Methylosinus trichosporium and its application in denitrification and deodorization

Abstract

This invention discloses a Massimomicrobial strain and its application in denitrification and deodorization, belonging to the field of microbial technology. The Massimomicrobial strain of this invention has been identified as a potential new species of the genus *Massilia*, named *Massilia sp.* X63. This strain was deposited at the Korean Center for Type Culture Collection on April 28, 2022, with accession number KCTC:92384. The Massimomicrobial strain of this invention possesses the 16S rDNA sequence shown in SEQ ID No. 1. Massimomicrobial strain X63 of this invention not only exhibits high heteroaerobic nitrification and aerobic denitrification capabilities but also strong nitrous oxide removal capabilities, showing promising application prospects in treating ammonia-containing odors, denitrifying wastewater, and inhibiting nitrous oxide production.

Description

Technical fields:

[0001] This invention relates to the field of microbial technology, and in particular to a Massébium and its application in denitrification and deodorization. Background technology:

[0002] With the rapid development of industrialization and urbanization, ammonia-containing odors are frequently generated in industrial and agricultural production environments and during domestic sewage treatment, posing a significant threat to human health and the environment. Currently, the main methods for deodorization include physical, chemical, and biological methods. Physical deodorization primarily utilizes the adsorption and masking effects of materials to reduce odor concentration, but its adsorption effect is limited and the deodorization speed is slow. Chemical deodorization mainly removes odors through chemical reactions between chemical reagents and malodorous substances, but chemical reagents are expensive and can easily cause secondary pollution. Biological deodorization mainly utilizes the metabolic processes of microorganisms to decompose and transform malodorous substances, thereby removing odors; it is characterized by high efficiency, environmental friendliness, and widespread economic applicability.

[0003] Bio-spraying and bio-trickling filtration are commonly used biological deodorization technologies. They purify gases by having harmful substances in the odorous gas absorbed by water and converted into a liquid phase, where they are then transformed into harmless substances by microorganisms. However, during biological deodorization, especially in ammonia and nitrogen removal, nitrous oxide (N2O) is often generated. For example, when waste gas enters a biological scrubbing tower or trickling filter, ammonia is adsorbed and dissolved into the circulating water, forming high concentrations of ammonium salts and nitrites (NO2). - ), nitrates (NO3) - Microorganisms, under anaerobic or low-oxygen conditions, reduce nitrates or nitrites in circulating water to nitrogen (N2) through biochemical reactions such as denitrification. This process is accompanied by the formation of the intermediate product N2O. N2O is a greenhouse gas with a global warming potential hundreds of times greater than carbon dioxide, making its emission control a crucial issue in environmental engineering. Therefore, developing effective ammonia and nitrogen removal technologies while simultaneously reducing N2O emissions is of great significance for achieving sustainable development and addressing global climate change.

[0004] This invention aims to provide microorganisms for treating ammonia-containing odorous gases using biospraying or biotrickling filtration technology. Simultaneously, the microorganisms enter the water circulation system of the biospraying / trickling filtration device to denitrify and remove nitrous oxide, providing technical support for the sustainable development of waste gas treatment. Summary of the Invention:

[0005] This invention provides a Massimo bacterium and its application in denitrification and deodorization. A new species of Massimo bacterium (Massilia sp.) X63 was isolated from environmental samples. This strain has a good ability to remove ammonia nitrogen, nitrate nitrogen and nitrous oxide. It can not only be used to treat wastewater containing ammonia nitrogen and nitrate nitrogen, but also is suitable for biological spraying and trickling filter reactors to reduce greenhouse gas emissions such as nitrous oxide.

[0006] This invention is achieved through the following technical solutions:

[0007] The first objective of this invention is to provide a strain of Massilia sp. X63, which was deposited on April 28, 2022, at the Korean Collection for Type Cultures (KCTC), located at 181 Ipsin-gil. Jeongeup-si, Jeollabuk-do 56212, Korea, with accession number KCTC: 92384.

[0008] Preferably, the 16S rDNA sequence of the Masseilles X63 is shown in SEQ ID No. 1.

[0009] A second objective of the present invention is to provide a microbial inoculant, characterized in that it comprises the above-mentioned Masseilles and biochar.

[0010] Preferably, the preparation steps of the microbial agent are as follows: immersing biochar particles in a bacterial solution of *Bacillus simonii*, culturing them in a shaker at 30–35°C at 150–200 r / min for 24–48 hours to allow the biochar to be loaded with microorganisms, filtering out the bacterial solution, drying them in an oven at 30–40°C until the mass is constant, and then removing them to obtain the microbial agent.

[0011] Further preferred, the preparation steps of the Massimo bacteria culture are as follows: 5% Massimo bacteria (Massilia sp.) are inoculated into a nutrient solution that has been sterilized at high temperature and cooled to room temperature, and cultured for 24 hours at 30°C and 180 r / min on a shaker to obtain the culture.

[0012] Further preferably, the nutrient solution comprises: glucose 5-8 g / L, ammonium chloride 0.8-1.6 g / L, sodium thiosulfate pentahydrate 5-8 g / L, potassium dihydrogen phosphate 0.1-0.5 g / L, disodium hydrogen phosphate 0.2-1.0 g / L, magnesium sulfate heptahydrate 0.2-1.0 g / L, calcium chloride 0.02-0.1 g / L, and a pH of 6-8.

[0013] Preferably, the preparation steps of the biochar particles are as follows: rice husks are placed in a tube furnace for pyrolysis and carbonization to obtain biochar, which is then ground into powder in a mortar and passed through a 50-mesh sieve to obtain biochar particles; the pyrolysis and carbonization conditions are: heating stage 25 to 500℃, time 1h; pyrolysis temperature 500℃, time 1.5h; cooling stage 500 to 25℃, time 0.5h, and nitrogen flow rate 100mL / min.

[0014] Preferably, the ratio of biochar particles to Masseille bacteria solution is 2g:(2-5)mL.

[0015] A third objective of this invention is to provide the application of the above-mentioned Masse bacteria or the above-mentioned bacterial agent in denitrification and deodorization.

[0016] Preferably, the use of Masseilles X63 with nitrate or ammonium salt as the sole nitrogen source in the removal of nitrate nitrogen and / or ammonia nitrogen.

[0017] Preferably, adding Bacillus simonii or its agent to a spray scrubbing tower or bio-trickling filter filled with packing material can remove nitrous oxide gas from the exhaust gas and pollutants such as nitrate nitrogen and ammonia nitrogen from the spray / trickling filter circulating water.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention provides a novel strain of Massilia sp. X63, which has been screened to efficiently remove ammonia nitrogen, nitrate, and nitrous oxide from water. High-density cells can be obtained by expanding the culture of Massilia sp. X63 using a carbon source nutrient solution. Alternatively, it can be loaded onto biochar to prepare a bacterial agent, which can be spread on the packing material of a nitrogen oxide spray / trickling filter or added to the circulating water recovery tank of a spray / trickling filter, making it well-suited for use in biological spray / trickling filters to achieve denitrification and deodorization.

[0020] Massilia sp. X63 was deposited on April 28, 2022, at the Korean Collection for Type Cultures (KCTC), located at 181 Ipsin-gil. Jeongeup-si, Jeollabuk-do 56212, Korea, with accession number KCTC:92384. Attached image description:

[0021] Figure 1 The colony morphology of *Masses X63* on activated culture medium in Example 2 is shown in (A) colony morphology on plate; (B) single colony morphology.

[0022] Figure 2 This is the genomic phylogenetic tree of Masseilles X63 in Example 3. Detailed implementation method:

[0023] Those skilled in the art will understand that the techniques disclosed in the following embodiments represent those discovered by the inventors that have proven effective in the practice of this invention. However, many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, still yielding the same or similar results. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional in the art.

[0024] Example 1: Screening and isolation of Massilia sp. X63

[0025] The *Massilia* sp. X63 strain of this invention was isolated and purified from the mangrove sediment in Hualong Town, Panyu District, Guangzhou City. The isolation and purification method is as follows:

[0026] (1) Culture medium:

[0027] Enrichment medium: potassium dihydrogen phosphate 1.5 g / L, disodium hydrogen phosphate 5.0 g / L, magnesium sulfate heptahydrate 0.1 g / L, potassium nitrate 1.0 g / L, sodium succinate 4.7 g / L, pH 7.2

[0028] Isolation medium: yeast extract 0.5 g / L, tryptone 1.0 g / L, NaCl 0.5 g / L, agar 1.5%.

[0029] Activation medium: glucose 5.0 g / L, ammonium chloride 0.8 g / L, sodium thiosulfate pentahydrate 5.0 g / L, potassium dihydrogen phosphate 0.1 g / L, disodium hydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.2 g / L, pH 7.0.

[0030] (2) Enrichment culture

[0031] Take 1g of mangrove mud from the bare beach and place it in an Erlenmeyer flask containing 100mL of enrichment medium. Incubate at 30℃ and 180r / min for 24h. After the culture medium becomes turbid, transfer 5mL of the culture medium to an Erlenmeyer flask containing 100mL of enrichment medium and incubate at 30℃ and 180r / min on a shaker for 24h. Repeat the enrichment process three times.

[0032] (3) Separation and purification

[0033] The enriched bacterial culture was diluted with sterile physiological saline to a concentration of 10. -2 10 -3 10 -4 10 -5 10 -6Different concentration gradients of bacterial suspension were prepared, and 0.2 mL of each concentration gradient was spread onto isolation medium and incubated at 30°C until distinct single colonies appeared. Single colonies with different morphologies were picked and transferred to isolation medium slant in test tubes and incubated at 30°C for 48 h. Then, they were transferred to a refrigerator at 4°C for storage, thus obtaining Massey bacteria X63.

[0034] Example 2: Identification of Nitrous Oxidizing Reducing Bacteria X63

[0035] Gram staining confirmed that strain X63 is a Gram-negative bacterium. After inoculating strain X63 onto activation medium and culturing at 30°C for 48 hours, morphological characteristics were observed. Single colonies were smooth, yellow, and rod-shaped. Figure 1 ).

[0036] DNA was extracted from strain X63, and its 16S rRNA gene fragment was amplified and sequenced using universal bacterial primers 1492R and 27F. The two anteroposterior sequences were assembled using Contig Express software to obtain the complete sequence, which is shown in SEQ ID No:1 and is 1352 bp in length. Homology analysis was performed using BLAST with sequences already registered in the GenBank database. The results showed that strain X63 is similar to the type strain Masslia yuzhufengenisiY1243-1. T It showed the highest similarity at 98.67%; however, it had lower 16S rRNA gene sequence similarity with other type strains of the genus *Masslia*. These results indicate that strain X63 is similar to the type strain *Masslia yuzhufengenisiY1243-1*. T The strain is most closely related to the bacteria. Based on the 16S rRNA gene sequence alignment, strain X63 belongs to the genus *Massezia*, and its taxonomic position needs to be determined by combining its genome analysis.

[0037] Example 3: Genomic analysis of Massilia sp. X63

[0038] The genome of strain X63 extracted in Example 2 was sent to Sangon Biotech (Shanghai) Co., Ltd. for genome sequencing using the Illumina NovaSeq platform. The genome sequence was then assembled using SPAdes v3.15.3 software, and finally, CheckM software was used to assess genome integrity and contamination levels. The results showed that the total genome length of strain X63 was 5.72 Mbp, the G+C content of the genomic DNA was 66.2 mol%, the genome integrity was 99.6%, and the contamination level was 1.4%.

[0039] A phylogenetic tree of the genome of strain X63 was constructed using UBCG software, and the results showed that ( Figure 2 Strain X63 is similar to the type strain Massilia yuzhufengenisiY1243-1 T and Massilia agri K-3-1 T Different branches were formed. The mean nucleotide identity (ANI) between the genome sequences of strain X63 and closely related model strains was calculated using the online tool ANI. The results showed that strain X63 and the closely related model strain *Massilia yuzhufengenisi* Y1243-1... T and Massilia agri K-3-1 T The ANI values ​​were 85.25% and 85.14%, respectively, which are lower than the ANI threshold (95%–96%) for defining new prokaryotic species. Therefore, the above results indicate that strain X63 is a potential new species of Massilia sp.

[0040] The strain X63 was named Massilia sp. X63 and was deposited on April 28, 2022, at the Korean Collection for Type Cultures (KCTC), located at 181 Ipsin-gil. Jeongeup-si, Jeollabuk-do 56212, Korea, with accession number KCTC:92384.

[0041] Example 4: Study on the growth characteristics of Massilia sp. X63

[0042] Masseuse X63 was inoculated onto R2A liquid medium (Qingdao Haibo) and incubated at 4, 10, 15, 20, 25, 30, 35, and 40°C for 3 days, and its growth was observed. Masseuse X63 was also inoculated into R2A liquid medium with pH values ​​of 3, 4, 5, 6, 7, 8, 9, and 10 and incubated at 30°C on a shaker at 180 rpm / min for 3 days. Furthermore, Masseuse X63 was inoculated into R2A liquid medium with NaCl additions of 0, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0% (w / v) and incubated at 30°C on a shaker at 180 rpm / min for 3 days. After incubation, the OD600 of the culture solution was measured to determine the growth of Masseuse X63 under different temperature, pH, and NaCl conditions.

[0043] Experimental results show that the growth temperature range of the Masseilles X63 is 15–35℃, with the optimal growth temperature being 30℃; the growth pH range is 4–10, with the optimal growth pH being 6.5; and the maximum NaCl concentration for growth is 3% (w / v).

[0044] Example 5: Study on the nitrous oxide reduction efficiency of Massimodium sp. X63

[0045] Masseilles X63 was inoculated into activation medium and incubated at 30°C and 180 rpm. -1 Incubate on a shaker at a constant temperature for 24 hours. Once the culture medium becomes turbid, transfer 8 mL of the bacterial suspension to a 10 mL centrifuge tube, centrifuge at 8000 rpm for 5 min, wash twice with PBS buffer, and then add 5 mL of PBS buffer to fully resuspend the culture. Transfer the suspension to a headspace vial containing 30 mL of potassium nitrate-free enrichment medium and seal. Simultaneously, add 35 mL of potassium nitrate-free enrichment medium to the headspace vial as a blank control. Finally, purge with 893.6 μg / L nitrous oxide gas and incubate at 30℃ and 180 rpm. -1 After incubation on a shaker at a constant temperature for 24 hours, the mixture was allowed to stand. The concentration of nitrous oxide gas in the headspace vial was determined using gas chromatography, and the removal rate of nitrous oxide in the treatment group was calculated compared with the blank control. The results showed that *Masses X63* achieved a nitrous oxide removal rate of 74.2%.

[0046] Example 6: Study on the denitrification performance of Massilia sp. X63

[0047] Masseilles X63 was inoculated with 5% ammonia nitrogen (NH4) solution. + -N) and nitrate nitrogen (NO3) - The culture medium, with chloroform (N-N) as the sole nitrogen source, was incubated at 30°C and 180 rpm. Samples were taken after 24 hours of incubation; a portion was used to measure the OD of the bacterial culture. 600 The other portion of the culture medium was centrifuged at 6000 r / min for 10 min, and the supernatant was collected and filtered through a 0.45 μm filter membrane. NO3 was then determined using ultraviolet spectrophotometry. - The -N content was determined by Nessler's reagent spectrophotometry for NH4+. + -N content. The removal rate was calculated by measuring the change in the concentration of the substance in the culture medium before and after culture: Removal rate (%) = (Initial concentration - Final concentration) / Initial concentration × 100%.

[0048] Culture medium with nitrate nitrogen as the sole nitrogen source: 1.1 g sodium succinate, 0.36 g KNO3, 0.2 g KH2PO4, 0.1 g MgSO4·7H2O, 0.28 g CaCl2, with 0.2% trace element mixture added by volume, pH adjusted to 7.2, and brought to a final volume of 1 L with deionized water. Sterilize at 121℃ for 20 min by high temperature and autoclaving.

[0049] Culture medium with ammonia nitrogen as the sole nitrogen source: 1.1 g sodium succinate, 0.19 g NH4Cl, 0.2 g KH2PO4, 0.1 g MgSO4·7H2O, 0.28 g CaCl2, add 0.2% trace element mixture by volume, adjust pH to 7.2, bring volume to 1 L with deionized water, and autoclave at 121℃ for 20 min.

[0050] Masseilles X63 at 50 mg / L NH4 + Under the condition that -N is the only nitrogen source, OD 600 Reaching 0.15, for NH4 + The removal rate of -N reached 71.7%; at 50 mg / L NO3 - Under the condition that -N is the only nitrogen source, OD 600 Reaching 0.11, for NO3 - The removal rate of -N reached 39.9%. These results indicate that *Masses* X63 is a novel heterotrophic nitrification-aerobic denitrification strain capable of efficiently removing ammonia nitrogen from water and nitrous oxide from gas. It has great application potential in the removal of pollutants such as ammonia nitrogen and nitrate nitrogen from waste gas and spray / trickling filter circulating water.

[0051] Example 7: Application of Bacillus simonii in denitrification and deodorization

[0052] 5% Massilia sp. was inoculated into a nutrient solution that had been sterilized at high temperature and cooled to room temperature, and cultured for 24 h at 30℃ and 180 rpm to obtain a Massilia sp. culture. The nutrient solution consisted of: glucose 5 g / L, ammonium chloride 0.8 g / L, sodium thiosulfate pentahydrate 5 g / L, potassium dihydrogen phosphate 0.5 g / L, disodium hydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, calcium chloride 0.05 g / L, and pH 6.5.

[0053] Rice husks were pyrolyzed and carbonized into biochar in a tube furnace, then ground into powder in a mortar and passed through a 50-mesh sieve to obtain biochar granules. The tube furnace conditions were as follows: heating stage 25 to 500℃, time 1h; pyrolysis temperature 500℃, time 1.5h; cooling stage 500 to 25℃, time 0.5h; nitrogen flow rate 100mL / min.

[0054] Biochar granules were immersed in a Bacillus simonii bacterial solution at a ratio of 2g:5mL and cultured in a shaker at 30-35℃ at 150-200r / min for 24-48 hours to allow the biochar to be loaded with microorganisms. The bacterial solution was then filtered off and dried in an oven at 30-40℃ until the mass was constant. The resulting Bacillus simonii inoculum was then obtained.

[0055] 0.1 g of *Bacillus simonii* inoculum was weighed into a 100 mL serum bottle, sealed, and then injected with 893.6 μg / L nitrous oxide gas. After 18 hours, a sample was taken and the nitrous oxide gas concentration was measured using gas chromatography. The change in concentration before and after treatment was determined, and the removal rate was calculated. The results showed that the *Bacillus simonii* inoculum removed 87.4% of the nitrous oxide gas.

[0056] 0.1 g of *Bacillus simonii* inoculant was added to an artificial wastewater culture medium containing ammonia nitrogen and nitrate nitrogen (0.5 g / L glucose, 0.15 g / L sodium acetate, 0.05 g / L NaCl, 0.15 g / L MgSO4·7H2O, 0.2 g / L NH4Cl, 0.04 g / L KH2PO4, 0.4 g / L KNO3). After incubation at 30℃ and 180 r / min for 24 h, samples were taken to determine the contents of total nitrogen, ammonia nitrogen, and nitrate nitrogen. The changes in contents before and after treatment were measured, and the removal rate was calculated. The results showed that the removal rates of total nitrogen, ammonia nitrogen, and nitrate nitrogen by *Bacillus simonii* inoculant reached 40.9%, 67.2%, and 50.5%, respectively.

[0057] SEQ ID NO.1 (16S rRNA sequence of Masseilles X63):

[0058]

Claims

1. A strain of Massilia sp. X63, characterized in that, The accession number is KCTC:92384.

2. A microbial inoculant, characterized in that, It contains the Masseilles and biochar as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The preparation steps are as follows: biochar particles are impregnated in the bacterial solution of Bacillus simonii, and then placed in a shaker at 30-35℃ and cultured at 150-200 r / min for 24-48 hours to allow the biochar to be loaded with microorganisms. After the bacterial solution is filtered off, the biochar is dried in an oven at 30-40℃ until the mass is constant, and then the bacterial agent is obtained.

4. The microbial agent according to claim 3, characterized in that, The steps for preparing the bacterial culture of *Masses* are as follows: 5% *Masses* is inoculated into a nutrient solution that has been sterilized at high temperature and cooled to room temperature, and then cultured at 30°C and 180 r / min for 24 h to obtain the bacterial culture.

5. The microbial agent according to claim 4, characterized in that, The nutrient solution is composed of: glucose 5-8 g / L, ammonium chloride 0.8-1.6 g / L, sodium thiosulfate pentahydrate 5-8 g / L, potassium dihydrogen phosphate 0.1-0.5 g / L, disodium hydrogen phosphate 0.2-1.0 g / L, magnesium sulfate heptahydrate 0.2-1.0 g / L, calcium chloride 0.02-0.1 g / L, and pH 6-8.

6. The microbial agent according to claim 3, characterized in that, The preparation steps of the biochar particles are as follows: rice husks are placed in a tube furnace for pyrolysis and carbonization to obtain biochar, which is then ground into powder in a mortar and passed through a 50-mesh sieve to obtain biochar particles. The pyrolysis and carbonization conditions are as follows: heating stage 25 to 500℃, time 1h; pyrolysis temperature 500℃, time 1.5h; cooling stage 500 to 25℃, time 0.5h; nitrogen flow rate 100mL / min.

7. The microbial agent according to claim 3, characterized in that, The ratio of biochar particles to Masse bacteria solution is 2g:(2-5)mL.

8. The application of the *Morchella esculenta* of claim 1 or the bacterial agent of claim 2 in denitrification and deodorization.

9. The application according to claim 8, characterized in that, Nitrate or ammonium salts are used as the sole nitrogen source to convert and remove nitrate nitrogen and / or ammonia nitrogen.

10. The application according to claim 8, characterized in that, Masseille bacteria or bacterial agents can be added to a spray scrubbing tower or biotrickling filter filled with packing material.

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