[Preparation of microcapsules containing denitrifying bacteria]
While stirring the organic phase at room temperature (20 ° C.), the inner aqueous phase is added and mixed to prepare a W / O emulsion, and the W / O emulsion is stirred at a liquid temperature of 20 ° C. To prepare a W / O / W emulsion and then coalesce the inner aqueous phase droplets in the dispersed W / O emulsion droplets by stirring at 150 rpm at atmospheric pressure for 30 minutes. While maintaining the stirring speed at 150 rpm, in the first stage, the liquid temperature is 25 ° C. and the atmospheric pressure is 800 hPa for 1 hour, in the second stage, the liquid temperature is 35 ° C., and the atmospheric pressure is 500 hPa for 1 hour, and in the third stage, the liquid temperature is 35 ° C. A three-stage in-liquid drying process for 2 hours at an atmospheric pressure of 300 hPa is performed, and the produced denitrifying bacteria-encapsulated microcapsules are separated by filtration, washed with a 0.5 molar aqueous hydrochloric acid solution to remove tricalcium phosphate, and distillation It was recovered in the wash to.

For the denitrifying bacteria-encapsulated microcapsules obtained in each production example, the average particle diameter, the recovery rate, the calculated specific surface area S1, the measured specific surface area S2, and the relative specific surface area S2 / S1 were examined. The amount used and the sodium alginate concentration in the inner aqueous phase are shown in Table 1 below. The calculated specific surface area S1 is the sum of the external specific surface area and the internal specific surface area per unit volume calculated based on the average particle diameter and the average lumen diameter of the capsule particles.

  Moreover, the cross-section of the denitrifying bacteria-encapsulating microcapsules obtained in Production Examples a to c and the entire scanning electron micrographs are represented by corresponding symbols (a) to (c) in FIG. The cross section of the denitrifying microcapsule-encapsulated microcapsule and the entire scanning electron micrograph are shown as corresponding symbols (d) to (g) in FIG.

  2 and 3, the denitrifying bacteria-encapsulating microcapsules of the present invention have a complete core-shell type and a large lumen, so that the denitrifying bacteria have a large immobilization volume and a capsule wall. Since it is extremely porous, it is clear that the diffusion of nitrate nitrogen into the capsule is very high. The very high porosity of the capsule wall is 46 even in Production Example e having the lowest relative specific surface area S2 / S1 shown in Table 1 (the measured specific surface area S2 is 46 times the calculated specific surface area S1). Is also demonstrated. Moreover, it turns out that the particle diameter tends to become smaller as the amount of n-hexane added to the organic phase increases from the comparison of Production Examples a to c in Table 1. Further, from the comparison of Production Examples d to g and c in Table 1, it can be seen that the higher the sodium alginate concentration in the inner aqueous phase, the greater the porosity of the capsule wall. However, in Production Example h having a sodium alginate concentration of 5% by weight, agglomeration occurs. Therefore, when the concentration is too high, it is presumed that the viscosity of the inner aqueous phase is too high to deteriorate the dispersion stability of the emulsion. As in Production Example d, microcapsules with good morphology can be obtained even if sodium alginate is not included in the inner aqueous phase. However, in order to protect the denitrifying bacteria to be encapsulated and prevent their dissipation, the presence of a protective polymer is required.

[Batch denitrification test]
0.12 g of NaNO ₃ and 0.2 mg of KH ₂ PO ₄ were added to 1 L of distilled water to prepare simulated contaminated water having a nitrogen concentration of 20 mg / L. In a 200 ml Erlenmeyer flask, 100 ml of simulated waste water contaminated with hydrogen gas in advance and 7 g (wet mass) 7 g of denitrifying bacteria-encapsulated microcapsules obtained in Production Example g were placed. The denitrification test was conducted under a hydrogen gas atmosphere (H ₂ flow rate 30 ml / min) in a shaking constant temperature bath at 100 ° C. and 100 rpm. During this denitrification test, simulated contaminated water is sampled over time, and the nitrogen nitrate concentration and nitrogen nitrite concentration are quantified with an ion chromatograph (Hitachi L-2470 conductivity detector) and denitrified. When the ability was examined, the results shown in FIGS. 4 and 5 were obtained.

  From the results shown in FIG. 4 and FIG. 5, it can be seen that nitrogen nitrate and nitrogen nitrite, which are contaminants in water, can be completely removed by the denitrifying bacteria-encapsulating microcapsules of the present invention. The total nitrogen removal rate determined by the least square method was 0.23 mg / L per hour.

Cs capsule outer shell Ed W / O emulsion droplet MC microcapsule Op outer organic phase Wd inner aqueous phase droplet Wp single aqueous phase

Claims (9)

  Denitrifying bacteria-encapsulated micro, in which an aqueous solution of a protective agent polymer containing autotrophic denitrifying bacteria is encapsulated in a core-shell type lumen having a porous capsule wall mainly composed of a hydrophobic polymer and polyalkylene glycol capsule.   The denitrifying bacteria-encapsulating microcapsules according to claim 1, wherein the mass ratio of hydrophobic polymer to polyalkylene glycol in the capsule wall is in the range of 1: 0.5 to 5.   The denitrifying bacteria-encapsulated microcapsule according to claim 1 or 2, wherein the hydrophobic polymer of the capsule wall is polymethyl methacrylate, and the polyalkylene glycol is polyethylene glycol having an average molecular weight of 500 to 500,000.   The relative specific surface area S2 / S1, which is the ratio of the calculated specific surface area S1 based on the average lumen diameter and the average particle diameter to the actually measured specific surface area S2, is 40 or more. The denitrifying bacteria inclusion microcapsule according to any one of the above.   By adding and mixing a protective polymer aqueous solution containing an autotrophic denitrifying bacterium into an organic phase in which a wall polymer mainly composed of a hydrophobic polymer and a polyalkylene glycol is dissolved in a low-boiling organic solvent, After preparing a W / O emulsion in which droplets of a protective polymer aqueous solution containing an autotrophic denitrifying bacterium are dispersed as an inner aqueous phase in an organic phase, the W / O emulsion is added to the aqueous phase and mixed to obtain a predetermined content. By stirring for a time, a W / O / W emulsion in which the droplets of the W / O emulsion are dispersed in the outer aqueous phase is prepared, and the dispersed droplets are brought into the inner side by coalescence of the inner aqueous phase droplets. The aqueous phase is converted into droplets having a structure covered with the outer organic phase, and then the organic solvent in the organic phase is removed by heating or / and drying under reduced pressure to gel the wall material polymer. Before Denitrificans method for producing microcapsules containing, characterized in that to produce the denitrifying bacteria containing microcapsules according to any one of claims 1 to 4.   The method for producing microcapsules containing denitrifying bacteria according to claim 5, wherein the low-boiling organic solvent of the organic phase contains dichloromethane as a main component.   The low boiling point organic solvent of the organic phase is a good solvent for the hydrophobic polymer, and the poor solvent having a higher boiling point than the good solvent and a low solubility for the hydrophobic polymer in the organic phase is 4% by mass or less in the solvent component. The method for producing microcapsules containing denitrifying bacteria according to claim 5 or 6, wherein the microcapsules are contained in a range of.   The method for producing microcapsules containing denitrifying bacteria according to any one of claims 5 to 7, wherein the aqueous solution of a protective agent polymer added and mixed in the organic phase is an aqueous solution of sodium alginate having a concentration of 0.5 to 10% by mass.   The method for producing microcapsules containing denitrifying bacteria according to any one of claims 5 to 8, wherein the aqueous phase used as the outer aqueous phase contains tricalcium phosphate as a dispersion stabilizer.