JP2018176127A - Method for removing nitrate nitrogen and nitrate nitrogen remover - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for removing nitrate nitrogen and a nitrate nitrogen remover capable of effectively and safely and efficiently removing environmental water such as groundwater and nitrate nitrogen in soil.SOLUTION: A method for removing nitrate nitrogen includes yeast belonging to Rhodotorula graminis, Pichia capsulata or Rhodosporidium sphaerocarpum and capable of assimilating nitrate nitrogen, and contacting the culture or extract thereof with environmental water containing nitrate nitrogen or soil to remove nitrate nitrogen.SELECTED DRAWING: Figure 11

Description

  TECHNICAL FIELD The present invention relates to a method for removing nitrate nitrogen and a nitrate nitrogen removing agent, and in particular, a strain for which the safety has already been recognized for nitrate nitrogen contained in environmental water or soil containing nitrate nitrogen. The present invention relates to a method for removing nitrate nitrogen and a nitrate nitrogen remover that can be removed using a yeast belonging to Rhodotorula, Pichia or Rhodosporidium.

In recent years, the pollution of environmental water such as ground water that spreads nationwide is said to be due to the side effects of modern intensive agriculture.
This is because nitrogen contained in improperly treated livestock excrement or excessively applied fertilizer or domestic water becomes ammonia nitrogen, nitrite nitrogen or nitrate nitrogen and penetrates underground.
Ammonia nitrogen is easily fixed to the soil, nitrous acid is unstable, and it is oxidized immediately to nitric acid, so mainly only nitrate nitrogen is transferred to groundwater, causing nitrate nitrogen contamination in groundwater. linked.

  Specifically, the amount of nitrogen used by fertilizers to be sprayed in agriculture etc. exceeds the natural nitrogen recycling capacity of soil microorganisms, and nitrate nitrogen accumulated in excess in soil by promoting distribution of compost is used in the soil. It leaches into infiltrating water and shifts to groundwater. As a result, the phenomenon that the nitrate nitrogen more than the environmental standard value set in each area etc. is contained in groundwater and pollutes groundwater occurs.

In addition, as a result, agricultural products and grass grown using ground water containing excess nitrate nitrogen will contain excess nitrate as a result, and it will be safe as a food and drink material for future animals and animals, etc. It has become a concern that causes a decline.
Therefore, the social significance of constructing effective measures for the removal of nitrate nitrogen in groundwater is great.

Methods of denitrifying nitrogen contained in waste water, which have been conventionally studied, are roughly classified into ion exchange methods and microbial methods.
Since denitrification by ion exchange only adsorbs nitrate ions to a strongly basic ion exchange resin, and generates a high salt concentration waste liquid containing NO ₃ ⁻ at the time of regeneration of the ion exchange resin, its treatment becomes a problem, and it is not environmentally friendly. Consideration is not enough, and it is necessary to introduce equipment for processing.

  For example, in Japanese Patent Application Laid-Open No. 2003-205289 (Patent Document 1), a series of reducing (sub) nitrite ion of water to be treated by electrochemical reaction and removing generated ammonia from the water to be treated instead of nitrogen gas And a processing device used for the nitrogen removal treatment.

On the other hand, in the denitrification method using microorganisms, nitrifying bacteria that perform nitrification reaction that oxidizes ammonium ion (NH ₄ ⁺ ) to nitrate ion (NO ₃ ⁻ ) and anaerobically reduce generated nitrate ion and finally nitrogen gas (N ₂₎ and to processes combining denitrifying bacteria or ammonium nitrite ions, (NO 2 ^_-) by anaerobic ammonium oxidation to electron donors, active research on the treatment process by anammox bacteria to nitrogen gas It has been done.
However, in these methods, since the presence of NH ₄ ⁺ or NO ₂ ⁻ is an essential condition, it is difficult to apply to denitrification in groundwater.
This is because NH ₄ ⁺ is relatively strongly retained in the soil, and NH ₄ ⁺ is leached into the ground water as oxidized NO ₃ ⁻ , so nitrogen in the groundwater is significantly biased to NO ₃ ⁻ It is.

In view of this point, as a nitrogen removal method by microorganisms, for example, in JP-A-2016-77954 (Patent Document 2), an ammonia nitrogen-containing wastewater containing a substance that inhibits anaerobic ammonia oxidation reaction by Anamox bacteria is treated As a biological nitrogen removal method, ammonia nitrogen-containing wastewater is brought into contact with ammonia-oxidizing bacteria to oxidize a portion of ammonia nitrogen to Ammonia nitrogen is oxidized to nitrite nitrogen in the nitrification tank using a fixed bed or fluidized bed having a biological sludge amount of 1.6 to 8.0 g-VSS / m ² In addition, a biological nitrogen removal method is disclosed that removes inhibitory substances by biological sludge retained on a carrier.

  However, a simple method of effectively denitrifying or denitrifying nitrate nitrogen in ground water or soil is expected.

Japanese Patent Application Publication No. 2003-205289 JP, 2016-77954, A

  It is an object of the present invention to solve the above problems and to effectively and safely and efficiently remove nitrate nitrogen in environmental water such as ground water and soil, a nitrate nitrogen removal method and a nitrate nitrogen remover It is to provide.

The present inventors, as a result of earnest research, have found that a specific microorganism strain can directly remove NO ₃ ⁻ contained in environmental water such as ground water and soil, and reached the present invention.

  The method for removing nitrate nitrogen according to claim 1 belongs to Rhodotorula graminis, Pichia capsulata, or Rhodosporidium sphaerocarpum, and assimilates nitrate nitrogen. A method for removing nitrate nitrogen, which comprises removing a competent yeast, a culture thereof or an extract thereof by contacting it with environmental water or soil containing nitrate nitrogen.

  The method for removing nitrate nitrogen according to claim 2 is the method for removing nitrate nitrogen according to claim 2, wherein the yeast belonging to Rhodotorula graminis is Rhodotorula graminis NBRC 0190, and the yeast belonging to Pichia capsulata is Pichia The method for removing nitrate nitrogen is characterized in that the yeast belonging to the genus Rhodosporidium sphaerocarpam is Capschulata (Pichia capsulata) NBRC 1770 and is Rhodosporidium sphaerocarpum (Rhodosporidium sphaerocarpum) NBRC 1939. is there.

  The method for removing nitrate nitrogen according to claim 3 is the same as the method for removing nitrate nitrogen described above, wherein the Rhodotorula graminis (Rhodotorula graminis), Pichia capsulata (Pichia capsulata) or Rhodosporidium sphaerocarpum (Rhodosporidium sphaerocarpum) is used. And a culture thereof or an extract thereof is supported on a carrier and immobilized, and brought into contact with environmental water containing nitrate nitrogen or the soil, and the method for removing nitrate nitrogen.

  The method for removing nitrate nitrogen according to claim 4 is the method for removing nitrate nitrogen, wherein the environmental water or soil containing nitrate nitrogen contains carbon at a C / N ratio (mass ratio) of 15 to 30. It is a method for removing nitrate nitrogen characterized in that

  The nitrate nitrogen removing agent according to claim 5 belongs to Rhodotorula graminis, Pichia capsulata or Rhodosporidium sphaerocarpum, and has an ability to assimilate nitrate nitrogen. It is a nitrate nitrogen removing agent in environmental water or soil, characterized in that it comprises the following yeast, its culture or its extract.

  The nitrate nitrogen removing agent in environmental water or soil according to claim 6 is the nitrate nitrogen removing agent according to claim 6, wherein the yeast belonging to Rhodotorula genus Glaminis is Rhodotorula graminis NBRC 0190 and belongs to Pichia Capsulata The nitrate nitrogen is characterized in that the yeast is Pichia capsulata NBRC 1770, and the yeast belonging to the genus Rhodosporidium sphaerocarpum is Rhodosporidium sphaerocarpum NBRC 1939. It is a remover.

  The nitrate nitrogen removing agent according to claim 7 is the above-mentioned nitrate nitrogen removing agent, wherein the Rhodotorula graminis (Rhodotorula graminis), Pichia capsulata (Pichia capsulata) or Rhodosporidium sphaerocarpum is contained. The belonging yeast, a culture thereof or an extract thereof is a nitrate nitrogen removing agent characterized in that it is supported on a carrier and immobilized.

According to the method for removing nitrate nitrogen of the present invention, it is possible to effectively remove nitrate nitrogen contained in environmental water such as ground water, rivers, ponds, lakes and marshes, ocean water, and soil.
In particular, since the nitric acid assimilation according to the present invention can directly remove only NO ₃ ⁻ , it can function effectively as a denitrification treatment under aerobic conditions such as environmental water such as ground water.
Further, in the present invention, the nitrate nitrogen removing agent is a known bacteria derived from naturally occurring acid rain which contains nitrate nitrogen, stem rain such as kuunagi and cedar and its rhizosphere soil, Also, since there is no problem with the safety of the cells, it can be extremely effectively applied to the removal of nitrate nitrogen in environmental water such as ground water and soil.

It is a graph which shows the time-dependent reduction rate of the nitrate ion in a culture medium at the time of changing the sodium chloride density | concentration contained in an example culture medium. It is a graph which shows the time-dependent reduction rate of the nitrate ion in a culture medium at the time of changing the sodium chloride density | concentration contained in another example culture medium. It is a graph which shows the time-dependent decreasing rate of the nitrate ion contained in a culture medium at the time of changing pH in an example culture medium. It is a graph which shows the time-dependent decreasing rate of the nitrate ion contained in the culture medium of another example when pH in another culture medium is changed. It is a graph which shows the time-dependent decreasing rate of the nitrate ion contained in a culture medium at the time of changing carbon nitrogen content in a culture medium. It is a graph which shows the time-dependent reduction rate of the nitrate ion contained in the example fresh water at the time of using 3 types of yeast. It is a graph which shows the time-dependent decrease rate of the nitrate ion contained in artificial seawater of an example at the time of using 3 types of yeast. It is a graph which shows the time-dependent reduction rate of the nitrate ion contained in a culture medium at the time of changing the density | concentration of the nitrate nitrogen contained in a culture medium. It is a graph which shows the time-dependent reduction rate of the nitrate ion contained in a culture medium at the time of changing the density | concentration of the nitrate nitrogen contained in a culture medium. FIG. 6 is a diagram showing a temporal phenomenon of nitrate ions contained in a culture medium when immobilized nitric acid assimilable yeast is immobilized on a carrier and used. It is a graph which shows the time-dependent reduction rate of the nitrate ion contained in groundwater. It is a diagram which shows the time-dependent decrease rate of the residual sugar concentration contained in groundwater.

The present invention will be described by the following embodiments, but is not limited thereto.
The method for removing nitrate nitrogen of the present invention is a yeast belonging to the genus Rhodotorula, which is capable of assimilating nitrate nitrogen, a culture thereof or an extract thereof, and contacting with soil water or other environmental water such as groundwater containing nitrate nitrogen. A method of removing nitrate nitrogen, specifically, a yeast having the ability to assimilate nitrate nitrogen, which belongs to Rhodotorula genus Graminis, Pichia genus Capsulata, or Rhodosporidium sphaerocarpam, A method for removing nitrate nitrogen, which comprises removing the culture or its extract by contacting it with environmental water or soil containing nitrate nitrogen.

  The yeast capable of assimilating nitrate nitrogen of the present invention is a yeast belonging to Rhodotorula graminis, Pichia capsulata or Rhodosporidium sphaerocarpum, Preferably, the yeast belonging to the genus Rhodotorula graminis is, as Rhodotorula graminis, Rhodotorula graminis NBRC 0190, NBRC 10747, the yeast belonging to the genus Pichia capschulata, Pichia capsulata NBRC 1770, NBRC 0721, NBRC 0974, NBRC 1768, NBRC 1769, NBRC 100352, the yeast belonging to Rhodosporidium sphaerocarpa, Rhodosporidium sphaerocarpam NBRC 1939, NBRC 1 38, can be exemplified NBRC 1937, NBRC 1938, or the like.

Since the above-mentioned yeast is a known yeast and can be distributed from the National Institute of Technology and Evaluation Product Technology Biotechnology Technology Center (NBRC) and has been confirmed to be safe, it can be easily obtained, It can be used suitably.
These yeasts are capable of assimilating nitrate nitrogen as they are capable of taking up nitric acid under aerobic condition and assimiling as amino acids and proteins of the yeast.

  In the present invention, a yeast having the ability to assimilate nitrate nitrogen, a culture thereof or an extract thereof (hereinafter referred to as "yeast" etc.) may be applied as a nitrate nitrogen removing agent which assimilates nitrate nitrogen. It is possible.

  By bringing the above-mentioned yeast capable of assimilating nitrate nitrogen into contact with water contaminated with nitrate nitrogen, or mixing it with soil contaminated with nitrate nitrogen, etc. Reduce nitrate nitrogen.

  Examples of environmental water to which the method of removing nitrate nitrogen according to the present invention and the application are applied include nitrate-contaminated ground water and rivers, ponds, lakes, ocean waters, etc. Soils such as residential areas and mountains can be targeted.

The present invention is a method capable of removing nitrate nitrogen contained in environmental water such as ground water by mixing the above-mentioned yeast with environmental water such as ground water containing nitrate nitrogen.
Either method can be used as long as yeast or the like can be added as it is to environmental water such as groundwater containing nitrate nitrogen or fixed to a carrier as long as it can be sufficiently contacted with environmental water such as groundwater. It may be used in a fixed bed or in a fluidized state or in any method.

  In addition, as a carrier for covering or adsorbing and immobilizing the above-mentioned yeasts and the like, any of various porous material available on the market such as polyvinyl alcohol (PVA), sodium alginate, carrageenan potassium, charcoal, bamboo charcoal, polyurethane etc. Etc. can be used.

  As a method of solidification as an example, it is possible to apply any known method such as immobilization by encapsulating in a polymer spherical gel or supporting on a porous body, for example, the former As an example of the case, a 1.5 w / w% aqueous solution (150 mL) of sodium alginate prepared by dissolving in ion-exchanged water is autoclaved (for example, 121 ° C., 15 minutes), cooled, and then 50 mL of preculture Add bacteria, mix uniformly, aspirate a fixed amount with a syringe, drop 1 drop (about 30 to 50 μL) into 1 w / w% calcium chloride aqueous solution (sterilized) prepared separately, and leave for 15 to 30 minutes And solidify into beads. The immobilized beads are collected and washed with sterile water to remove the adhering potassium chloride solution, thereby making it possible to immobilize the nitrate nitrogen removing agent of the present invention.

  The present invention is applicable to ordinary groundwater or the like if it is environmental water or soil such as groundwater containing nitrate nitrogen, and the pH thereof is within the range of about 4.0 to 9.0. For example, the contained nitrate nitrogen can be effectively removed, for example, 98% or more can be removed.

  Moreover, since environmental water to be treated may contain sodium chloride, there is no problem if the content concentration is about 0 to 3.0 w / w%, and the contained nitrate nitrogen is It is also possible to remove 98% or more.

  Furthermore, in order to remove nitrate nitrogen, it is desirable to require the presence of a carbon source, and the ratio of nitrogen to carbon is not particularly limited as long as the ground water contains an organic substance that becomes a carbon source, but preferably C / When the ratio of N is 15 to 30, more preferably 18 to 25 and still more preferably about 20, the effects of the present invention can be exhibited more effectively in a short period of time.

The nitrate nitrogen agent and removal method of the present invention can also be used as a soil improver for denitrification in soil zones such as upland fields and pastures.
For example, the nitrate nitrogen removing agent of the present invention is added to a soil with excess nitrate, and optionally mixed with the soil to remove nitrate nitrogen contained in the soil. Even if the soil and the above yeast are directly sprayed and mixed, or those immobilized on a carrier, any method may be used as long as the soil can be sufficiently contacted.

  The nitrate nitrogen removal agent of the present invention is added to the soil with excess nitrate to measure the reduction of nitrate nitrogen in the soil over time, and the excess nitrate nitrogen in the agricultural land is reduced. For example, it will be possible to grow crops safely. Thus, by functioning as a soil modifying agent, it becomes possible to prevent the source of nitrate nitrogen contamination of environmental water such as ground water.

The present invention is specifically described by the following examples and test examples.
(Use reagent)
・ Glucose (formula weight = 180.16) Wako Pure Chemical Industries, Ltd.
・ Peptone Wako Pure Chemical Industries, Ltd.
・ Yeast extract Wako Pure Chemical Industries, Ltd.
・ Malt extract Wako Pure Chemical Industries, Ltd.
-Magnesium sulfate (formula weight = 246.47) (reagent) Katayama Chemical Industry Co., Ltd.
-Potassium chloride (formula weight = 74.55) (reagent) Katayama Chemical Industry Co., Ltd.
· Ferrous sulfate · heptahydrate (formula weight = 278.02) (reagent) Kanto Chemical Co., Ltd.
· Magnesium sulfate · heptahydrate (formula weight = 246.47) (reagent) Katayama Chemical Industry Co., Ltd.
Sodium chloride (formula weight = 58.44) (reagent) Nacalai Tesque, Inc.
-Sodium nitrate (formula weight = 84.99) (reagent) Wako Pure Chemical Industries, Ltd.
-Potassium dihydrogen phosphate (formula weight = 141.96) (reagent) Nacalai Tesque, Inc.
-Dipotassium hydrogen phosphate (formula weight = 174.18) (reagent) Wako Pure Chemical Industries, Ltd.
・ Somogie copper liquid Wako Pure Chemical Industries, Ltd.
・ Nelson liquid Wako Pure Chemical Industries Ltd.
-Sterilized ion exchange water (CPW-200 made by Advantec Co., Ltd.)
・ Aquarium artificial sea water element (product name Red Sea Salt, made by Red Sea Co., Ltd.)
-Polyvinyl alcohol (PVA) Wako Pure Chemical Industries, Ltd. [160-11485] (Saponification degree: about 98%, polymerization degree: 1600 to 1800)

(Used strain)
Yeast: Rhodotorula graminis NBRC 0190
Yeast: Rhodosporidium sphaerocarpam (Rhodosporidium sphaerocarpum)
NBRC 1939
Yeast: Pichia capsulata NBRC 1770

(Measurement of nitrate ion concentration)
In each example, the nitrate ion concentration was measured by ion chromatography using an ion analyzer (IA-100, manufactured by Toa DKK Co., Ltd., or IA-200, manufactured by Toa DKK Co., Ltd.).
First, prepare each standard solution of [NO _3- ] = 1, 2, 5, 10, 20 ppm, inject it into the ion analyzer so that the sample injection amount is 20 μL, and hold the nitrate ion retention time of the obtained chromatogram. A calibration curve was created from the conductivity values at.

Subsequently, the measurement of the nitrate ion concentration in the culture solution to be evaluated was evaluated using the calibration curve.
Specifically, the culture solution sample to be evaluated was filtered through a membrane filter, an appropriate amount was taken using a syringe, and the mixture was injected into the above-described ion analyzer in the same manner as described above.
The nitrate ion concentration in the culture solution sample was determined from the conductivity value at the retention time of nitrate ion and the calibration curve prepared above.

(Measurement of residual sugar (glucose) concentration)
The measurement of the glucose (residue sugar) concentration contained in the culture solution was measured by an absorptiometric analysis method by the somogie-Nelson method using an absorptiometer (UV mini 1240 Shimadzu Corp.). Specifically, it is as follows.
First, prepare standard solutions with glucose concentrations of 0.01, 0.05, 0.10, 0.15, 0.20 g / L, put 1 mL of each solution in a test tube, and add 1 mL of somogyous copper solution. Add and cap with marbles and heat in a boiling water bath for 10 minutes.
Then, it was quenched with running water for 5 minutes, developed color by adding 1 mL of Nelson solution, allowed to stand for 8 minutes, diluted to a total volume of 25 mL using a measuring flask, and allowed to stand for 15 minutes.
The absorbance at a wavelength of 660 nm, control solution: ion-exchanged water, optical path length: 1 cm was measured using the absorptiometer, and a calibration curve was prepared using the values of absorbance for each glucose concentration.

The measurement of the glucose concentration in the culture solution to be evaluated was evaluated using the prepared calibration curve.
Specifically, the culture solution sample to be evaluated was filtered through a membrane filter, and the filtrate was diluted with sterile ion exchange water so that the glucose concentration was within the calibration curve concentration range.
One mL of this diluted sample was developed in the same manner as the above standard solution, and absorbance was measured.
The obtained absorbance value was compared with the calibration curve prepared above and multiplied by a dilution factor to determine the glucose (residual sugar) concentration in the culture solution sample.

(1) Regeneration of yeast The above three types of yeast (Yeast: Rhodotorula genus Glaminis NBRC 0190, Yeast: Rhodosporidium sphaerocarpam NBRC 1939, distributed by the National Institute of Technology and Product Technology, Biotech Center (NBRC)) A lyophilized preparation of yeast: Pichia sp. Capsulata NBRC 1770) was recovered and used.
The restoration method can be restored by applying a known method, and specifically, for example, it was restored as follows.

The central part of a cotton plug enclosed in each freeze-dried preparation ampoule is scratched with a double-edged flat file, and the scratched ampoule is disinfected with gauze containing 75 w / w% alcohol and then wrapped in sterile gauze, I broke the ampoule and opened it.
Immediately after opening, using a Pasteur pipette, add the reagents shown in Table 1 below (add each reagent shown in Table 1 below and dilute to 1 L with ion-exchanged water, autoclave sterilize at 121 ° C for 15 minutes About 0.2 mL of the post-cooled YM liquid medium) was added to each ampoule.
Thereafter, the cells were sufficiently stirred to suspend each cell, and then YM culture medium (each reagent except for powder agar in Table 2 below was added and diluted to 1 L with ion-exchanged water and prepared) Inoculate into YM liquid medium), or YM agar slant medium and YM agar plate medium (prepared by adding each reagent in Table 2 below and diluting to 1 L with ion exchange water), 80 rpm in a 25 ° C water bath After shaking (in the case of a liquid) or standing for a fixed period (about 2 to 7 days) under the following conditions, the growth of the bacteria was confirmed and stored in a refrigerator at 5 to 8 ° C.

(2) Seed yeast used for main culture As a pre-culture medium, dissolve YM liquid medium prepared in the above (1) or mineral salt and glucose shown in the following Table 3 per liter of sterile ion-exchanged water, and autoclave sterilization (121 Using Tzapeck's liquid medium, which has been allowed to cool down for 15 minutes, and inoculating appropriate amounts (approximately 1% volume if liquid) of each of the yeasts restored in (1) into these mediums, Each culture broth cultured with shaking at 80 rpm for several days at .degree. C. was centrifuged at 3000 rpm for 15 minutes, the supernatant was removed, and the precipitated cells were suspended in sterile ion-exchanged water.
The removal of the liquid phase by centrifugation and the operation of suspension with sterile ion-exchanged water were repeated about one to two more times to remove the medium and wash the cells. The obtained final suspension was used as an inoculum (yeast) of the main culture.

(3) Influence of Sodium Chloride Concentration As shown in Table 6 below, a liquid medium simulating a Tzapek liquid medium (containing neither carbon nor nitrogen) prepared by dissolving various inorganic salts per liter of ion-exchanged water Sodium nitrate so that the nitrogen concentration ([NO ₃ ^-- N]) is 25.5 ppm or 99.8 ppm, and further adding glucose as a carbon source so that the C / N ratio is 20 Each liquid medium was prepared by adding sodium chloride such that the sodium chloride concentration was 0 to 3.0 w / w%.

The seed suspension of Rhodotorula yeast obtained in the above (2) is inoculated into 60 mL of each liquid culture medium having different sodium chloride concentrations, each at a volume of 1% of each liquid culture medium. The shaking culture was carried out at 65 rpm and the nitrate ion in each liquid medium was analyzed using the above ion analyzer to evaluate the nitrate ion removing ability.
The results are shown in FIG. 1 and FIG.

In Fig. 1, [NO ₃ ^-- N] was initially 25.5 ppm, but it decreased rapidly at all salt concentrations, and became less than 0.5 ppm which is the quantitation limit of ion chromatograph in 3 days .
Also, as shown in FIG. 2, when [NO ₃ ^-- N] is a high concentration of 112 ppm, the removal rate is slow even though the sodium chloride concentration which is the salt concentration of seawater is 3.0 w / w% It is found that the ion concentration decreases, and the nitrate ion removal rate is 95% or more at day 9 at other concentrations, and it becomes clear that this bacterium expresses nitrate ion removal ability in environmental water with a wide salt concentration. The

(4) Influence of pH As shown in Table 6 below, nitrate nitrogen concentration (in a liquid culture medium imitating a Tzapek liquid culture medium (containing neither carbon nor nitrogen) prepared by dissolving various inorganic salts per liter of ion-exchanged water) Sodium nitrate is added so that [NO ₃ ^-- N]) is 25.5 ppm or 99.8 ppm, and glucose which is a carbon source is further added so that the C / N ratio is 20, and pH of each culture medium is added. Each liquid culture medium was prepared by adding a pH buffer solution prepared from potassium dihydrogenphosphate and dipotassium hydrogenphosphate to a pH of 4.5 to 8.5.

Inoculate the seed suspension of Rhodotorula genus yeast obtained in the above (2) into 60 mL of the liquid culture medium having different pH from each at a volume of 1% of each liquid culture medium, and adjust the temperature to about 20 ° C, 65 rpm The shaking culture was carried out at the same time, and the nitrate ion in each liquid medium was analyzed using the above ion analyzer to evaluate the nitrate ion removing ability.
The results are shown in FIG. 3 and FIG.

3 and 4 than initially - 3 days in the case _[NO ³ -N] is 25.5 ppm, - at 8 days even when _[NO ³ -N] is a high concentration of 112 ppm, all ranges discussed It was found that the removal rate of nitrate ion is 98% or more at a pH of 1, and the nitrate ion removing ability is effectively exhibited at a wide pH assumed as general environmental water.

(5) Influence of carbon nitrogen (C / N) ratio (mass ratio) As shown in Table 6 below, Tzapek liquid culture medium (containing neither carbon nor nitrogen) prepared by dissolving various inorganic salts per liter of ion-exchanged water The simulated liquid medium contains sodium nitrate so that the nitrate nitrogen concentration ([NO ₃ ^-- N]) is 25, 50, 100 and 200 ppm, and glucose so that the carbon concentration is 500, 1000, 2000 and 4000 ppm. Was added to prepare each liquid medium.

Inoculate 100 mL of the liquid medium with different nitrate nitrogen concentration and glucose carbon concentration as described above with the inoculum suspension of Rhodotorula genus yeast obtained in the above (2) in 1% volume of each liquid medium. The strain was cultured at about 20 ° C. with shaking at 65 rpm, and nitrate ions in each liquid medium were analyzed using the above ion analyzer to evaluate the nitrate ion removing ability.
The results are shown in FIG.

  From FIG. 5, it is shown that the nitrate ion removing ability is effectively exhibited in all the carbon and nitrogen content ranges, and even if the concentration of nitrate nitrogen is the same, the glucose concentration is high and it is better to use a large excess to nitrate ions. The initial decrease of nitrate ion tends to increase because the initial growth rate of bacteria increases. However, since there is an upper limit to the growth density, it was found that the nitrate ion removing effect was not improved as much as the difference in carbon nitrogen ratio where the nitrate ion concentration was high.

  Based on these results, the difference between nitrate nitrogen concentration and glucose carbon concentration, and nitrate nitrogen concentration and glucose carbon concentration at the point at which the nitrate ion removal rate was around 90% was initially included in the medium. The amount of nitrogen, the amount of carbon consumed, and the carbon / nitrogen mass ratio (C / N ratio) thereof are shown in Table 4. Since the C / N ratio converges to about 20, it is understood that the C / N ratio is preferably about 18 to 30, particularly about 20 as the amount of organic carbon necessary for the nitric acid assimilation reaction. .

(6) Assimilation of nitrate ion in fresh water and artificial seawater using the above-mentioned Rhodotorula, Rhodosporidium and Pichia nitrate assimilation yeast. The Tzapek modified liquid medium (C / N ratio = 32, pH ≒ 6.9) of Table 3 prepared in (2) was prepared and used as a fresh water medium.

  Sodium nitrate and glucose are dissolved in 1 L of a solution of 33.4 g of artificial seawater for aquarium dissolved in sterile ion-exchanged water as shown in Table 5 below, and this is used as an artificial seawater medium (C / N ratio = 32, pHpH6.9).

After autoclave sterilization of 60 mL of each liquid culture medium of the above-mentioned fresh water culture medium and artificial seawater culture medium, then it was allowed to cool, 600 μL of various yeast suspensions obtained in the above (2) washed with sterile ion exchange water were inoculated respectively .
Subsequently, while shaking culture was performed at 25 ° C. and 65 rpm, the time-dependent change of the nitrate ion concentration of the culture medium was analyzed by the ion chromatograph using the above-mentioned ion analyzer.
These results are shown in FIG. 6 for the fresh water medium and in FIG. 7 for the artificial seawater medium, respectively.

It can be seen from FIGS. 6 and 7 that all three types of yeast show high nitrate assimilation.
In particular, Rhodosporidium sp. Is excellent in nitrate assimilation rate in fresh water, and in Rhodotorula sp. No change in nitrate assimilation rate is found between freshwater and seawater. In addition, Pichia can express nitrate assimilation ability in fresh water and seawater, but it is understood that Rhodotorula is more excellent in nitrate assimilation rate and higher in removing nitrate ion.

From these results, it was found that the above three types of yeast can be used for assimilation of nitrate nitrogen in ground water.
In particular, the application of Rhodotorula graminis NBRC 0190 of the genus Rhodotorula is preferred.

(7) Assimilation of nitrate ion using the above-mentioned Rhodotorula (Rhodotorula) nitrate assimilable yeast
i) Composition of Tzapek liquid medium Inorganic salts were dissolved in 1 L of ion-exchanged water as shown in Table 6 below to prepare a liquid medium containing no nitrate ion, organic component and agar. NaNO ₃ is added to this liquid culture medium so that the nitrate ion concentration becomes a desired concentration ([NO ₃ ⁻ ] = 885, 443, 221, 111, 44.3, 22.1 ppm), and nitrate nitrogen is further added. Glucose was added so that the molar ratio of organic carbon to organic carbon was C / N = 20 to prepare each tzapeck medium (pH ≒ 6.7 to 7.0).

ii) After autoclave sterilization of 60 mL of each Tzapek medium prepared in this way and then allowing to cool, 600 μL of Rhodotorula yeast suspension obtained in the above (2) was washed with sterile ion exchange water and planted Fungus.
While shaking culture was carried out at 25 ° C. and 65 rpm, the time course of the nitrate ion concentration of the culture medium was analyzed using the above-mentioned ion analyzer.
The results are shown in FIG. 8 and FIG.

  As shown in FIGS. 8 and 9, the above-mentioned Rhodotorula yeast can highly remove nitrate ions with a removal rate of 99% or more even at a low nitrate ion concentration assuming a polluted groundwater level from the high nitrate ion concentration of the polluted wastewater level I understand that.

(8) Assimilation of nitrate ion by Rhodotorula (Rhodotorula) immobilized nitrate assimilable yeast
i) Composition of Tzapek liquid medium Tzapek liquid medium similar to Table 3 prepared in the above (2) was prepared.

ii) Carrier for Immobilizing Yeast As a carrier for immobilizing the Rhodotorula genus yeast obtained in (2) above, polyvinyl alcohol (PVA) was used.

iii) Preparation of immobilized PVA disc and assimilation of nitrate ion The Rhodotorula yeast suspension obtained in (2) above is added to the Tzapek liquid culture medium in Table 3 above, and shake culture (25 ° C.) , 100 rpm).
A 10 w / w% aqueous solution of autoclaved PVA at a temperature of 115 ° C. for 15 minutes was added and mixed with a 20 v / v% amount of the aqueous PVA solution, and the 96-well microplate ( 200 μL of each was dispensed into wells of 5 mm in diameter.

Next, the above-mentioned PVA solution containing yeast suspension in a microplate was frozen at -20 ° C for 2 hours while being dispensed to a 96-well microplate, and then thawed at room temperature for 1 hour.
The procedure of freezing and thawing was repeated five times to produce the above-mentioned Rhodotorula yeast-immobilized PVA disc (about φ5 mm × 8 mm, cylindrical) having elasticity.

Twenty-five such PVA disks are placed in a 300 mL Erlenmeyer flask containing 100 mL of the above-mentioned i) Tzapek liquid medium, shake cultured (25 ° C., 100 rpm), and the medium (C / N ratio = 32, pH ≒). The time course of nitrate ion concentration in 6.9) was measured using an ion analyzer.
The results are shown in FIG.

  From FIG. 10, it can be confirmed that Rhodotorula yeast is fixed to PVA, and it is effective for reduction of nitrate nitrogen, and a removal rate of 95% or more was obtained.

(9) Assimilation of Nitrate Nitrogen in Groundwater Two types of groundwater (Kumamoto Prefecture, well water) were collected, and a removal test of nitrate nitrogen contained in the ground water was conducted.

The water quality of two types of groundwater (groundwater A and groundwater B) is as shown in Table 7 below.
The pH of each sewage is measured using a glass electrode pH meter (D-71 manufactured by Horiba, Ltd.), and the cation concentration is measured by an atomic absorption spectrometer (AA-6200 manufactured by Shimadzu Corporation), The anion concentration was measured using an ion analyzer (IA-100, manufactured by Toa DKK Co., Ltd.). Also, TOC (organic carbon) was measured using a TOC meter (TOC-5000A, manufactured by Shimadzu Corporation).

Rhodotorula (Rhodotorula) yeast was used as a yeast that assimilates nitrate nitrogen.
As a pre-culture medium, the Todopec liquid culture medium of Table 3 above is inoculated with the Rhodotorula genus yeast suspension obtained in the above (2) at a volume of 1% of the culture medium, and it is about 4 days at 20 ° C. The shaking culture was centrifuged at 3000 rpm for 15 minutes. Then, the supernatant was removed, and the precipitated cells were suspended in sterile ion-exchanged water. The removal of the liquid phase by centrifugation and the operation of suspension with sterile ion-exchanged water were repeated once or twice to remove the medium and wash the cells. The final suspension was used as an inoculum for the main culture.
In the main culture, 100 mL of groundwater A and groundwater B were treated with the following 1) to 3), and then 1 mL of inoculum suspension was inoculated respectively.

1) Each sewage was autoclaved (121 ° C, 15 minutes) and then allowed to cool.
2) After weighing in glucose so that C / N = 20 with respect to nitrate concentration in each sewage, autoclave sterilization (121 ° C., 15 minutes) was performed, and then allowed to cool.
3) On the other hand, glucose was weighed in so that C / N = 20 with respect to nitrate concentration in each sewage, and it was in a released state without sterilization.

About 100 mL of regional sewage A and B treated with the above 1) to 3), 1 mL of Rhodotorula yeast seed suspension is inoculated respectively, and shake culture is carried out at about 20 ° C. and 65 rpm. The time course of nitrate ion concentration in the medium was analyzed by the above ion analyzer. The results are shown in Table 8 (the numerical values are in ppm) and in FIG.
In addition, the residual sugar concentration was analyzed by the Somogyi-Nelson method, and the results are shown in Table 9 (the numerical values are ppm) and FIG.

From Table 8 and FIG. 11, as shown by A-1 and B-1, even when the carbon source glucose is not positively added, the carryover component of the carbon source inside the cells at the time of preculture is obtained. Although the concentration of nitrate ions decreases due to the presence of a slight amount of nitrate, the rate of nitrate ions removal slows down as the carbon source gradually becomes insufficient as a result.
On the other hand, it is understood that the nitrate ion concentration is reduced to less than 0.5 ppm in 1 to 2 days by adding glucose at a ratio of C / N = 20.

Furthermore, since the nitrate assimilation of this bacterium is an aerobic reaction, it shows an effect comparable to that in the non-sterile release state, which makes it easy to divert to a groundwater purification system.
Also, according to Table 9 and FIG. 12, glucose charged at C / N = 20 is also almost consumed as the nitrate ion is removed, and the residual sugar concentration becomes less than 3 ppm even when converted to the organic carbon concentration (TOC value). Know that

The present invention can be effectively used for the purification of nitrate nitrogen-contaminated ground water and rivers, ponds, lakes, ocean waters, etc., and it can be applied to soils polluted with nitrate nitrogen, thereby causing nitrate in the soil. It is possible to apply also to the reduction of nitrogen.

Claims (7)

  A yeast having the ability to assimilate nitrate nitrogen, a culture thereof or an extract thereof belonging to Rhodotorula graminis, Pichia capsulata or Rhodosporidium sphaerocarpum, and capable of assimilating nitrate nitrogen A method for removing nitrate nitrogen, which comprises contacting nitrate nitrogen with environmental water or soil containing nitrate nitrogen to remove nitrate nitrogen.   In the method for removing nitrate nitrogen according to claim 1, the yeast belonging to Rhodotorula graminis is Rhodotorula graminis NBRC 0190, and the yeast to which Pichia capsulata belongs is Pichia capsulata NBRC 1770, which is Rhodosporium. A method for removing nitrate nitrogen, wherein Sphaerocarpa (Rhodosporidium sphaerocarpum) is Rhodosporidium sphaerocarpam NBRC 1939.   3. The method for removing nitrate nitrogen according to claim 1, wherein the yeast belonging to Rhodotorula graminis, Pichia capsulata or Rhodosporidium sphaerocarpum, and a culture thereof A method for removing nitrate nitrogen, which comprises immobilizing the substance or its extract on a carrier and bringing it into contact with environmental water or soil containing nitrate nitrogen.   The method for removing nitrate nitrogen according to any one of claims 1 to 3, wherein environmental water or soil containing nitrate nitrogen contains carbon at a C / N ratio (mass ratio) of 15 to 30. A method of removing nitrate nitrogen, characterized in that   A yeast belonging to the genus Rhodotorula graminis, a genus Pichia capsulata or a genus Rhodosporidium sphaerocarpum and capable of assimilating nitrate nitrogen, a culture thereof or an extract thereof A nitrate nitrogen remover for environmental water or soil characterized in that   The nitrate nitrogen-removing agent according to claim 5, wherein the yeast belonging to Rhodotorula graminis is Rhodotorula graminis NBRC 0190, and the yeast belonging to Pichia capsulata is Pichia capsulata NBRC 1770, A nitrate nitrogen remover for environmental water or soil, characterized in that the yeast belonging to the genus Rhodosporidium sphaerocarpum is Rhodosporidium sphaerocarpum NBRC 1939. The nitrate nitrogen-removing agent according to claim 5 or 6, which is a yeast belonging to Rhodotorula graminis, Pichia capsulata or Rhodosporidium sphaerocarpum, and a culture thereof. Alternatively, a nitrate nitrogen removing agent for environmental water or soil characterized in that the extract is supported on a carrier and immobilized.

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