Fibricated fiber microbacteria and applications thereof

Abstract

This invention discloses a fibrous microbacterium and its applications. The fibrous microbacterium, named IURM R23 and taxonomically named *Cellulosimicrobium cellulans*, was deposited on January 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 29729. The fibrous microbacterium provided by this invention exhibits highly efficient ammonia nitrogen degradation performance under high-salt conditions. The optimal degradation temperature for ammonia nitrogen by this fibrous microbacterium is 30–35°C, and the degradation effect is best under neutral pH conditions. The fibrous microbacterium provided by this invention tolerates low C / N ratios and has a wide tolerance range, thus possessing highly efficient nitrogen removal capabilities.

Description

Technical Field

[0001] This invention relates to the fields of water treatment and environmental microbiology, and in particular to a fibrous microbacterium and its applications. Background Technology

[0002] With the deepening of urbanization and industrialization, large amounts of domestic sewage and industrial wastewater containing high concentrations of nitrogen and organic matter are discharged into water systems. Excessive nitrogen and phosphorus emissions are a significant factor leading to eutrophication and water pollution, which severely restricts the sustainable development of related industries. Large nitrogen emissions not only pollute drinking water sources, threatening the safety of drinking water for humans and livestock, but also reduce dissolved oxygen in water bodies, exacerbating eutrophication, damaging aquatic ecosystems, harming the habitats and reproduction of aquatic organisms, releasing pungent odors that cause black and smelly water, and ultimately affecting human health.

[0003] The treatment of nitrogen-containing wastewater mainly includes physical / chemical methods and biological methods. Physical / chemical or biological treatment methods are costly and require additional energy. Microbial methods are gradually being adopted due to their advantages such as mild and efficient operation, high cost-effectiveness, and no secondary pollution. However, ammonia-oxidizing bacteria are intolerant to salt, resulting in slow reactions, low denitrification rates, and high oxygen requirements, making them ineffective in treating saline wastewater and often failing to effectively treat nitrogen-containing wastewater. Therefore, screening safe, salt-tolerant denitrifying bacteria for the treatment of nitrogen-containing wastewater holds significant promise. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention provides a fibrous microbacterium and its application in denitrification treatment.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] On the one hand, the present invention provides a fibrous microbacterium, named IURM R23, taxonomically named Cellulosimicrobium cellulans, which was deposited on January 23, 2024 at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.29729.

[0007] In another aspect, the present invention provides a nitrogen-containing compound degradation agent, comprising the above-mentioned fibrotic microbacteria.

[0008] Preferably, the nitrogen-containing compound includes ammonium salts and / or nitrates.

[0009] In another aspect, the present invention provides the application of the above-mentioned fibrous microbacteria or the above-mentioned nitrogen-containing compound degrading agents in denitrification treatment.

[0010] Preferably, its application in the denitrification treatment of nitrogen-containing wastewater.

[0011] Preferably, the nitrogen-containing wastewater includes ammonia nitrogen and / or nitrate nitrogen.

[0012] Preferably, the temperature of the denitrification treatment is 30–35°C.

[0013] Preferably, the denitrification treatment is carried out under conditions of pH 6 to 9, and more preferably under conditions of neutral acidity or alkalinity.

[0014] Preferably, the C / N ratio of the denitrification treatment is 1 to 5.

[0015] The above technical solution has the following advantages or beneficial effects:

[0016] This invention obtained the fibrotic microbacterium IURM R23 through soil screening. This bacterium has good ammonia nitrogen degradation ability and high tolerance to high concentrations of salt, and can be used for ammonia nitrogen treatment in high-salinity wastewater. Furthermore, this bacterium has the ability to degrade ammonia nitrogen in different environments, achieving a degradation rate of 75.26% in pig farm manure. Attached Figure Description

[0017] Figure 1 The images show plate colony diagrams and micrographs of the fibrotic microbacterium IURM R23 from Example 1 of this invention.

[0018] Figure 2 This is the molecular evolutionary tree of fibrotic microbacteria constructed in Example 1 of the present invention.

[0019] Figure 3 The image shows the removal effect of fibrous microbacteria IURM R23 at different salt concentrations on ammonia nitrogen in Example 2 of this invention.

[0020] Figure 4 The graph shows the removal effect of fibrous microbacteria IURM R23 on ammonia nitrogen at different temperatures in Example 3 of this invention.

[0021] Figure 5 This is a graph showing the removal effect of fibrous microbacteria IURM R23 on ammonia nitrogen at different pH levels in Example 3 of the present invention.

[0022] Figure 6 This is a graph showing the removal effect of fibrous microbacteria IURM R23 on ammonia nitrogen under different C / N ratios in Example 3 of the present invention.

[0023] Figure 7 This is a graph showing the changes in ammonia nitrogen degradation rate, nitrate nitrogen concentration, and nitrite nitrogen concentration in pig farm manure over time in Example 4 of the present invention.

[0024] Preservation Instructions

[0025] The fibrotic microbacterium IURM R23 of this invention, classified as Cellulosimicrobium cellulans, was deposited on January 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.29729, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0026] The following embodiments are merely some, not all, of the embodiments of the present invention. Therefore, the detailed descriptions of the embodiments provided below are not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0027] In this invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. The methods described in the following embodiments are conventional methods in the art, unless otherwise specified.

[0028] The pig farm wastewater in the following examples was sourced from a farm in Changping District, Beijing. It was sampled on November 21, 2023, using a disposable sterile sampling box and frozen at -20°C for later use.

[0029] Example 1: Isolation, purification, identification, and performance testing of the strain

[0030] (1) Isolation and purification of strains

[0031] 1) Enrichment culture of strains

[0032] Take 5g of Hainan red soil sample and add it to a 250mL Erlenmeyer flask. In a clean bench, add 100mL of MSM liquid medium (g / L: 1.0g Na3C6H5O7·2H2O, 2.0g (NH4)2SO4, 1.5g K2HPO4, 0.5g KH2PO4, 6.0g NaCl, 0.2g MgSO4·7H2O, pH 7.0–8.0) to the Erlenmeyer flask for enrichment culture. Transfer the Erlenmeyer flask to a shaker at 28–32℃ and 180rpm for culture. After 4 days of culture, the first subculture enrichment solution is obtained. Then, the second subculture enrichment is performed: add 5mL of the first subculture enrichment solution to fresh enrichment medium and culture for 4 days under the same shaker conditions to obtain the second subculture enrichment solution. Repeat the enrichment process to obtain the fourth subculture enrichment solution.

[0033] 2) Screening and isolation of strains

[0034] Add 9 mL of sterile water to 1 mL of the fourth passage enrichment solution, vortex to mix, and then dilute the fourth passage enrichment solution to 10 mL using a serial dilution method. -6 Take 150 μL of each of the 10 samples. -3 10 -4 10 -5 The diluted solution was spread onto MSM solid salt medium (obtained by adding 20 g / L agar powder to the above MSM liquid medium) with 20 g / L agar powder. The medium was spread in a clean bench, and after the moisture evaporated, it was transferred to a constant temperature inverted culture at 30°C. After 48 hours, various single colonies appeared.

[0035] Select single colonies with different morphologies and colors and inoculate them onto LB solid medium (g / L: 5.0g yeast extract, 10.0g peptone, 5.0g NaCl, 20g agar powder, pH 7.2–7.4). Perform the first streak and incubate at 30°C for about 24 hours. Single colonies will grow on the plates. Then, pick single colonies and inoculate them onto MSM solid salt medium. Streak them again and incubate at 30°C for about 48 hours. Single colonies will grow on the plates. After three generations of purification, seal the plates with film and temporarily store them at 4°C.

[0036] 4) Secondary screening

[0037] The strains obtained from the initial screening were streaked on LB solid medium and incubated at an incubator to obtain pure single colonies. These single colonies were then inoculated into LB broth (obtained from the above LB solid medium without 20 g / L agar powder) and incubated at 30°C with shaking for 24 h. 2 mL of the broth was then centrifuged at 12000 rpm for 2 min, the supernatant was discarded, and sterile physiological saline was added to adjust the OD. 600 The ammonia nitrogen concentration was approximately 1.0. 1% of the culture medium was inoculated into MSM salt culture medium with an initial ammonia nitrogen concentration of 412.00 mg / L. The medium was cultured at 30°C with shaking. Each group had three replicates. Sterile water was added to replace the bacterial culture as a blank control (CK). After 48 hours, the change in residual ammonia nitrogen in each culture medium was detected. The ammonia nitrogen concentration was detected by the phenol-sodium hypochlorite colorimetric method and the ELISA reader-hydrazine sulfate method to detect the nitrite nitrogen content and nitrate nitrogen content in the ammonia nitrogen degradation solution.

[0038] (2) Identification of strain IURM R23

[0039] 1) Morphological observation

[0040] The strain was inoculated into LB liquid medium and cultured at 30°C with shaking to obtain a bacterial suspension. A small amount of the suspension was stained with crystal violet to observe the morphological characteristics of the strain. Images of the strain on LB plates and under a light microscope are shown below. Figure 1 Its colonies are pale yellow, dotted, smooth, and opaque. When stained with crystal violet, the strains appear round or oval under a microscope.

[0041] 2) Molecular biological identification

[0042] Bacterial cell walls were disrupted through physical grinding to obtain a cell-wall-breaking solution containing dissolved genomic DNA. Primers were designed, and the cell-wall-breaking solution was used as a template to amplify 16S rDNA. Image formation was achieved using gel electrophoresis, followed by DNA sequencing. The DNA sequencing sequences were compared with the Standard databases on the National Center for Biotechnology Information website to obtain information on species most closely related to the sequence being analyzed. A molecular phylogenetic tree was constructed using MEGA7 software. Figure 2 .

[0043] The primer sequences used in the amplification are:

[0044] 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID NO.2;

[0045] 533F: 5′-GTGCCAGCMGCCGCGGTAA-3′, SEQ ID NO. 3.

[0046] Analysis revealed that the strain was most closely related to Cellulosimicrobium cellulans, and it was identified as such. It was named IURM R23 and submitted to NCBI, with GenBank accession number PP217355.1. Its nucleotide sequence is shown in SEQ ID NO.1, specifically:

[0047]

[0048] Example 2: Salt tolerance experiment of fibrous microbes

[0049] Using MSM liquid culture medium without NaCl as the basal medium, gradient salt cultures with salt concentrations of 2, 4, 6, 8, 10, 20, and 30 g / L were prepared by adding different amounts of NaCl. The bacterial strain was then inoculated into each gradient salt culture medium at a 1% inoculum. The cultures were incubated at 30℃ and 180 rpm with shaking. Each group was replicated in triplicate, and ammonia nitrogen concentration was measured after 48 hours. Results are as follows: Figure 3 As shown. Analysis Figure 3 It can be seen that the fibrotic microbacteria can grow in salt concentrations of 2–30 g / L. Under a salinity of 30 g / L, the ammonia nitrogen degradation rate is 41.71%, indicating that the strain has high salt tolerance.

[0050] Example 3: Degradation characteristics of ammonia nitrogen by the fibrous microbacterium IURM R23

[0051] (1) Ammonia nitrogen removal effect at different temperatures

[0052] Denitrification experiments were conducted at different temperatures (20, 25, 30, 35, and 40°C) using MSM inorganic salt broth as the basal medium, inoculated with 1% IURM R23. Residual ammonia nitrogen concentration was measured after 48 hours, with three replicates per group. Significance analysis between groups was performed using the new multiple range method, with Latin letters assigned at the 5% significance level. Results are shown below. Figure 4 As shown, temperature has a significant impact on ammonia nitrogen removal efficiency, and there is no significant difference in ammonia nitrogen degradation rate between 30℃ and 35℃.

[0053] (2) Ammonia nitrogen removal efficiency at different pH levels

[0054] The pH of the basal medium was adjusted to 6, 7, 8, and 9 using Na2HPO4-NaOH alkaline buffer and hydrochloric acid-KH2PO4 acidic buffer, respectively. The inoculum size was 1%, and a blank control was set up. The medium was cultured at 30℃ and 180 rpm with shaking. Residual ammonia nitrogen concentration was measured after 48 hours. The results are as follows: Figure 5 As shown, the significance analysis using the new complex range method indicates that pH value has a significant impact on ammonia nitrogen removal efficiency, and the ammonia nitrogen degradation rate is highest when the pH value is 7, which is statistically significant compared with other values.

[0055] (3) Ammonia nitrogen removal efficiency at different C / N ratios

[0056] Gradient culture media with C / N ratios of 0.5, 1, 3, 5, 10, and 20 were obtained by adding different amounts of sodium citrate. The bacterial strains were then inoculated into the culture media at a 1% inoculum level. Sterile water was used as a blank control. The cultures were incubated at 30℃ and 180 rpm with shaking. Ammonia nitrogen concentrations were measured after 48 hours. The results are as follows: Figure 6 As shown, the significance analysis using the new complex range method indicates that the C / N ratio has a significant impact on the ammonia nitrogen removal efficiency, with no significant difference when the C / N ratio is between 1 and 5.

[0057] from Figures 4-6 It can be seen that the optimal degradation temperature of ammonia nitrogen by strain IURM R23 is between 30 and 35℃; the degradation effect of ammonia nitrogen is the best under neutral acid-base conditions; the degradation of ammonia nitrogen is the best when the C / N ratio is 1 to 5, and the degradation rate of ammonia nitrogen reaches 86.79% when the C / N ratio is 3. Moreover, it is tolerant to low C / N ratios and has a wide range of tolerance for C / N ratios, indicating that this strain has a highly efficient nitrogen removal ability.

[0058] Example 4: Degradation of ammonia nitrogen in pig farm wastewater by the fibrinous microbacterium IURM R23

[0059] Pig farm manure wastewater with an ammonia nitrogen concentration of approximately 500 mg / L was inoculated with IURM R23 bacterial culture at a 1% inoculum. The mixture was treated at 180 rpm and 35°C, with a control group included. Samples were taken at 12, 24, 36, 48, and 60 hours. After centrifugation at 12000 rpm for 5 minutes, the supernatant was filtered through a 0.45 μm filter membrane. The concentrations of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in the filtered samples were then measured. The results are shown in [Figure number missing]. Figure 7 .

[0060] from Figure 7 The data shows that the ammonia nitrogen degradation rate and nitrite nitrogen concentration increase with time, while the nitrate nitrogen concentration decreases, indicating that nitrate nitrogen is converted into nitrite nitrogen, suggesting a denitrification function. This is because the product of heterotrophic nitrification, NO3... - -N is reduced to NO2 by nitrate reductase during aerobic denitrification. - -N, NO2 - The accumulation of -N induces the synthesis of nitrite reductase, which reduces it to gaseous nitrogen that escapes from the water surface. In the treatment of actual pig farm wastewater, the degradation rate of IURM R23 reached 75.26% in 48 hours.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of fibrous microbacterium, characterized in that, The fibrotic microbacteria were named IURM R23, and their taxonomic name was... Cellulosimicrobium cellulans It was deposited on January 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.29729.

2. A nitrogen-containing compound degradation agent, characterized in that, It includes the fibrotic microbacteria of claim 1; the nitrogen-containing compound includes ammonium salts and / or nitrates.

3. The application of the fibrous microbacteria of claim 1 or the nitrogen-containing compound degradation agent of claim 2 in denitrification treatment, characterized in that, Application in nitrogen removal treatment of nitrogen-containing wastewater, wherein the nitrogen-containing wastewater includes ammonia nitrogen and / or nitrate nitrogen.

4. The application according to claim 3, characterized in that, The temperature for the denitrification treatment is 30~35℃.

5. The application according to claim 3, characterized in that, The denitrification treatment was carried out under conditions of pH 6-9.

6. The application according to claim 3, characterized in that, The denitrification treatment is carried out under neutral acid-base conditions.

7. The application according to claim 3, characterized in that, The C / N ratio of the denitrification treatment is 1 to 5.

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