JP2014200203A - Evaluation method of bacterial floras - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an evaluation method of bacterial floras by understanding metabolic pathways of nitrogenous compounds by ammonia-oxidizing bacteria (AOB) in bacterial floras, such as sludge and soil.SOLUTION: An evaluation method of the bacterial flora according to the invention comprises a first process of supplying ammonia and oxygen to a bacterial flora, a second process of supplying nitrous acid labeled withN to the bacterial flora after the ammonia is substantially exhausted in the first process, and a third process of supplying hydroxylamine labeled withN to the bacterial flora after the second process.

Description

  The present invention relates to a method for evaluating flora such as sludge and soil.

Nitrous oxide (N ₂ O) released into the atmosphere is a substance having a greenhouse effect that is 300 times that of carbon dioxide (CO ₂ ). N ₂ O is known as one of the largest ozone-depleting substances in the 21st century that replaces chlorofluorocarbons, and the reduction of its release has become a global concern.

Until now, wastewater treatment facilities have been considered to have a low contribution as a hotbed for the release of N ₂ O. However, in recent years, it has been reported that the amount of N ₂ O released has been increasing due to progress in energy saving and cost reduction in the wastewater treatment process. For example, for N _{2 O} emission rating from wastewater treatment facilities, integrated values in the IPPC report is pointed out that not correct, N _{2 O} emissions from wastewater treatment facilities so far has been underestimated However, attention has been drawn that the level is not negligible (for example, Non-Patent Document 1).

  On the other hand, in wastewater treatment facilities, polluted components of wastewater are rendered harmless by bringing a wide variety of microorganisms into contact with the wastewater. Usually, the waste water treatment process is performed by bringing various microorganisms suspended as sludge or immobilized on a carrier into contact with waste water.

In general, the waste water contains a nitrogen compound such as ammonia (NH ₃ ). Nitrogen compounds are oxidized in wastewater treatment. In particular, ammonia oxidizing bacteria (AOB) having the property of oxidizing NH ₃ play a central role in the oxidation of nitrogen compounds in wastewater treatment.

However, AOB converts NH ₃ into ammonia nitrogen (NH ₄ ⁺ —N), hydroxylamine (NH ₂ OH), nitrite nitrogen (NO ₂ ⁻ ), nitric oxide (NO), nitrate nitrogen (NO ₃ ) ^- ) and other enzymes that oxidize and reduce, as well as enzymes involved in the pathway for producing N ₂ O from NH ₂ OH and NO, and may produce N ₂ O as a secondary. Therefore, AOB is regarded as a problem as a source of N ₂ O.

N ₂ O is also known to be released by denitrifying bacteria, and it is also generated by non-biological pathways by chemical reactions in addition to being released by biological pathways by microorganisms. Has also been said.

Until now, basic research on the release route of N ₂ O in wastewater treatment and the like has been performed. For example, Non-Patent Document 2 reports a comparison between prediction of an N ₂ O production pathway of autotrophic ammonia-oxidizing bacteria and actual research by simulation. Also, experimentally, research related to material circulation and metabolic mechanisms of microorganisms are being conducted. In material circulation, for example, Non-Patent Document 3 reports an analysis using an isotopomer utilizing a combination of isotope ratios existing in nature. On the other hand, regarding the metabolic pathway of microorganisms, for example, Non-Patent Document 4 has a report on the metabolic pathway and nitrogen circulation based on the transcriptional activity of messenger RNA. Further, Patent Document 1 discloses a waste water treatment apparatus and a waste water treatment method using AOB.

JP 2012-245479 A

Foley J., de Hass D., de Haas D., Yuan ZG., Lant P. Water Research 2010, 44 (3), 831-844 Ni BJ, Yuan ZG, Chandran K, Vanrolleghem PA, Murthy S., Biotechnology and Bioengineering 2013, 110 (1), 153-163 Wunderlin P, Lehmann MF, Siegrist H, Tuzson B, Joss A, Emmenegger L, Mohn J., Environmental Science & Technology 2012, 47 (3), 1339-1348 Yu R, Chandran K., Bmc Microbiology 2010, 10-

In recent years, partial nitrification has attracted attention as a low-cost, energy-saving treatment method for wastewater containing nitrogen. However, the partial nitrification method is achieved by finding favorable conditions (dissolved oxygen, pH, temperature, etc.) for the growth of AOB using the difference in physiological properties of AOB and nitrite-oxidizing bacteria (NOB). In comparison with the conventional method, it is difficult to suppress the generation of N ₂ O.

Furthermore, even in the above-described conventional research and examination results, the release route of N ₂ O is not necessarily grasped accurately, and it is not always sufficient how AOB contributes to the emission of N ₂ O. Not discussed.

One of the objects according to some aspects of the present invention is to provide a method for evaluating the flora by grasping the metabolic pathway of nitrogen compounds by AOB in flora such as sludge and soil. . In addition, one of the objects according to some aspects of the present invention is a method for characterizing (assessing) the flora by grasping the production route of N ₂ O, particularly by NOB, in the flora such as sludge and soil. Is to provide.

  SUMMARY An advantage of some aspects of the invention is to solve at least a part of the problems described above, and the invention can be implemented as the following aspects or application examples.

[Application Example 1] One aspect of the method for evaluating a flora according to the present invention is a first step of supplying ammonia and oxygen to the flora, and after the ammonia is substantially depleted in the first step, ¹⁵ A second step of supplying N-labeled nitrous acid to the flora, and a third step of supplying ¹⁵ N-labeled hydroxylamine to the flora after the second step.

  According to this application example, the metabolic pathway of nitrogen compounds by AOB in the flora can be grasped, and the flora can be evaluated.

  [Application Example 2] In Application Example 1, the method may further include a step of mass-analyzing a gas generated from the microflora.

  According to this application example, the metabolic pathway of the nitrogen compound by AOB in the flora can be grasped more accurately, and the flora can be evaluated.

Application Example 3 In Application Example 2, the gas may be nitrous oxide.
According to this application example, the metabolic pathway of N ₂ O by AOB in the flora can be grasped, and the N ₂ O production mechanism of the flora can be evaluated.

[Application Example 4] In any one of Application Examples 1 to 3, the method may further include a step of quantitatively analyzing genes expressed in the flora.

  According to this application example, the metabolic pathway of nitrogen compounds by AOB in the bacterial flora is analyzed by combining the measurement results by isotopes and the measurement results by the expression level of functional genes. It is possible to grasp which is dominant and to evaluate the flora more accurately.

  Application Example 5 In Application Example 4, the gene may encode an enzyme involved in nitrogen oxidation and / or reduction.

  According to this application example, in the flora, it is possible to grasp which metabolic pathway of nitrogen compounds by AOB has priority in the biological route and the chemical route, and more accurately evaluate the flora. can do.

According to the present invention, the above-described complicated N ₂ O production pathway can be identified by using stable isotopes. In addition, according to the present invention, it is possible to confirm the contribution of the generation of N ₂ O through a biological pathway by quantifying messenger RNA (mRNA) encoding an enzyme responsible for a reaction involving nitrogen. Therefore, effective information for taking measures for suppressing the occurrence of N ₂ O can be obtained for the flora evaluated according to the present invention.

The schematic diagram of the metabolic pathway of the nitrogen compound by AOB. The plot of the density | concentration for every kind of nitrogen compound which concerns on an experiment example. Plot for _{N 2} O of the fraction time including the ¹⁵ N according to the experimental example. Integration plots for ^{_{14 N 2 O, 15 N 14}} NO or ¹⁴ N ¹⁵ NO and ¹⁵ N ₂ O time according to experimental examples. Map showing schematically the N _{2 O} production pathway according to experimental examples. The plot with respect to time of the relative expression level of the functional gene which concerns on an experiment example.

  Several embodiments of the present invention will be described below. Embodiment described below demonstrates an example of this invention. The present invention is not limited to the following embodiments, and includes various modified embodiments that are implemented within a range that does not change the gist of the present invention. Note that not all of the configurations described below are essential configurations of the present invention.

1. Method for evaluating the flora The method for evaluating the flora according to this embodiment includes a first step of supplying ammonia and oxygen to the flora, and after the ammonia is substantially depleted in the first step, ¹⁵ N A second step of supplying labeled nitrous acid to the flora, and a third step of supplying hydroxylamine labeled with ¹⁵ N to the flora after the second step.

  FIG. 1 is a flowchart showing an example of specific steps of the evaluation method according to the present embodiment.

1.1. Bacterial flora The bacterial flora to which the evaluation method of the present embodiment is applied is not particularly limited. As used herein, a flora refers to a group of bacteria that grow in a specific environment, or a microbiota.
flora). Specific examples of the bacterial flora to which the evaluation method of the present embodiment is applied include sludge (activated sludge), soil, microbial flora of animal bodies and epidermis, and the like, which exist as a collection of bacteria in the global environment. If it is a thing, it will not specifically limit. Part or all of the flora can be collected, and this can be evaluated by the evaluation method of the present embodiment.

1.2. 1st process The evaluation method of the microflora which concerns on this embodiment contains a 1st process. The first step is a step of supplying ammonia and oxygen to the flora.

This step is performed, for example, in a state where the flora to be evaluated is introduced into an iterative batch type or continuous tank type bioreactor by an appropriate method. Such a bioreactor is not particularly limited, but if it is of the partial nitrification type, since AOB that plays a central role in the production of N ₂ O is accumulated, the accuracy of the evaluation method is improved. This is more preferable. There is no particular limitation on the volume of the bioreactor. The bacterial flora introduced into the bioreactor may be dispersed in water or appropriately stirred. Moreover, it is more preferable that the bioreactor of the present embodiment has a configuration in which a gas generated from the introduced flora or a dispersion thereof and a part of the flora can be collected. Moreover, it is more preferable that the bioreactor has appropriate airtightness from the viewpoint of improving the accuracy of analysis of collected gas and the like. The bioreactor may be provided with an aeration mechanism. When aeration is performed, oxygen, air, or the like can be used, and the flow rate is not particularly limited. Moreover, before this process is performed, the bacterial flora may be trained as appropriate in the bioreactor.

In this step, ammonia and oxygen are supplied to the bioreactor. Ammonia may be supplied as ammonia nitrogen (NH ₄ ⁺ -N). Oxygen may be supplied as high-purity oxygen gas or air. Ammonia and oxygen may be supplied to the bioreactor as they are, or may be supplied in a mode such as bubbling. Moreover, when performing this process, it is more preferable to stir the inside of a bioreactor as uniformly as possible from a viewpoint of improving the accuracy of evaluation.

  In the evaluation method of this embodiment, this step has a role of confirming whether ammonia is metabolized by the flora. The amount of ammonia supplied to the bioreactor in this step is, for example, 10 mg-N / L or more and 5,000 mg-N / L or less, preferably 10 mg-N / L or more and 1,000 mg- with respect to 1 g of the bacterial flora. N / L or less. Oxygen supplied to the bioreactor in this step may be supplied continuously, for example, 0.1 mg / L or more and 7.0 mg / L or less, preferably 1.0 mg / L with respect to 1 g of the bacterial flora. The density may be adjusted to L or higher. Furthermore, the volume fraction of oxygen with respect to ammonia supplied to the bioreactor in this step is, for example, 2.0 or more and 5.0 or less. If the volume fraction is in the range of 3.43 or more and 4.32 or less, a stoichiometric analysis may be performed.

  The temperature in this step is not particularly limited as long as metabolism by the bacterial flora can be expected. For example, the temperature can be 25 ° C. or more and 35 ° C. or less. Similarly, the pH during this step is not particularly limited as long as metabolism by the flora can be expected, but it can be set to, for example, 6.0 or more and 8.8 or less. Further, when the predetermined pH fluctuates in this step, the pH may be adjusted with, for example, a buffering agent (buffer). In this step, the dissolved oxygen (DO) concentration may be appropriately adjusted as long as metabolism by the flora can be expected.

  When the bacterial flora has the ability to metabolize ammonia, the supplied ammonia is reduced in the system in this step.

1.3. Second Step The method for evaluating a flora according to the present embodiment includes a second step. The second step is a step of supplying nitrous acid labeled with ¹⁵ N to the bacterial flora.

  The second step is performed after ammonia is substantially depleted in the first step. In the present specification, the state in which ammonia is substantially depleted means that the state in which ammonia has completely disappeared and the state in which metabolism by the flora does not occur or ammonia remains to a late degree are included.

As described above, when the bacterial flora has the ability to metabolize NH ₄ , in the first step, the supplied NH ₄ is reduced in the system, and NH ₄ is substantially depleted. Thereafter, the second step is performed.

In this step, nitrous acid labeled with ¹⁵ N may be supplied to the flora in the state of nitrite nitrogen ( ¹⁵ NO ₂ ⁻ ). ^As nitrous acid labeled with ¹⁵ N, for example, those commercially available from OO Co., Ltd. can be used. Nitrous acid may be supplied as it is into the bioreactor, or may be supplied in a manner such as bubbling. Moreover, when performing this process, it is more preferable to stir the inside of a bioreactor as uniformly as possible from a viewpoint of improving the accuracy of evaluation.

  In the evaluation method of this embodiment, this step has a role of confirming whether nitrous acid is metabolized by the flora. The amount of nitrous acid supplied to the bioreactor in this step is, for example, 10 mg-N / L or more and 5,000 mg-N / L or less, preferably 10 mg-N / L or more and 1,000 mg with respect to 1 g of the bacterial flora. -N / L or less.

  The temperature of this step is not particularly limited as long as metabolism by the flora can be expected, but can be, for example, 25 ° C. or more and 35 ° C. or less. Similarly, the pH during this step is not particularly limited as long as metabolism by the flora can be expected, but it can be set to, for example, 6.0 or more and 8.8 or less. Further, when the predetermined pH fluctuates in this step, the pH may be adjusted with, for example, a buffering agent (buffer). In this step, the dissolved oxygen (DO) concentration may be appropriately adjusted as long as metabolism by the flora can be expected.

  In the case where the flora has the ability to metabolize nitrous acid, the supplied nitrous acid is reduced in the system in this step. Further, when the bacterial flora does not have the ability to metabolize nitrous acid, the supplied nitrous acid remains in the system in this step.

1.4. Third Step The method for evaluating a flora according to the present embodiment includes a third step. The third step is a step of supplying hydroxylamine labeled with ¹⁵ N to the flora. The third step is performed after the second step.

  This step can be performed even when the nitrous acid supplied in the second step is substantially depleted or when the nitrous acid remains.

In this step, as the hydroxylamine ( ¹⁵ NH ₂ OH) labeled with ¹⁵ N, for example, a reagent commercially available from OO Co., Ltd. can be used. Hydroxylamine may be supplied as it is into the bioreactor or may be supplied in a manner such as bubbling. Moreover, when performing this process, it is more preferable to stir the inside of a bioreactor as uniformly as possible from a viewpoint of improving the accuracy of evaluation.

  In the evaluation method of this embodiment, this step has a role of confirming whether hydroxylamine is metabolized by the bacterial flora. The amount of hydroxylamine supplied to the bioreactor in this step is, for example, 0.001 mg-N / L or more and 0.1 mg-N / L or less, preferably 0.001 mg-N / L with respect to 1 g of the bacterial flora. The above is 0.02 mg-N / L.

  The temperature of this step is not particularly limited as long as metabolism by the flora can be expected, but can be, for example, 25 ° C. or more and 35 ° C. or less. Similarly, the pH during this step is not particularly limited as long as metabolism by the flora can be expected, but it can be set to, for example, 6.0 or more and 8.8 or less. Further, when the predetermined pH fluctuates in this step, the pH may be adjusted with, for example, a buffering agent (buffer). In this step, the dissolved oxygen (DO) concentration may be appropriately adjusted as long as metabolism by the flora can be expected.

  When the flora has the ability to metabolize hydroxylamine, in this step, the supplied hydroxylamine is reduced in the system. Moreover, when the bacterial flora does not have the ability to metabolize hydroxylamine, the supplied hydroxylamine remains in the system in this step.

1.5. Other steps 1.5.1. Step of Mass Spectrometry The method for evaluating the flora of this embodiment may further include a step of mass spectrometry. This step is performed by introducing gas and liquid generated from the flora into an apparatus having a mass spectrometry function. This step can be performed in at least one of the first step, the second step, and the third step. Moreover, this process may be performed simultaneously with all the processes through the 1st process, the 2nd process, and the 3rd process. Furthermore, this step may be performed continuously, for example, when the first to third steps are performed in a continuous tank type bioreactor, or performed in a repetitive batch type bioreactor. In some cases, it may be performed intermittently. When this step is performed intermittently (intermittently), it is more preferable that the step be performed a plurality of times at least in each step of the first step, the second step, and the third step. In this way, it becomes easier to track the evolution of the generated gas in each step of the first step, the second step, and the third step.

This step can be performed, for example, by collecting the gas phase in the bioreactor by an appropriate sampling method and introducing it into an apparatus having a mass spectrometry function. The mass spectrometric function is not particularly limited as long as it has a mass resolution capable of separately detecting a nitrogen compound labeled with ¹⁵ N and a nitrogen compound that is the same type of nitrogen compound but not labeled. In addition to the mass analysis function, the apparatus having the mass analysis function may have a fractionation function (for example, chromatography). According to such an apparatus, quantitative analysis can be performed in addition to mass spectrometry. Specific examples of the apparatus used in this step include gas chromatography / mass spectroscopy (GC / MS). In addition, as a device used in this step, a device having a quadrupole type MS of GC / MS is more preferable because it can perform quantitative analysis of multiple components simultaneously with high sensitivity.

Information on the identification of chemical species and their abundance obtained by this step is plotted against time, for example. Thereby, the change of the chemical species in each process of a 1st process, a 2nd process, and a 3rd process can be tracked. As the chemical species to be measured in this step is not particularly limited, the chemical species of the type measured in at least the second and third steps, contains N 2 _O, 15 ^NNO and ^{15 N} 2 _O More preferably. That is, by measuring nitrous oxide having a molecular weight (mass number) of 44, 45 and 46, nitrous oxide contained in the gas generated from the flora is derived from nitrous acid supplied in the second step. Information on whether nitrogen is contained or whether nitrogen is derived from the hydroxylamine supplied in the third step can be obtained. Thereby, more useful information for specifying the production route of nitrous oxide can be obtained.

Regarding the quantification of dissolved N ₂ O obtained in this step, for example, a 5 mL water sample collected from a liquid sampling port is placed in a 50 mL gas chromatography vial pre-sealed with 0.1 mL of chloroform and stirred vigorously. Then, after standing at 30 ° C. for 2 hours, it can be quantified by measuring the N ₂ O concentration in the gas phase that has reached vapor-liquid equilibrium by gas chromatography.

1.5.2. Step of quantitatively analyzing genes The method for evaluating the flora of this embodiment may further include a step of quantitatively analyzing genes. This step is a step of performing quantitative analysis by amplifying a gene generated in the bacterial flora by a PCR method (Polymerase Chain Reaction). The gene of interest is not particularly limited, but is preferably a gene encoding an enzyme involved in nitrogen oxidation and / or reduction. Further, the gene may be DNA or RNA as long as it encodes the enzyme. In the case of amplifying RNA, a reverse transcription PCR method can be employed. Furthermore, among the genes encoding enzymes, messenger RNA (mRNA) has a positive correlation between its amount and the expression level of the enzyme, so the amount of enzyme expressed in the flora Can be measured from the quantification result of mRNA.

  The step of quantitatively analyzing a gene such as mRNA can be performed in at least one of the first step, the second step, and the third step. In addition, this step may be performed simultaneously with all the steps through the first step, the second step, and the third step. Furthermore, this step may be performed continuously, for example, when the first to third steps are performed in a continuous tank type bioreactor, or performed in a repetitive batch type bioreactor. In some cases, it may be performed intermittently (intermittently). When this process is performed intermittently, it is more preferable to perform this process a plurality of times at least in each process of the first process, the second process, and the third process. This makes it easier to track the expression level of the enzyme in each step of the first step, the second step, and the third step.

The enzymes to be quantified include AMO: ammonia oxidase (an enzyme that oxidizes ammonia to hydroxylamine), HAO: hydroxylamine oxidoreductase (an enzyme that oxidizes hydroxylamine to nitrite), NIR: nitrite reductase (suboxide) From enzymes that reduce nitric acid to nitric oxide), NOR: nitric oxide reductase (an enzyme that reduces nitric oxide to nitrous oxide), and NXR: nitrite oxidase (an enzyme that oxidizes nitrite to nitric acid) And at least one selected from the group consisting of: Among these enzymes, AMO: ammonia oxidase (enzyme that oxidizes ammonia to hydroxylamine), HAO: hydroxylamine oxidoreductase (enzyme that oxidizes hydroxylamine to nitrite), NIR: nitrite reductase (nitrite) By obtaining quantitative information about an enzyme that reduces NO to nitric oxide) and NOR: nitric oxide reductase (an enzyme that reduces nitric oxide to nitrous oxide), for example, N ₂ O is a biological pathway It can be determined by which of the chemical pathways it occurs.

  This step can be performed, for example, by collecting a part of the bacterial flora in the bioreactor by an appropriate sampling technique and amplifying DNA, RNA, or mRNA contained therein by the PCR method. Primers and reagents used in the PCR method are complementary to the base sequence to be amplified (base sequence corresponding to the target enzyme) and specific for the functional gene of AOB. be able to. For example, if the base sequence corresponding to the enzyme to be quantified is known, an oligonucleotide having a sequence corresponding to this can be used as a primer. The amplified gene can be quantified as appropriate by spectroscopic analysis or the like.

  In this method for evaluating the flora, if this step is adopted, RNA is extracted from the flora sample, RNA is converted into DNA by a reverse transcription reaction, and the amount of expressed mRNA is quantified. It can be measured by PCR or the like.

  Information about the expression level of the enzyme obtained by this step is plotted against time, for example. Thereby, the change of the activity of each enzyme in each process of a 1st process, a 2nd process, and a 3rd process is traceable. Further, the gene quantified in this step is more preferably mRNA encoding the above-mentioned AMO, HAO, NIR and NOR. By quantifying these mRNAs, it is possible to identify the biological production pathway of nitrous oxide in the flora and to determine the activity of the biological production pathway (relative difference from the activity of the chemical production pathway). ) Can be obtained.

1.5.3. Process order, etc. In the evaluation method of this embodiment, the first process, the second process, and the third process described above are not necessarily performed in this order. For example, the third step may be performed after the first step, or the first step may be performed after the second step and the third step. In the evaluation method of the present embodiment, the first step, the second step, and the third step may each be performed a plurality of times. These steps can be performed in combination in an appropriate order so that desired information can be obtained.

1.6. Effects According to the method for evaluating the flora according to the present embodiment, a complex N ₂ O production pathway can be identified by using stable isotopes. In addition, according to the evaluation method of the present embodiment, the contribution of the generation of N ₂ O through the biological pathway is quantified using a messenger RNA (mRNA) encoding an enzyme responsible for a reaction involving nitrogen. Can be confirmed. By analyzing these information in combination, a dominant route can be specified in the N ₂ O generation route. Therefore, effective information for taking measures for suppressing the occurrence of N ₂ O can be obtained for the flora evaluated according to the present invention.

2. Experimental Examples The experimental examples are shown below to further explain the present invention, but the present invention is not limited to the following experimental examples.

2.1. Sample Activated sludge was collected from a laboratory-scale reaction tank where ammonia was removed for a long period of time using a reaction tank in which biological treatment of landfill leachate was biologically treated as seed sludge. The activated sludge was used after long-term training of AOB. Artificial wastewater was used as the liquid treated by each activated sludge. As drainage, (NH ₄ ) ₂ SO ₄ 2830 mg, MgSO ₄ · 7H ₂ O 280 mg, KH ₂ PO ₄ 27 mg, CaCl ₂ · 2H ₂ O 120 mg, NaCl 600 mg, FeSO ₄ · 7H ₂ O 3.3mg, MnSO ₄ · _{H 2} O and 3.3mg, CuCl ₂ · 2H ₂ O and 0.8mg, ZnSO ₄ · 7H ₂ O and 1.7 mg, and 0.3mg of NiSO ₄ · 6H ₂ O, What was dissolved in 1 L of distilled water was used. Moreover, before making it contact with activated sludge, it confirmed that the said wastewater contained ammonia.

2.2. Bioreactor A self-made batch bioreactor was made. The bioreactor was equipped with a stirring device, a gas inlet, an aeration mechanism, and a gas phase and liquid phase sampling mechanism. The volume of the bioreactor was 5 L, and a temperature controller was installed to keep the system at 28 ° C. throughout the following experiment.

2.3. Analysis The sampled gas phase was introduced into Shimadzu Corporation model: GCMS-QP2010 PPplus, and the components were qualitatively quantified. The sampled liquid phase was subjected to solid-liquid separation, RNA was extracted from sludge, reverse transcription reaction was performed to synthesize DNA, and then the functional gene of mRNA was quantified by quantitative PCR. And mRN encoding each enzyme
The relative expression level of A was calculated with reference to before the start of the experiment.

2.4. Nitrogen Compound Metabolic Path FIG. 1 shows the nitrogen compound reaction path when AOB is present in the activated sludge. In FIG. 1, arrows indicate biological metabolic pathways by AOB, wavy arrows indicate chemical reaction pathways, and alternate long and short dash arrows indicate the flow of electrons involved in the reaction. Abbreviations in the figure are AMO: ammonia oxidase (enzyme that oxidizes ammonia to hydroxylamine), HAO: hydroxylamine oxidoreductase (enzyme that oxidizes hydroxylamine to nitrite), NIR: nitrite reductase (suboxide) With an enzyme that reduces nitrate to nitric oxide), NOR: Nitric oxide reductase (an enzyme that reduces nitric oxide to nitrous oxide), and NXR: Nitrite oxidase (an enzyme that oxidizes nitrite to nitric acid) is there. The parentheses attached to these abbreviations are functional genes corresponding to the respective enzymes. Referring to FIG. 1, as a mechanism of N ₂ O generation, focusing on where two Ns come from, (1) 2NH ₂ OH → N ₂ O (aerobic conditions), (2) 2NO ₂ ⁻ Focusing on the three routes of → N ₂ O (anaerobic conditions) and (3) NH ₂ OH + NO ₂ ⁻ → N ₂ O, N ₂ from an activated sludge tank intended for partial nitrification using a stable isotope It will be understood that there is a possibility that the generation route of O can be grasped.

2.5. Experimental conditions First, activated sludge was put into artificial wastewater (400 mg-N / L) containing ammonia, and a nitrification batch test was conducted. Ammonia oxidation was performed while supplying oxygen (corresponding to the first step described above) (Phase I in FIG. 2). During this time, the gas phase is sampled approximately every hour to qualitatively determine the generated gas (ammonia (NH ₄ ⁺ ), nitrous acid (NO ₂ ⁻ ), nitric acid (NO ₃ ⁻ ), nitrous oxide (N ₂ O)). Analysis was carried out.

Next, in the result of qualitative quantitative analysis, after confirming that NH ₄ ⁺ was substantially depleted, ¹⁵ NO ₂ labeled with ¹⁵ N was supplied to the system (corresponding to the second step described above). (Phase II in FIG. 2). Similarly to Phase I, the gas phase is sampled approximately every hour during this time to generate gas (ammonia (NH ₄ ⁺ ), nitrous acid (NO ₂ ⁻ ), nitric acid (NO ₃ ⁻ ), nitrous oxide (N ₂ O )) Qualitative quantitative analysis was conducted.

Next, in the result of qualitative quantitative analysis, after the amount of nitrogen compound in the system was stabilized, ¹⁵ NH ₂ OH labeled with ¹⁵ N was supplied to the system (corresponding to the third step described above) ( Phase III in FIG. 2). In the same manner as Phase I and II, the gas phase is sampled about every 0.5 hour during this period to generate gas (ammonia (NH ₄ ⁺ ), nitrous acid (NO ₂ ⁻ ), nitric acid (NO ₃ ⁻ ), nitrous oxide. Qualitative quantitative analysis of (N ₂ O)) was performed.

  During these steps, activated sludge is collected approximately every 1 hour, RNA extraction, DNA synthesis by reverse transcription reaction, quantitative PCR are performed, and the mRNA level of the functional gene of the enzyme that catalyzes each pathway. The expression level of was monitored.

  The batch test as described above was conducted under the respective conditions with the dissolved oxygen concentration being 7.0 mg / L, 1.5 mg / L, and 0.3 mg / L. In addition, the pH of the sample in each test was continuously monitored, and automatic adjustment was performed as needed so that each test might be 7.8.

2.6. Experimental Results FIG. 2 to FIG. 4 are the results of plotting changes in the concentration of each nitrogen compound against time in a batch test with a dissolved oxygen concentration of 1.5 mg / L. FIG. 2 is a plot of the concentration for each nitrogen compound type, FIG. 3 is a plot of the fraction of N ₂ O containing stable isotope ¹⁵ N in the generated N ₂ O versus time, and FIG. , ¹⁴ N ₂ O (molecular weight 44), ¹⁵ N ¹⁴ NO or ¹⁴ N ¹⁵ NO (molecular weight 45) and ¹⁵ N ₂ O (molecular weight 46) in detected N ₂ O are integrated plots with respect to time. In addition, in FIG. 2 to FIG.

Looking at Figure 2, in phase I, in any of the activated sludge AOB are integrated (batch tests other than the dissolved oxygen concentration 1.5 mg / L are not shown.), NO ₂ ^- accumulation observed Thus, it can be seen that the generation of NO ₃ ⁻ is about 10% of the whole.

Further, FIG. 2 revealed that N ₂ O was released in the process of consuming NH ₄ ⁺ . Since the supply of ¹⁵ N is not performed at the time of Phase I, the generated molecular weight is 44 of ¹⁴ N ¹⁴ NO. NH ₄ ⁺ after depletion, NO ₂ labeled with ¹⁵ N in Phase II ^- was added and the peak of _{N 2} O is not observed, _{N 2} O at this time were found not to occur. From this, it can be understood that there is almost no generation of N ₂ O (2NO ₂ ⁻ → N ₂ O) by the denitrification route.

Next, in Phase III, ¹⁵ NH ₂ OH was added in a state where NO ₂ ⁻ was sufficiently accumulated and NH ₂ OH was consumed, and a large peak of N ₂ O was rapidly observed. The rapid generation of N ₂ O in Phase III was similarly observed in all experimental systems having dissolved oxygen concentrations of 7.0 mg / L, 1.5 mg / L, and 0.3 mg / L.

Referring to FIG. 3, the mass percentage of ¹⁵ N of N ₂ O generated when ¹⁵ NH ₂ OH was added was 58.9%. On the other hand, the ¹⁵ N ratio of NH ₂ OH at that time was 100% by mass, and the ¹⁵ N ratio of NO ₂ ^— was 11% by mass. This result regarding the mass% of ¹⁵ N can be explained rationally assuming that the generation of N ₂ O is generated by the reaction of ¹⁵ NH ₂ OH and ¹⁴ NO ₂ ^— one molecule at a time. Therefore, it was found that N ₂ O from a reactor intended for partial nitrification as a target can be explained as being produced by reacting N with a hybrid from one molecule each of NH ₂ OH and NO ₂ ^— . In addition, in the figure, although N ₂ O other than the molecular weight 44 was not detected until Phase II, ¹⁵ NH ₂ OH was supplied at the start of Phase III, and N ₂ O having a molecular weight of 45 and 46 was detected. I found out. The above results are summarized in FIG.

FIG. 5 is a map schematically showing an N ₂ O production path for explaining the above results. As shown in FIG. 5, the proportion of the N ₂ O production pathway is 55.0% for the hydroxylamine nitrite oxidation pathway (non-biological pathway), the hydroxylamine O ₂ oxidation pathway and the N ₂ O ₂ H It was found that the total of ₄ oxidation or NOH oxidation pathways was 33.1%, and NO O ₂ oxidation pathway was 11.9%. These values could be calculated by integrating the amounts of N ₂ O having molecular weights of 44, 45, and 46 in Phase III shown in FIG.

  By grasping such a map, it was possible to grasp the outline of the reaction route of the activated sludge used.

On the other hand, the formation of hybrid N ₂ O was confirmed by evaluation with a stable isotope, but the judgment as to whether this was through a biological route or a chemical route was as described above. It is still not clear enough in the experimental system. That is, in the above experimental system, although the flow of a substance can be traced by analysis using a stable isotope, further information is required to classify chemical and biological reactions.

Therefore, the functional gene (mRNA) of AOB was quantified. FIG. 6 (a) shows the relative expression level of each functional gene in a batch test with a dissolved oxygen concentration of 7.0 mg / L (mRNA expression level before starting the experiment by adding ammonia artificial waste water is 1). It is the graph plotted against. FIG. 6 (b) shows the relative expression level of each functional gene in a batch test with a dissolved oxygen concentration of 7.0 mg / L (mRNA expression level before the start of the experiment by adding ammonia artificial drainage is 1) over time. It is the graph plotted against. 6 (a) and 6 (b) are tests of the same dissolved oxygen concentration, but are intended for activated sludge having different types and activities of AOB. In the case of the test of FIG. 6 (b), AOB Systems with low activity were targeted. The mRNA corresponding to each enzyme is shown in the figure as amoA for AMO, haoA for HAO, irK for NIR, and norB for NOR. FIG. 6 shows the above-described phases.

Looking at FIG. 6 (a), when comparing the relative value of mRNA related to the functional gene of AOB (mRNA expression level before starting the experiment by adding ammonia artificial drainage was 1), the mRNA expression level of activated sludge was It was found that the fluctuation was more dynamic (maximum: relative expression level 148 times at mirK), and the high N ₂ O generation amount and behavior were consistent. On the other hand, in the case where the activity of AOB is extremely low (FIG. 6 (b)), the movement of mRNA was extremely slow and no significant change was observed. Thus, there is a high correlation between the function of mRNA and the production of N ₂ O, and whether the biological pathway or non-biological pathway (chemical pathway) is the more dominant experimental system. It turned out to be a tool for evaluation.

Based on the above experimental results, an accurate N ₂ O generation pathway in the flora can be obtained by fusing analysis of the N ₂ O production pathway using stable isotopes and quantitative analysis of mRNA serving as an index representing the activity of ammonia oxidizing bacteria. It was found that it is possible to know the biological or non-biological N ₂ O production pathway accurately.

From the results of the above experimental examples, the present invention can clarify the release route of N ₂ O in all wastewater treatment facilities and the flora of soil and the like, and aimed at suppressing the release of N ₂ O into the environment. It turns out that it can show tactics. Further, the present invention can be applied to, for example, development of a mathematical model related to a generation path. According to the present invention, for example, it is possible to provide information to be fed back to the operation management of a wastewater treatment facility and the quality control of soil, and specific measures for reducing the generation of N ₂ O without requiring a long-term experiment. Can be found.

The present invention includes a technique that makes it possible to accurately grasp the N ₂ O production path by AOB. Therefore, it becomes possible to establish clear tactics for identifying the exact N ₂ O release route from wastewater treatment and reducing the release amount, and to contribute to the reduction of greenhouse gas emissions from wastewater treatment facilities. Can do. The present invention can be applied not only to wastewater treatment facilities but also to any environment in the natural environment. For example, it can be applied to various industrial, engineering, and agricultural fields such as soil (field) serving as a hotbed for N ₂ O generation, landfill final disposal site, paddy field, etc. it is possible to clarify the _{2 O} emission path, thereby contributing like assembly strategy inhibition of release of N _{2 O} to the environment.

  The present invention is not limited to the above-described embodiments, and various modifications can be made. For example, the present invention includes substantially the same configuration (for example, a configuration having the same function, method and result, or a configuration having the same purpose and effect) as the configuration described in the embodiment. In addition, the invention includes a configuration in which a non-essential part of the configuration described in the embodiment is replaced. In addition, the present invention includes a configuration that exhibits the same operational effects as the configuration described in the embodiment or a configuration that can achieve the same object. In addition, the invention includes a configuration in which a known technique is added to the configuration described in the embodiment.

Claims (5)

A first step of supplying ammonia and oxygen to the flora;
In the first step, after the ammonia is substantially depleted, a second step of supplying ¹⁵ N-labeled nitrous acid to the flora;
After the second step, a third step of supplying hydroxylamine labeled with ¹⁵ N to the flora;
A method for evaluating a bacterial flora. In claim 1,
A method for evaluating a flora further comprising a step of mass-analyzing a gas generated from the flora. In claim 2,
The method for evaluating a flora, wherein the gas is nitrous oxide. In any one of Claims 1 thru | or 3,
A method for evaluating a flora further comprising a step of quantitatively analyzing a gene expressed in the flora. In claim 4,
The method for evaluating a bacterial flora, wherein the gene encodes an enzyme involved in oxidation and / or reduction of nitrogen.