A novel microorganism capable of reducing the concentration of ammoniacal nitrogen in wastewater

Abstract

To provide a desired microorganism capable of processing waste water containing a high concentration of ammoniacal nitrogen, which has been difficult to treat with conventional microorganisms.SOLUTION: Provided is a microorganism deposited under Accession No. FERM P-22400. The microorganism has the ability to reduce the concentration of ammoniacal nitrogen in waste water.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a novel microorganism capable of reducing the concentration of ammoniacal nitrogen in wastewater. [Background technology]

[0002] In recent years, technologies for treating wastewater from livestock manure, food waste, and the like have been developed. However, such wastewater contains high concentrations of ammoniacal nitrogen, and therefore measures to deal with such high concentrations of ammoniacal nitrogen are required in the treatment of the wastewater. In addition to technologies for treating the wastewater itself, technologies have also been developed to generate electricity (biogas power generation) using methane gas extracted from wastewater through methane fermentation. However, because the liquid after methane fermentation (methane fermentation digestate) also contains high concentrations of ammoniacal nitrogen, measures to deal with the high concentrations of ammoniacal nitrogen are also required in the treatment of the methane fermentation digestate.

[0003] Here, because plants such as those of the Grass family can absorb ammoniacal nitrogen, one example of a measure against ammoniacal nitrogen is to use wastewater (methane fermentation digestive liquid) as liquid fertilizer and apply it to rice and feed crop fields. In particular, when applying fertilizer to paddy fields, the liquid fertilizer can be mixed in from the water inlet, making the process relatively simple. However, since the time and frequency of fertilization are limited, there are issues in terms of how to utilize the wastewater (methane fermentation digestate) throughout the year.

[0004] On the other hand, with regard to methane fermentation digestate (ammonia nitrogen), technology utilizing algae has been developed (Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2018-61452 A [Patent Document 2] JP 2019-150821 A Summary of the Invention [Problem to be solved by the invention]

[0006] However, since the technology described in Patent Document 1 involves culturing microalgae after diluting the methane fermentation digestive liquid, there is a problem that a large amount of dilution water of the methane fermentation digestive liquid will be generated when trying to put this technology into practical use (see [Claim 1] and

[0025] of Patent Document 1). Furthermore, this technology only describes that algae are cultivated in the methane fermentation digestive liquid, i.e., that they can survive in the methane fermentation digestive liquid, but there is no description regarding the treatment of ammonia nitrogen in the methane fermentation digestive liquid. Furthermore, the technology described in Patent Document 2 is a technology in which ammoniacal nitrogen (first digestive liquid) itself is treated (oxidized, nitrified) to convert it into nitrite nitrogen or nitrate nitrogen using nitrifying bacteria, and the only thing that is described is that algae are cultivated in the liquid after treatment (second digestive liquid) (see

[0024] to

[0028] in Patent Document 2).

[0007] In addition, since it is generally known that ammoniacal nitrogen inhibits the growth of microorganisms at high concentrations, it has been difficult to treat wastewater containing high concentrations of ammoniacal nitrogen in its undiluted form without diluting it.

[0008] As a result of extensive research, the present inventors have succeeded in isolating a novel microorganism (algae) that has the ability to reduce the concentration of ammonia nitrogen in wastewater in its undiluted, undiluted state. In other words, this invention has been made in consideration of the above-mentioned problems, and aims to provide a novel microorganism (algae) capable of treating wastewater containing high concentrations of ammonia nitrogen, which was difficult to treat using conventional microorganisms. [Means for solving the problem]

[0009] In order to achieve the above object, claim 1 of the present invention is a microorganism deposited under accession number FERM P-22400, which has the ability to reduce the concentration of ammoniacal nitrogen in wastewater.

[0010] A second aspect of the present invention relates to the microorganism according to the first aspect, wherein the wastewater is a methane fermentation digestive liquid.

[0011] A third aspect of the present invention relates to a method for treating wastewater, comprising supplying the microorganism according to the first or second aspect to the wastewater. Effect of the Invention

[0012] According to the present invention, by using the novel microorganism (algae) identified by the accession number FERM P-22400, it is possible to treat wastewater containing high concentrations of ammoniacal nitrogen, which was difficult to treat using conventional algae. Specifically, it is believed that the novel microorganism (algae) of the present invention reduces the concentration of ammoniacal nitrogen in wastewater by taking in the ammoniacal nitrogen into its body.

[0013] It is believed that a portion of the ammonia nitrogen taken into the body of the novel microorganism (algae) is synthesized into amino acids using the energy obtained by the photosynthesis performed by the novel microorganism (algae). Furthermore, it is believed that a portion of the amino acids will form proteins starting from these amino acids, which will then become part of the body of the novel microorganism (algae). Therefore, the novel microorganism (algae) of the present invention can also be used as a means for producing high value-added components such as amino acids from wastewater containing ammonia nitrogen.

[0014] Furthermore, when the wastewater is a methane fermentation digestate (in the case of biogas power generation), the ammonia nitrogen concentration in the methane fermentation digestate can be reduced, so the treated liquid can be returned to the methane fermentation tank and can be reused as recycled water during methane fermentation. Furthermore, during methane fermentation, if the novel microorganisms (algae) that have taken up ammonia nitrogen are removed from the methane fermentation digestive liquid system by solid-liquid separation or the like, inhibition of methane fermentation in the fermenter can be suppressed, and the production efficiency of methane gas (energy) can be improved.

[0015] The novel microorganisms (algae) that have absorbed ammonia nitrogen can also be used as nitrogen fertilizer by mixing them into soil. However, because the novel microorganisms (algae) of the present invention perform photosynthesis using chlorophyll contained in their bodies, just like common algae, they fix carbon dioxide contained in the atmosphere or in the exhaust gases from biogas power generation in the form of organic matter within their bodies. Therefore, the novel microorganisms (algae) that have been separated from the solid-liquid mixture can also be reused as a raw material for biogas power generation. [Brief description of the drawings]

[0016] [Figure 1] This is an image of a colony observed from the DH-MA02 strain (accession number FERM P-22400). [Diagram 2] This is a morphological observation image of the DH-MA02 strain (accession number FERM P-22400). [Diagram 3] Photographs showing the state of the DH-MA02 strain (accession number FERM P-22400) after liquid culture.

[0017] The microorganism according to the present invention belongs to the genus Chlorella, and more specifically, is presumed to belong to Chlorella sorokiniana, and has the strain name DH-MA02. The microorganism according to the present invention was deposited at the National Institute of Technology and Evaluation, Patent Organism Deposit Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) on December 15, 2020, and its deposit number is FERM P-22400. The microorganisms according to the present invention include mutant strains that have the ability to take up ammonia nitrogen and have been mutated by known techniques such as exposure to ultraviolet light or radiation, as well as mutant strains that have mutated in nature.

[0018] The method for isolating a microorganism according to the present invention is as follows.

[0019] (1) First screening First, agricultural wastewater, industrial wastewater, and environmental water were collected from Ishikawa Prefecture and other areas in Japan. These collected waters were inoculated into modified MBM liquid medium and cultured at 20°C under light irradiation. The light irradiation conditions were approximately 4,000 lux, with a light period of 24 hours and a dark period of 0 hours. Those in which algal growth was confirmed by visual inspection were used as samples for the first screening.

[0020] (2) Secondary screening Next, the above culture solution was inoculated into the serially diluted methane fermentation digestive liquid and cultured under the same light irradiation conditions. Samples in which algae growth was confirmed in the undiluted methane fermentation digestive liquid were selected as samples for the second screening. Twelve samples passed the second screening.

[0021] (3) Third Screening Next, each sample was inoculated into 10 L of modified MBM liquid medium and cultured outdoors for two weeks to prepare a preculture solution. 480 L of new modified MBM liquid medium and the preculture solutions of the 12 samples (total of 120 L) were placed in a 600 L treatment tank, stirred, and cultured for two weeks. After measuring turbidity and chlorophyll fluorescence to confirm sufficient proliferation, methane fermentation digestion liquid was administered at a rate of 5 to 15 L / day for acclimation. After that, continuous operation was performed for about one year. The culture solution was sampled periodically, and the time-dependent changes in the nitrogen components and algae concentrations in the culture solution were analyzed. DNA was extracted from this culture solution, and the microbial community structure was analyzed to determine the dominant species.

[0022] (4) Fourth Screening Finally, the culture was plated on modified MBM agar medium containing various antibiotics and cultured at 25°C for 14 days under light irradiation. The light irradiation conditions were approximately 4,000 lux, 24 h light / 0 h dark. Single colony isolation was performed to create a library of microalgae. Each strain was pre-cultured and inoculated into modified MBM liquid medium containing methane fermentation digestion liquid adjusted to various nitrogen concentrations or ammonium sulfate, and those that were found to have a reduced ammonia concentration were selected. From these, those that matched the dominant species mentioned above were selected as the final screening strains.

[0023] Next, the taxonomic and morphological properties of the microorganism DH-MA02 strain according to the present invention will be described.

[0024] (taxonomic properties) First, the base sequence of the microbial DH-MA02 strain according to the present invention is the 18S rRNA gene shown in SEQ ID NO:1. Next, a homology search of the base sequence was performed based on the international base sequence database (GenBank), yielding the results shown in Table 1, which showed a high homology of over 99% with the genus Chlorella, a type of green algae. Therefore, the microorganism DH-MA02 strain related to the present invention is presumed to be a Chlorella sp. closely related to Chlorella sorokiniana.

[0025] [Table 1]

[0026] (morphological properties) Next, morphological observation of the microorganism DH-MA02 strain according to the present invention was performed on modified MBM medium at 25°C for 14 days. The colony observation image and morphological observation image are shown in Fig. 1 and Fig. 2, respectively. Specifically, when cultured on modified MBM agar medium, circular colonies with a diameter of about 1 to 2 mm were formed. The cells were spherical in shape and slightly pale green in color. When the bacteria were statically cultured in modified MBM liquid medium, they dispersed and proliferated within the medium, turning a relatively uniform green and cloudy color.

[0027] (Culture conditions, storage conditions) Finally, the culture conditions and storage conditions for the microorganism DH-MA02 strain according to the present invention are shown below. Note that the culture conditions and storage conditions described below are merely examples, and are not particularly limited as long as the microorganism DH-MA02 strain according to the present invention can grow under them. [Culture conditions] Medium name: Modified MBM liquid medium Sterilization conditions for medium: Autoclave (121°C, 15 min) Culture temperature: 25℃ Culture period: 2 to 14 days Culture method: static culture Light requirement: Required (approximately 4,000 lux, 24h light / 0h dark or 12h light / 12h dark) [Storage conditions] Freezing method storage: Possible (around -80℃, protective material: 10% DMSO) Storage by subculture: Either solid culture or liquid culture is possible (subculture interval: 1 month, storage temperature: 4℃)

[0028] From the above results of taxonomic properties (base sequence analysis) and morphological properties (shape characteristics), the microorganism DH-MA02 strain according to the present invention is presumed to be a Chlorella sp. closely related to Chlorella sorokiniana. EXAMPLES

[0029] Next, the ability of the microorganism DH-MA02 strain according to the present invention to reduce the concentration of ammonia nitrogen in wastewater will be described based on examples.

[0030] First, wastewater (methane fermentation digestive liquid) and ammonium sulfate (reagent, Fujifilm Wako Pure Chemical Industries, Ltd.) collected from two biogas power generation facilities (Plants A and B) were diluted with water to various concentrations in addition to the undiluted state, and the ammoniacal nitrogen concentration was measured. The ammoniacal nitrogen concentration of each undiluted solution was 3000 mg / L (Plant A) and 3400 mg / L (Plant B), and the ammonium sulfate aqueous solution was 5000 mg / L as the undiluted solution.

[0031] Next, 1.5 mL of the original solution and each diluted solution were placed in the wells of a microplate, and then 1% culture solution of the microorganism DH-MA02 strain was added. The culture was left to stand for 5 days at 25°C under light irradiation, and the concentration of ammonia nitrogen after 5 days was measured, and the treatment rate was calculated from the concentration before addition (treatment). The light irradiation conditions were approximately 4,000 lux, with a light period of 14 h and a dark period of 10 h. The results are shown in Tables 2 to 4 (Table 2: Concentration measurement results of methane fermentation digested liquid collected from plant A, Table 3: Concentration measurement results of methane fermentation digested liquid collected from plant B, Table 4: Concentration measurement results of ammonium sulfate aqueous solution).

[0032] [Table 2]

[0033] [Table 3]

[0034] [Table 4]

[0035] As a result, the microorganism DH-MA02 strain according to the present invention showed a high ability to reduce the concentration of ammoniacal nitrogen in wastewater of all concentrations, and in particular, showed a high ability to reduce the concentration of ammoniacal nitrogen even in the raw solution (wastewater containing a high concentration of ammoniacal nitrogen).

[0036] From the above results, it was found that the microorganism DH-MA02 strain according to the present invention is a novel microorganism (algae) that has the ability to reduce the concentration of ammonia nitrogen, which is not found in conventional algae, and that the microorganism (algae) has an extremely high processing ability. Furthermore, it was found that the microorganism (algae) has processing ability even in water. Although the detailed mechanism by which the novel microorganism (algae) of the present invention reduces the concentration of ammoniacal nitrogen is unknown, it is believed that the novel microorganism (algae) of the present invention reduces the concentration of ammoniacal nitrogen by taking it into its body.

[0037] In addition, it is believed that the novel microorganism (algae) of the present invention can also synthesize amino acids using some of the ammonia nitrogen that it has taken into its body. It is further believed that some of the amino acids serve as the starting point for the formation of proteins, which then become part of the body of the novel microorganism (algae). Specifically, the novel microorganism (algae) of the present invention performs photosynthesis using chlorophyll contained within its body, just like common algae. That is, it takes in water from wastewater and carbon dioxide contained in the wastewater or the atmosphere into its body, and synthesizes carbohydrates using light energy. The novel microorganism (algae) of the present invention synthesizes part of the ammonia nitrogen into amino acids using the energy obtained by photosynthesis, and is further considered to use some of the amino acids as starting points for producing proteins. It is considered that the novel microorganism (algae) itself uses such proteins for its own survival and proliferation. [Industrial Applicability]

[0038] The microorganism according to the present invention can be used for treating wastewater containing a high concentration of ammonia nitrogen, particularly for treating methane fermentation digestate. [Accession number]

[0039] FERM P-22400

Claims

1. A microorganism deposited under accession number FERM P-22400 that has the ability to reduce the concentration of ammoniacal nitrogen in wastewater.

2. The microorganism according to claim 1, characterized in that the wastewater is a methane fermentation digestive liquid.

3. A method for treating wastewater, comprising supplying the microorganism according to claim 1 or 2 to the wastewater.

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