JP2008289424A - Method for producing microorganism carrier - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a microorganism carrier promoting the adhesion of microorganisms while keeping the biological activity of the microorganism to be carried. <P>SOLUTION: The method for producing a microorganism carrier comprises coating a surface of a substrate formed of at least one kind of substance selected from the group consisting of metals, ceramics and glass with an extracellular polymer formed by a microorganism forming an extracellular polymer. The microorganism carrier obtained by the method is suitable for a bioreactor. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing a microbial carrier for coating a substrate surface with an extracellular polymer.

  In recent years, various bioreactors (biological reactors) utilizing the biological activity (metabolism, assimilation, enzyme reaction, etc.) of microorganisms have been developed. For example, functional sugars, peptides, natural pigments, vitamins in the food field In the pharmaceutical field, anti-tumor agents, antibiotics, monoclonal antibodies, and agricultural chemicals are manufactured. In the chemical field, fine chemicals, enzymes, surfactants, fats and oils are manufactured, and in the energy field, In the field of environmental hygiene, the production of fuel is being used for wastewater, waste gas, waste treatment, and the like.

  In the development and operation of bioreactors, it is required to stably carry microbial cells in the reactor reaction section in a short period of time with high density and stability. For the purpose of facilitating the loading of microorganisms, microorganisms are attached. A process for increasing the surface area by roughening the physical shape of the solid support, a process for chemically modifying the surface characteristics of the solid support, and the like are also performed.

  For example, Japanese Patent Application Laid-Open No. 10-150982 discloses 5-400 μm pores that are open to the surface by heating with zeolite as a bioreactor carrier that has high mechanical strength, is inexpensive, and has a large amount of immobilized microorganisms. A method for producing porous particles having the following is disclosed. However, the loading of microorganisms on the carrier is an immobilization method that utilizes the spontaneous adhesion phenomenon of microorganisms, and it takes a relatively long time to carry a certain number of microorganisms. There is a problem that attached microorganisms are easily detached in an environment where a mechanical force acts.

  In addition, JP-A-7-99960 discloses that the surface of a ceramic monolith having a bead-like, pellet-like, or horseshoe-like ceramic carrier or a honeycomb structure is used to stably immobilize microorganisms and enzymes on the ceramic carrier for a long period of time. Long-term stable biotechnology in which needle-like or columnar mullite crystals having a length of 1 to 100 μm and a thickness of 0.1 to 10 μm are grown at a high density in a lawn shape and then a chitosan film is embedded between the crystals by the glutaraldehyde method A method for obtaining a reactor support is disclosed. In the crosslinking method such as the glutaraldehyde method used in this carrier surface modification method, amino groups must be introduced in advance on the carrier surface, and microorganisms and enzymes can be immobilized without extremely complicated pretreatment. I couldn't. Furthermore, the bond by the cross-linking method may be hydrolyzed and cleaved by a biological reaction in an aqueous solution, and it is inevitable that the amount of immobilization decreases with time. There were many cases where improvement was desired even when this was not possible.

  Furthermore, JP 2003-202329 A discloses a lipopolysaccharide adsorbent for immobilizing a compound having an amino group or imino group such as polyethyleneimine (PEI) on a carrier, and adsorbing lipopolysaccharide of gram-negative bacteria, and And a detection method for detecting lipopolysaccharide by immunoassay using the same. However, when producing a lipopolysaccharide adsorbent, complicated pretreatment is required to chemically immobilize polycations such as PEI on the surface of the carrier, or the growth of adsorbed gram-negative bacteria is suppressed. In particular, when used as a bioreactor, it was difficult to maintain biological activity.

  Here, microorganisms existing in nature live in a state of adhering to a certain solid surface, and these often form a film-like aggregate called a biofilm (biofilm). Microorganisms in biofilms are known to be significantly different physiologically compared to cells when they exist in suspension, and become biostable by forming biofilms, It is considered to exhibit more biological functionality, such as high resistance to drugs. In addition, the biofilm formed on the solid surface is characterized by accumulation of adhesive polymer-like substances in addition to microbial cells.

  It has been conventionally known that bacteria produce a polymer-like substance extracellularly. For example, Japanese Patent Application Laid-Open No. 2004-208563 acquires new microorganisms belonging to the genus Enterobacter from domestic and foreign soils and groundwater by screening. In addition, a method for producing a water-soluble polysaccharide composed mainly of fucose, galactose, glucose and glucuronic acid is disclosed. It is said that the water-soluble polysaccharide obtained by this method can be used for applications such as biocompatible materials, drug delivery system (DDS) media, functional foods, pharmaceuticals, and absorbent water retention agents. However, when exposed to acidic conditions in the presence of polyvalent metal ions, the free carboxyl groups of the polysaccharide molecular chains are masked, resulting in gelation and insolubility, or a significant decrease in adsorption affinity for solid surfaces. Depending on environmental conditions, the properties as a surface modifier may be lost.

  JP 2002-527072 A discloses at least one Agrobacterium radiobacter I-2001 (or DSM 12095) strain, a recombinant thereof, or a mutant thereof, and the strain, a recombinant thereof. Or a heteropolysaccharide obtainable by fermentation in a medium containing a carbon source that can be assimilated by a mutant thereof. This heteropolysaccharide can be used as a thickener or gelling agent in, for example, petroleum, agricultural chemicals, foods, cosmetics, and industrial detergents. However, there was no description about using such a heteropolysaccharide as a microbial carrier.

  Furthermore, Japanese translations of PCT publication No. 2004-500806 describes microorganisms that are non-toxic and non-antigenic and produce exopolysaccharides composed of neutral sugars, acidic sugars, and amine sugars. This exopolysaccharide is said to be usable as, for example, a soil conditioner, a food or drug additive, a plasma extender, or a biological growth inhibitor. However, there was no description about using such exopolysaccharide as a microbial carrier.

  Japanese Patent Application Laid-Open No. 6-239608 describes a microorganism having the ability to produce a viscous substance and activated carbon obtained by supporting the viscous substance produced by the microorganism. According to this, when microorganisms having the ability to produce viscous substances and other microorganism cells are immobilized on activated carbon as necessary, activated carbon with immobilized microorganism cells can be produced at low cost. The activated carbon obtained is said to have a high substance removal ability. However, it has been required to immobilize microorganisms on the surface of a substrate other than activated carbon.

JP-A-10-150982 Japanese Patent Laid-Open No. 7-99960 JP 2003-202329 A JP 2004-208563 A Japanese translation of PCT publication No. 2002-527072 Special Table 2004-500806 JP-A-6-239608

  The present invention has been made to solve the above problems, and by coating the surface of a substrate with an extracellular polymer, the adhesion of microorganisms can be promoted while maintaining the biological activity of the supported microorganisms. An object of the present invention is to provide a method for producing a microbial carrier.

  The object is characterized in that a substrate surface formed from at least one selected from the group consisting of metals, ceramics and glass is coated with an extracellular polymer produced by a microorganism that produces an extracellular polymer. It is solved by providing a method for producing a microbial carrier.

At this time, it is preferable that the surface of the base material is formed of a metal, and it is preferable that the coating amount of the extracellular polymer is 0.1 to 10 mg / m ² as the total organic carbon amount. The extracellular polymer is preferably a polysaccharide or protein, and a mixture thereof, and the extracellular polymer is preferably electrostatically amphoteric in an aqueous solution. In addition, it is preferable that the apparent effective charge of the extracellular polymer is 1 C / g or more when the pH is 3, and -1 C / g or less when the pH is 12, and the extracellular polymer is produced. It is preferable that the microorganism to be used is a microorganism belonging to the genus Agrobacterium.

At this time, it is preferable that the microorganism producing the extracellular polymer has the following mycological properties. A particularly suitable strain is the Agrobacterium genus strain 11-4 (accession number FERM AP-21298). It is also preferable that the microorganism that produces the extracellular polymer has resistance to fosfomycin and ampicillin.
(Cell morphology)
Neisseria gonorrhoeae Size: 0.5-1.0 × 0.8-2.0μm
Motility + (peripheral)
Gram staining −
Endospores −
(Physiological properties)
Gelatin liquefaction −
Litmus milk No change Indole production −
Degradation of esculin −
Nitrite production reduction from nitrate ＋
Catalase +
Starch hydrolysis −
OF test-/ +
Sugar breakdown Glucose +
Galactose +
Arabinose +
Sucrose +
Lactose −
Maltose +
Mannitol +
N-acetyl-glucosamine assimilation +
Potassium gluconate assimilation +
n-Capric acid assimilation −
Adipic acid assimilation −
dl-malic acid assimilation +
Sodium citrate assimilation −
Assimilation of phenyl acetate (PAC) −
Cytochrome oxidase +
Attitude toward oxygen Aerobic Growth temperature 20-37 ℃
Optimal pH 7.2

  A preferred embodiment of the present invention is a method for immobilizing microorganisms, characterized in that a microorganism is supported on a microorganism carrier obtained by the above-described method for producing a microorganism carrier, and another preferred embodiment of the present invention is The wastewater treatment method is characterized in that wastewater treatment is performed using the microorganism carrier obtained by the above-described method for producing a microorganism carrier. Moreover, a microbial carrier for a bioreactor obtained by the above method for producing a microbial carrier is also a preferred embodiment of the present invention.

  According to the method for producing a microbial carrier of the present invention, the adhesion of microorganisms can be promoted while the biological activity of the supported microorganisms is maintained by coating the substrate surface with an extracellular polymer.

  The present invention relates to a method for producing a microbial carrier characterized in that a substrate surface is coated with an extracellular polymer produced by a microorganism that produces an extracellular polymer.

  The method for producing a microbial carrier of the present invention is characterized in that the substrate surface is coated with an extracellular polymer. By this, adhesion of the microbial cell to the microorganism carrier obtained can be promoted. Here, the extracellular polymer is produced by a microorganism that produces an extracellular polymer, and is not particularly limited as long as it has an ability to support the microorganism. For example, polysaccharides, polyamino acids (peptides) From the viewpoint of adsorption affinity, polysaccharides or proteins, and mixtures thereof are preferable. Examples of monosaccharides constituting the polysaccharide include neutral sugars such as glucose, galactose, xylose, mannose, rhamnose, fucose, arabinose, and ribose; galacturonic acid, alginic acid, gluconic acid, iduronic acid, mannuronic acid, glucuronic acid, and the like Uronic acid; amino sugars such as glucosamine, galactosamine, mannosamine and the like.

  Among them, it is preferable that the polysaccharide constituting the extracellular polymer contains uronic acid having a carboxyl group, and this enables the extracellular surface of the base material formed from a metal, ceramics, glass, etc. that are hydrophilic. The polymer adsorbs irreversibly. In particular, it is preferable because it adsorbs well to a substrate surface made of a metal having a large positive surface charge with an extremely strong interaction. In the present invention, the uronic acid is preferably contained in an amount of 20% by weight or more, more preferably 30% by weight or more in terms of galacturonic acid. On the other hand, the content of uronic acid is usually 90% by weight or less.

  The extracellular polymer obtained by the method for producing a microbial carrier of the present invention preferably contains 0.5% by weight or more protein in terms of bovine serum albumin, and more preferably contains 1% by weight or more protein. As a result, the extracellular polymer becomes electrostatically amphoteric, and the extracellular polymer is well coated on various substrate surfaces having positive or negative electrostatic characteristics. On the other hand, the protein content is usually 60% by weight or less.

  The microorganism that produces the extracellular polymer is not particularly limited as long as it is a microorganism capable of producing an extracellular polymer, and includes the genus Pseudomonas, the genus Bacillus, the genus Alcaligenes, and the Agrobacterium. Microorganisms belonging to the genus, Euglena genus, Xanthomonas genus, Acetobacter genus, Gluconobacter genus and the like can be used. Among these, from the viewpoint of safety, the genus Agrobacterium is preferable.

A specific strain found by the present inventor is Agrobacterium genus 11-4 (reception number FERM AP-21298, hereinafter may be abbreviated as “11-4 strain”), and has the following mycological properties: I have it. In the following description, + means positive and-means negative.
(Cell morphology)
Neisseria gonorrhoeae Size: 0.5-1.0 × 0.8-2.0μm
Motility + (peripheral)
Gram staining −
Endospores −
(Physiological properties)
Gelatin liquefaction −
Litmus milk No change Indole production −
Degradation of esculin −
Nitrite production reduction from nitrate ＋
Catalase +
Starch hydrolysis −
OF test-/ +
Sugar breakdown Glucose +
Galactose +
Arabinose +
Sucrose +
Lactose −
Maltose +
Mannitol +
N-acetyl-glucosamine assimilation +
Potassium gluconate assimilation +
n-Capric acid assimilation −
Adipic acid assimilation −
dl-malic acid assimilation +
Sodium citrate assimilation −
Assimilation of phenyl acetate (PAC) −
Cytochrome oxidase +
Attitude toward oxygen Aerobic Growth temperature 20-37 ℃
Optimal pH 7.2

  The above Agrobacterium genus 11-4 strain was deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center on May 16, 2007, and assigned the receipt number FERM AP-21298.

  In the present invention, the microorganism that produces the extracellular polymer is preferably one that has resistance to antibiotics and antibacterial agents, and specifically, one that has resistance to fosfomycin and ampicillin. . In this way, when the microorganism that produces extracellular polymer has resistance to antibiotics, antibacterial agents, etc., the produced extracellular polymer itself has a protective function against antibiotics, antibacterial agents, etc. It is preferable because the biological activity of the microorganism attached to the microorganism carrier obtained in the present invention can be maintained. This point is also shown in a comparison test with a mutant strain that does not produce an extracellular polymer in Examples described later.

  The present invention is characterized in that the substrate surface is coated with an extracellular polymer, and the substrate surface is formed from at least one selected from the group consisting of metals, ceramics and glass. From the viewpoint of irreversibly adsorbing the extracellular polymer by electrostatic interaction or coordination bond, the substrate surface preferably has a positive or negative surface charge.

  The metal used for the substrate is not particularly limited, and examples thereof include stainless steel, titanium, aluminum, and an aluminum alloy from the viewpoint of corrosion resistance, chemical resistance, and durability. Further, the ceramic or glass used for the substrate is not particularly limited, and is preferably a metal oxide such as alumina, zirconia, and titania from the viewpoints of corrosion resistance, chemical resistance, and durability. Borosilicate glass, soda Glass, quartz glass and the like are preferable.

  Especially, it is preferable that the base-material surface is formed from a metal. When the surface of the substrate is formed of a metal, it is usually difficult for microorganisms to be adsorbed, and the biological activity of the adsorbing microorganisms may be reduced due to the antibacterial action of the metal. The microorganism carrier obtained by the present invention can promote the adhesion of microorganisms without reducing the biological activity even when the substrate surface is formed of metal, and the adhered microorganisms can be washed with water. It is preferable because it is not easily detached.

  In addition, the substrate body can be made of a different material and the surface can be plated, and a substrate on which a plating film such as electro nickel plating, electroless nickel plating, electroless nickel composite plating, etc. is formed can be used. it can.

  The shape of the substrate is not particularly limited. For example, a flat plate type such as a plate having a smooth surface or an uneven surface; a tubular type such as a pipe, a nozzle, or a column; a groove type such as a microchannel; a non-woven fabric, a fiber, or the like Fibrous molds: particulate molds such as fine particles, crushed pieces, spheres, etc .; porous molds such as sintered bodies and compacted bodies, etc. Among them, from the viewpoint of reaction efficiency, a shape such as a tubular mold or a groove mold Is preferred. Specific products having such a substrate surface include bioreactors, biosensors, microreactors, analytical instruments and the like.

The method for producing a microbial carrier according to the present invention is characterized in that the surface of a substrate is coated with an extracellular polymer, and the amount of the extracellular polymer coated on the surface of the substrate is 0.1 as the total amount of organic carbon. It is preferable that it is -10 mg / m < ² >. Here, the coating amount of the extracellular polymer is calculated from the amount of CO ₂ when the extracellular polymer is forcibly detached from the substrate surface by strong alkali washing and the recovered liquid is burned. When the coating amount of the extracellular polymer is less than 0.1 mg / m ² , there is a possibility that the adhesion amount of microorganisms may be insufficient, more preferably 0.2 mg / m ² or more, and even more preferably 0.8. 5 mg / m ² or more. On the other hand, the adsorption force between the substrate surface and the extracellular polymer is large, but the interaction between the extracellular polymers is weak. Therefore, when the coating amount of the extracellular polymer exceeds 10 mg / m ² , the multi-layer coating is obtained. There is a possibility that the extracellular polymer adsorbed on the upper part may be detached, more preferably 8 mg / m ² or less, and further preferably 5 mg / m ² or less.

  In the method for producing a microbial carrier of the present invention, an aqueous solution containing an extracellular polymer is preferably applied to the surface of a substrate. At this time, the concentration of the aqueous solution containing the extracellular polymer is preferably 0.1 to 5 g / L. If the concentration of the aqueous solution containing the extracellular polymer is less than 0.1 g / L, the surface of the base material may not be sufficiently covered, and more preferably 0.5 g / L or more. On the other hand, when the concentration of the aqueous solution containing the extracellular polymer exceeds 5 g / L, the extracellular polymer layer may be excessively adsorbed on the surface of the base material, so that the extracellular polymer layer adsorbed on the uppermost part may be detached. Preferably it is 3 g / L or less.

  The pH of the aqueous solution containing the extracellular polymer is preferably in the range of 3-10. When the pH of the aqueous solution containing the extracellular polymer is less than 3, since the extracellular polymer aggregates, there is a possibility that the substrate surface cannot be uniformly coated. More preferably, the pH is 6 or more. On the other hand, when the pH of the aqueous solution containing the extracellular polymer exceeds 10, the amount of adsorption of the extracellular polymer may decrease, and the pH is more preferably 9 or less. In addition, microorganisms that produce extracellular polymers can be reused by separating the strains by centrifugation after producing the extracellular polymers.

  In this invention, it is preferable that the extracellular polymer which coat | covered the base-material surface is amphoteric electrostatically in aqueous solution. This provides a good coverage of the extracellular polymer on various substrate surfaces having positive or negative electrostatic properties. The apparent effective charge of the extracellular polymer is preferably 1 C / g or more when the pH is 3 and -1 C / g or less when the pH is 12. Thus, since the apparent effective charge fluctuates more than a certain amount across zero C / g in the pH range of 3 to 12, the extracellular polymer coated on the substrate surface is electrostatically absorbed in the aqueous solution. It turns out that it is amphoteric.

  The aqueous solution containing the extracellular polymer may contain other components as long as the effects of the present invention are not impaired. Moreover, you may sterilize with respect to the aqueous solution containing an extracellular polymer. In particular, when a microorganism that produces an extracellular polymer is reused, it is preferable to perform sterilization after separating the strain. In addition, the step of purifying the resulting extracellular polymer can be omitted.

  In this invention, it is preferable that the temperature at the time of coat | covering an extracellular polymer on the base-material surface is 10-40 degreeC. When the temperature is lower than 10 ° C., molecular shrinkage of the extracellular polymer occurs, and there is a possibility that the coating area per molecule of the extracellular polymer is decreased, and more preferably 20 ° C. or more. On the other hand, when the temperature at the time of coating exceeds 40 ° C., the amount of adsorption of the extracellular polymer may decrease, and more preferably 30 ° C. or less.

  Moreover, in this invention, it is preferable that the processing time at the time of coat | covering an extracellular polymer on the base-material surface is 5 minutes or more. When the treatment time is less than 5 minutes, the adsorption amount may not be sufficient since the adsorption equilibrium is not reached, and more preferably 30 minutes or more. On the other hand, the processing time is usually 48 hours or less.

  In the present invention, the method for coating the surface of the substrate with the extracellular polymer is not particularly limited, but it is preferable to uniformly contact the surface of the substrate and the aqueous solution containing the extracellular polymer. Specifically, stationary immersion, shaking immersion, fluid contact, etc. are mentioned.

  As described above, the microbial carrier obtained by the method for producing a microbial carrier of the present invention may be circulated as a product as it is, that is, in the form of a gel in which the extracellular polymer contains moisture. You may distribute | circulate as a product, after drying the water | moisture content which an extracellular polymer contains in the range which does not inhibit.

  A preferred embodiment of the microbial carrier obtained by the production method of the present invention is a microbial immobilization method characterized in that a microorganism is supported on a microbial carrier. In particular, the microbial carrier obtained by the present invention is preferable because it has an effect of promoting adhesion while maintaining the biological activity of the supported microorganism. The microorganism to be carried is not particularly limited, and various microorganisms can be appropriately selected, and examples thereof include the genus Alcaligenes.

  In the present invention, a preferred embodiment is a wastewater treatment method characterized in that wastewater treatment is performed using a microorganism carrier obtained by the production method of the present invention. Since the microorganism carrier obtained by the production method of the present invention has a large amount of adhered microorganisms, the wastewater treatment can be performed efficiently.

  Further, the microbial carrier obtained by the production method of the present invention can be used for, for example, a bioreactor, a biosensor, a microreactor, an analytical instrument, etc. Among them, a preferred embodiment is a microbial carrier for a bioreactor. is there. The microbial carrier obtained by the production method of the present invention can be suitably used as a bioreactor having a large amount of immobilized microorganisms, since the amount of microorganisms to be carried is large.

  Hereinafter, the present invention will be described more specifically with reference to examples.

[Isolation of strains]
Dozens of samples are collected from soil, wastewater, etc. in order to isolate high extracellular polymer-producing bacteria, and each sample is suspended in sterile water to a concentration of 0.1 g / ml, and then diluted with sterile water. A series was created and smeared on nutrient agar. After culturing at 32 ° C. for several days, bacteria with high production of extracellular polymer were isolated by visual observation. As a result of selection by simple property investigation (culture temperature, growth, selectivity for medium) among the isolated bacteria, Agrobacterium genus strain 11-4 (reception number FERM AP-21298) having a good property was obtained. did. Moreover, when the obtained 11-4 stock | strain was cultured at 32 degreeC for 96 hours, when the colony formed was observed, it was white.

[Mycological properties]
When the morphological observation using the optical microscope with respect to 11-4 stock | strain and the physiological and biochemical test were investigated, the result shown below was obtained. In the following description, + means positive and-means negative.
(Cell morphology)
Neisseria gonorrhoeae Size: 0.5-1.0 × 0.8-2.0μm
Motility + (peripheral)
Gram staining −
Endospores −
(Physiological properties)
Gelatin liquefaction −
Litmus milk No change Indole production −
Degradation of esculin −
Nitrite production reduction from nitrate ＋
Catalase +
Starch hydrolysis −
OF test-/ +
Sugar breakdown Glucose +
Galactose +
Arabinose +
Sucrose +
Lactose −
Maltose +
Mannitol +
N-acetyl-glucosamine assimilation +
Potassium gluconate assimilation +
n-Capric acid assimilation −
Adipic acid assimilation −
dl-malic acid assimilation +
Sodium citrate assimilation −
Assimilation of phenyl acetate (PAC) −
Cytochrome oxidase +
Attitude toward oxygen Aerobic Growth temperature 20-37 ℃
Optimal pH 7.2

[Identification test]
Based on the taxonomic nature of the 11-4 strains, Bergey's Manual of Systematic Bacteriology, Vol. 1, NR Krieg, JG Holt (ed), Williams & Wilkins, Baltimore (1984) and Bergey's Manual of Determinative Bacteriology, ( 9th edition), JG Holt, NR Krieg, PHA Sneath, JT Staley, ST, Williams (ed), Williams & Wilkins, Baltimore (1994) Was judged to be appropriate.

[Generation of extracellular polymer]
A 20 ml LB solid medium (tryptone 1%, yeast extract 0.5%, sodium chloride 1%, agar 1.5%) placed in a petri dish in a culture solution of Agrobacterium 11-4 strain (0.1 ml / dish ) Was uniformly spread and cultured in a thermostat at 32 ° C. for about 3 days to produce an extracellular polymer. 20 ml of 10 mM PBS buffer (phosphate-sodium chloride, pH 7.4) was added per petri dish to suspend the cell-extracellular polymer mixture on the solid medium, and this suspension was collected. In order to separate the extracellular polymer fraction from the cell-extracellular polymer mixed suspension obtained from the 30 plates, the mixture was stirred with a stirrer at room temperature for 1 hour. After stirring, the cells and crude extracellular polymer were separated by centrifugation at 20,000 × g for 30 minutes. The crude extracellular polymer in the upper layer was heated at 80 ° C. for 30 minutes to eliminate the contamination of the bacterial cells, and then the bacterial cells were killed, and then the bacterial cell residues were removed by centrifugation (20,000 × g, 30 minutes). . Cold ethanol was added to the supernatant fraction to a final concentration of 80% to precipitate the crude extracellular polymer fraction. After standing at −20 ° C. overnight, an extracellular polymer fraction was obtained by centrifugation (30,000 × g, 30 minutes, 4 ° C.). This extracellular polymer fraction was dissolved in PBS buffer, DNase and RNase were added so as to be 10 μg / ml each, and kept at 37 ° C. for 3 hours to decompose nucleic acid residues. Thereafter, the enzyme was inactivated at 80 ° C. for 30 minutes, the protein residue was removed by centrifugation, and the extracellular polymer fraction was separated again by ethanol precipitation. The resulting precipitate was dried at 80 ° C. to obtain a crude extracellular polymer extract. By this method, 600 mg of crude extracellular polymer was obtained as a dry weight from 30 petri dishes.

[Confirmation of sugar]
In order to confirm the presence of sugar in the crude extracellular polymer isolated from the Agrobacterium 11-4 strain, simple quantification was performed by the phenol sulfate method and the carbazole sulfate method. As a result, it was estimated that about 5% by weight of neutral sugar in terms of mannose and about 40% by weight of acidic sugar in terms of galacturonic acid were contained. In addition, as a result of conducting a biuret reaction to confirm the presence of peptide bonds, it was found that about 2% by weight of protein was contained in terms of bovine serum albumin. The crude extracellular polymer was dissolved in ion-exchanged water so as to have a concentration of about 1 mg / ml, and its absorption spectrum was measured, but no special absorption maximum peak indicating the presence of nucleic acid was observed.

[Effective charge measurement of extracellular polymer]
10 mg of crude extracellular polymer was suspended in 100 ml of 0.1 M KNO ₃ solution, and the net charge of the crude extracellular polymer was measured by potentiometric titration. The relationship between the obtained pH and the effective charge is shown in FIG. It was found that the zero charge point of the crude extracellular polymer is in the vicinity of pH 8, and has a property of being positively charged at a pH lower than pH 8 and negatively charged at a pH higher than pH 8. Thus, it was found that the extracellular polymer produced by the Agrobacterium 11-4 strain is electrostatically amphoteric.

[Resistance to antibiotics]
In order to examine the susceptibility of Agrobacterium 11-4 strains to various antibiotics, susceptibility tests to fosfomycin, ampicillin and chloramphenicol were performed. The obtained results are summarized in Table 1.

A mutant strain that does not produce an extracellular polymer is produced by irradiating the cultured bacterial solution (about 1 × 10 ⁸ cells / ml) of Agrobacterium genus 11-4 with ultraviolet rays under the condition that 99.9% or more of the bacteria die. This was designated as Agrobacterium genus Fos1-1 strain. Hereinafter, it may be abbreviated as “Fos1-1 strain”. Then, the sensitivity test with respect to fosfomycin, ampicillin, and chloramphenicol was done like the above using the Fos1-1 strain | stump | stock. The obtained results are summarized in Table 1.

  As can be seen from Table 1, Agrobacterium genus strain 11-4 was found to be resistant to fosfomycin and ampicillin. From this, it was shown that the presence of the extracellular polymer has a function of protecting cells from the action of antibiotics. Moreover, as can be seen from the results of the sensitivity test using the Fos1-1 strain, by losing the ability to produce extracellular polymers, the Agrobacterium 11-4 strain has resistance to all the antibiotics tested. It can be seen that the drug resistance ability is clearly different depending on the presence or absence of extracellular polymer.

[Example 1]
An aqueous solution (pH 7.2) having a concentration of 2 g / L was prepared using 100 mg of crude extracellular polymer isolated from Agrobacterium genus 11-4, and 1 mg of total organic carbon was added to titanium particles having a particle size of 30 μm. / M ² . 0.5 g of surface-modified titanium particles, which are microbial carriers obtained in this way, are added to the culture solution of Fos1-1 strain (1 × 10 ⁸ cells / ml) and contacted at 32 ° C. for a certain period. It was. After contact, the titanium particles to which the bacterial cells adhered were washed twice by centrifugation using 10 ml of sterilized water, and then sonicated in sterile water to which 1% non-surfactant (Tween 80) was added for 10 minutes. The microbial cells adsorbed on the particles were detached. The number of bacteria in the detachment solution was measured by a plate dilution method, and the number of attached bacteria was calculated. The obtained results are summarized in Table 2.

[Comparative Example 1]
In Example 1, the Fos1-1 strain was used in the same manner as in Example 1 except that 0.5 g of untreated titanium particles having a particle size of 30 μm were used instead of using 0.5 g of surface-modified titanium particles as a microorganism carrier. And the number of attached bacteria was calculated. The obtained results are summarized in Table 2.

  As can be seen from Table 2, it can be seen that in Example 1 in which the surface of the titanium particles was coated with an extracellular polymer, the number of adherent bacteria was about two orders of magnitude higher than in Comparative Example 1 using untreated titanium particles.

[Example 2]
An aqueous solution (pH 7.2) having a concentration of 2 g / L was prepared using 100 mg of a crude extracellular polymer isolated from Agrobacterium 11-4 strain, and this was used to prepare a stainless steel flat plate (vertical 20 mm × width 50 mm). Adsorption was carried out at 1 mg / m ² as the amount of organic carbon. The plate of surface modified stainless steel, which is the microbial carrier thus obtained, was added to the culture solution of Fos1-1 strain (1 × 10 ⁸ cells / ml) and contacted at 32 ° C. for 5 days. . After the contact, the surface of the stainless steel plate to which the bacterial cells adhered was immersed and washed with sterilized water, and then excess water was removed. A suitable amount of Living / Dead fluorescent reagent (manufactured by Molecular Probe) was dropped thereon to stain the bacteria, and the state of bacteria adsorption on the stainless steel plate surface was observed with a confocal laser microscope. In this case, the living cells maintaining the biological activity are stained green and the dead cells are stained red, but in Example 2, a large amount and uniform adhesion of the cells, almost all of the attached bacteria The body was stained green. Thus, it can be seen that coating the surface of the substrate with an extracellular polymer has an effect of promoting adhesion while maintaining the biological activity of the microorganism.

[Comparative Example 2]
In Example 2, Fos1-1 was used in the same manner as in Example 2 except that an untreated stainless steel plate (vertical 20 mm × width 50 mm) was used instead of using a surface-modified stainless steel plate as a microorganism carrier. The strain was brought into contact and the bacteria were observed for adsorption. In Comparative Example 2, the cells were sparsely attached, and some of the attached cells were stained red.

It is a figure which shows the relationship between pH and an apparent effective charge.

Claims (13)

  Production of a microorganism carrier characterized in that a substrate surface formed from at least one selected from the group consisting of metals, ceramics and glass is coated with an extracellular polymer produced by a microorganism producing an extracellular polymer. Method.   The method for producing a microbial carrier according to claim 1, wherein the substrate surface is formed of a metal. The method for producing a microbial carrier according to claim 1 or 2, wherein the coating amount of the extracellular polymer is 0.1 to 10 mg / m ² as the total amount of organic carbon.   The method for producing a microbial carrier according to any one of claims 1 to 3, wherein the extracellular polymer is a polysaccharide or a protein, and a mixture thereof.   The method for producing a microbial carrier according to any one of claims 1 to 4, wherein the extracellular polymer is electrostatically amphoteric in an aqueous solution.   The apparent effective charge of the extracellular polymer is 1 C / g or more when the pH is 3, and is -1 C / g or less when the pH is 12, The microorganism carrier according to any one of claims 1 to 5 Production method.   The method for producing a microbial carrier according to any one of claims 1 to 6, wherein the microorganism that produces the extracellular polymer is a microorganism belonging to the genus Agrobacterium. The method for producing a microbial carrier according to any one of claims 1 to 7, wherein the microorganism producing the extracellular polymer has the following mycological properties.
(Cell morphology)
Neisseria gonorrhoeae Size: 0.5-1.0 × 0.8-2.0μm
Motility + (peripheral)
Gram staining −
Endospores −
(Physiological properties)
Gelatin liquefaction −
Litmus milk No change Indole production −
Degradation of esculin −
Nitrite production reduction from nitrate ＋
Catalase +
Starch hydrolysis −
OF test-/ +
Sugar breakdown Glucose +
Galactose +
Arabinose +
Sucrose +
Lactose −
Maltose +
Mannitol +
N-acetyl-glucosamine assimilation +
Potassium gluconate assimilation +
n-Capric acid assimilation −
Adipic acid assimilation −
dl-malic acid assimilation +
Sodium citrate assimilation −
Assimilation of phenyl acetate (PAC) −
Cytochrome oxidase +
Attitude toward oxygen Aerobic Growth temperature 20-37 ℃
Optimal pH 7.2   The method for producing a microbial carrier according to claim 7 or 8, wherein the microorganism that produces the extracellular polymer is Agrobacterium genus 11-4 (reception number FERM AP-21298).   The method for producing a microorganism carrier according to any one of claims 1 to 9, wherein the microorganism that produces the extracellular polymer has resistance to fosfomycin and ampicillin.   A method for immobilizing a microorganism, comprising supporting a microorganism on a microorganism carrier obtained by the method according to any one of claims 1 to 10.   A wastewater treatment method, wherein wastewater treatment is performed using the microbial carrier obtained by the method according to claim 1.   A microbial carrier for a bioreactor obtained by the method according to any one of claims 1 to 10.

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