Actinomycete from plutella xylostella for high efficient utilization of ammonium nitrogen and application thereof

Abstract

The application discloses an Actinomadura sp. HUBU1 strain derived from a diamondback moth, which can efficiently utilize ammonium nitrogen. The strain has been preserved in the China Center for Type Culture Collection on April 24, 2026, and the preservation number is CCTCC NO: M 2026802. The strain can grow with ammonium nitrogen as the only inorganic nitrogen source, and the growth rate in the ammonium nitrogen culture medium is significantly higher than that in the nitrate nitrogen and nitrite nitrogen culture medium. After being cultured in the ammonium nitrogen liquid culture medium for 60 hours, the ammonia nitrogen removal rate reaches 17.5%. Key enzyme activity determination shows that the GDH activity reaches a peak value at 24-36 hours of culture, and the GS activity significantly increases at 48 hours of culture, indicating that the strain has a dynamic and synergistic ammonia assimilation enzyme system. The application also provides a microbial inoculum containing the strain and an application of the microbial inoculum in preparing an assimilation ammonium nitrogen and treating an ammonium-containing liquid system. The strain has a unique source, high ammonia assimilation efficiency and clear enzymatic characteristics, and has a good industrial application prospect.

Description

Technical Field

[0001] This application relates to the field of environmental microbiology technology, specifically to an actinomycete derived from the diamondback moth that efficiently utilizes ammonium nitrogen and its applications. Background Technology

[0002] Nitrogen is a crucial nutrient element in agricultural ecosystems, closely related to crop growth, soil fertility, and ecological functions. In the resource utilization of organic solid waste, the effective utilization rate of nitrogen is a key factor determining product quality and environmental burden. Large quantities of organic waste, such as livestock and poultry manure, kitchen waste, and crop straw, are generated in urban and rural areas across China, containing abundant nitrogen resources. However, due to performance limitations of existing treatment technologies, nitrogen loss rates remain high, hindering the development of high-value utilization of organic waste.

[0003] In traditional composting and bioconversion systems, nitrogen is mainly produced in the form of organic nitrogen and ammonium nitrogen (NH4+). 4+ Ammonium nitrogen exists in the form of volatile ammonia (NH3). When the compost temperature, pH value, and aeration increase, ammonium nitrogen is easily lost through volatile ammonia (NH3), which is the main pathway for nitrogen loss in composting systems. Simultaneously, the nitrification-denitrification process also produces greenhouse gases such as nitrous oxide (N2O), causing nitrogen to dissipate in gaseous form, thus reducing the nitrogen use efficiency of resource-based products. Studies show that in conventional composting systems, total nitrogen loss due to NH3 volatilization can reach 20% to 50%, sometimes even higher, significantly reducing the fertilizer efficiency of the product. To reduce nitrogen loss, existing studies have employed physicochemical interventions such as particle structure optimization, mineral additives (such as phosphogypsum), biochar addition, and aeration and moisture management. These methods have reduced ammonia volatilization and greenhouse gas emissions to some extent, but fundamentally improving nitrogen biointegration efficiency remains challenging.

[0004] In recent years, inoculation with functional microbial agents has been considered an effective and environmentally friendly method to reduce nitrogen loss and improve the resource utilization efficiency of organic waste. Current screening studies on ammonia-assimilating microorganisms mainly focus on high-temperature composting systems, activated sludge from wastewater treatment, or the guts of saprophytic insects such as black soldier flies. For example, the heat-resistant bacterium LL-8 isolated from chicken manure compost can reduce ammonia volatilization by 42.9%; while Klebsiella pneumoniae isolated from the gut of black soldier flies showed strong ammonia utilization capacity in pig manure substrates. However, existing research has significant limitations in terms of screening sources and microbial groups: on the one hand, it relies excessively on bacteria or fungi in nitrogen-rich environments. Although some fungi possess ammonia assimilation capabilities under laboratory conditions, they often face bottlenecks such as long growth cycles, sensitivity to environmental fluctuations, weak competitiveness, and poor operational stability in large-scale organic waste treatment, making industrial application difficult; on the other hand, strains with highly efficient ammonia assimilation capabilities are rarely reported in the important microbial group of actinomycetes, especially those derived from the guts of agricultural pests in low-nitrogen environments, which are currently almost nonexistent.

[0005] In fact, the exploitation of biological resources in low-nitrogen environments in nature remains insufficient. Agricultural pests such as corn borers, diamondback moths, and aphids, which feed on low-nitrogen plant sap, have evolved highly efficient nitrogen acquisition and recycling mechanisms in their gut microbiota. Studies have shown that Morganella morganii and Klebsiella acidogenetica in the gut of the Oriental fruit fly play key roles in urea hydrolysis and ammonia assimilation, helping the host synthesize essential amino acids in low-nitrogen environments; the symbiotic bacterium Arsenophonus of the cotton aphid can still regulate host needs even when amino acids are lacking. This indicates the existence of excellent bacterial resources in the guts of agricultural pests, which can rapidly convert inorganic nitrogen into organic nitrogen, providing a starting point for the development of novel nitrogen-retaining bacterial agents. However, existing research has mostly focused on bacterial groups, and the potential of actinomycetes, which are also widely distributed in the insect gut, in ammonia assimilation has not yet been fully explored.

[0006] Furthermore, the assimilation efficiency and environmental adaptability of existing strains urgently need improvement: when treating high-concentration organic waste, existing strains have narrow adaptability and low nitrogen assimilation rates in complex matrices, resulting in unstable nitrogen retention. More importantly, existing studies lack systematic determination and time-series analysis of the activities of key ammonia assimilation enzymes (glutamine synthase GS, glutamate dehydrogenase GDH, and glutamate synthase GOGAT), making it difficult to provide reliable process parameters for industrial applications.

[0007] Therefore, the existing technology has the following problems: First, the screening of ammonia assimilation microorganisms relies too much on bacteria or fungi in nitrogen-rich environments, and there is a serious lack of exploration of actinomycetes, especially those from the gut of agricultural pests in low-nitrogen environments; Second, the existing strains lack systematic determination and time-series analysis of the activities of key enzymes (GS, GDH, GOGAT) in ammonia assimilation, and cannot provide reliable process parameters for industrial applications. Summary of the Invention

[0008] In view of this, the purpose of this application is to provide a highly efficient ammonia nitrogen-utilizing actinomycete derived from the diamondback moth and its application. In this application, an actinomycete (Actinomadura sp. HUBU1) was isolated from the intestine of the diamondback moth. Through systematic verification of the strain's ammonia nitrogen assimilation capacity and the activity changes of key enzymes (GS, GDH, GOGAT), it was found that this effectively addresses the shortcomings of existing strains in terms of nitrogen biointegration efficiency, environmental adaptability, and process feasibility. This provides a novel, efficient, stable, and industrially viable ammonia assimilation microbial solution for organic waste treatment and agriculture.

[0009] To achieve the above objectives, this application provides the following technical solution:

[0010] In the first aspect, this application provides a strain of Actinomadura sp. HUBU1, which is a highly efficient ammonium nitrogen-utilizing actinomycete. It was deposited on April 24, 2026, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2026802 and address 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0011] The aforementioned Actinomadura sp. HUBU1 was isolated from the intestine of the diamondback moth (Plutellaxylostella).

[0012] In some embodiments, the glutamate dehydrogenase (GDH) activity of the Actinomadura sp. HUBU1 reaches its peak between 24 and 36 hours of culture, and the glutamine synthase (GS) activity is significantly higher at 48 hours of culture than at 12 hours.

[0013] In some embodiments, the Actinomadura sp. HUBU1 strain, after being cultured in ammonium nitrogen liquid medium for 60 hours, achieves a removal rate of ammonia nitrogen of not less than 17%.

[0014] Secondly, this application provides a microbial agent comprising Actinomadura sp. HUBU1 as described in the first aspect.

[0015] In some embodiments, the microbial agent is used to remove ammonium nitrogen from a liquid environment.

[0016] Thirdly, this application provides the use of Actinomadura sp. HUBU1, as described in the first aspect, in the preparation of microbial preparations for assimilating ammonium nitrogen.

[0017] In some embodiments, the microbial preparation is used to treat wastewater containing ammonia nitrogen or organic solid waste.

[0018] Fourthly, this application provides a method for reducing ammonia nitrogen loss during the treatment of organic waste, comprising: inoculating the organic waste with Actinomadura sp. HUBU1 as described in the first aspect, or the microbial agent as described in the second aspect, for treatment.

[0019] In some preferred embodiments, the inorganic nitrogen source in the culture medium used to culture Actinomadura sp. HUBU1 is ammonium nitrogen.

[0020] Compared with existing technologies, it has at least the following advantages and beneficial effects:

[0021] 1. This application describes the isolation of an actinomycete from the gut of the diamondback moth, an agricultural pest that has been exposed to a low-nitrogen environment for a long period. The strain was identified molecularly as *Actinomadura* sp. HUBU1. This strain differs from existing technologies that rely excessively on bacteria or fungi from nitrogen-rich environments (such as compost or activated sludge), filling a resource gap in ammonia assimilation by actinomycetes derived from the gut of low-nitrogen agricultural pests.

[0022] 2. The strain isolated in this application grows well in a medium with ammonium nitrogen as the sole inorganic nitrogen source, reaching a growth plateau in approximately 36 hours. Its growth rate in ammonium nitrogen medium is significantly higher than that in nitrate nitrogen and nitrite nitrogen medium, demonstrating a clear preference for ammonium nitrogen. This characteristic makes it particularly suitable for treatment systems where ammonium nitrogen is the main nitrogen form.

[0023] 3. The Madura actinomycetes provided in this application possess highly efficient ammonia nitrogen assimilation capabilities. After culturing in ammonium nitrogen liquid medium for 60 hours, this strain significantly reduced the ammonia nitrogen concentration in the culture medium from the initial 263.95 ± 2.99 mg / L to 217.82 ± 2.05 mg / L, achieving an ammonia nitrogen removal rate of 17.5% (P<0.001). It can be used as a functional strain for the treatment of ammonia nitrogen-containing wastewater or for the biological retention of nitrogen in organic waste.

[0024] 4. This application is the first to systematically determine the activity changes of key ammonia assimilation enzymes (GDH, GS, GOGAT) in the actinomycete HUBU1 at different culture times, revealing its dynamic and synergistic ammonia assimilation mechanism: GDH activity peaked at 24–36 h (approximately 0.43–0.44 U / mL), significantly higher than after 12 h and 48 h; GS activity significantly increased to 0.523 U / mL at 48 h, higher than 0.419 U / mL at 12 h. This dynamic mechanism enables the strain to adapt to fluctuations in ammonium concentration and maintain continuous assimilation capacity.

[0025] 5. The Madura actinomycete strain provided in this application grows stably in ammonium nitrogen medium and can be further prepared into a microbial agent, which can be widely used in ammonia nitrogen wastewater treatment, organic solid waste composting for nitrogen retention, agricultural non-point source pollution control and other scenarios, and has good prospects for industrial promotion.

[0026] Instructions for strain preservation:

[0027] Classification and nomenclature: Actinomadura sp. HUBU1;

[0028] Preservation institution: China Center for Type Culture Collection;

[0029] Preservation institution code: CCTCC;

[0030] Address of the depository: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province;

[0031] Deposit date: April 24, 2026;

[0032] The accession number is CCTCC NO: M 2026802. Attached Figure Description

[0033] Figure 1 The images show the colony morphology of the target strain HUBU1 provided in this application embodiment, where A is the colony morphology of the target strain HUBU1 on LB solid medium and B is the colony morphology of the target strain HUBU1 on ammonia assimilation solid medium.

[0034] Figure 2 A phylogenetic tree based on the 16S rRNA gene sequence provided in this application embodiment.

[0035] Figure 3 The growth curves of strain HUBU1 provided in the embodiments of this application under different nitrogen source conditions.

[0036] Figure 4 A standard curve of ammonia nitrogen provided for an embodiment of this application.

[0037] Figure 5 The graph shows the change in ammonia nitrogen concentration during the cultivation of strain HUBU1 provided in this application embodiment.

[0038] Figure 6 The graph shows the changes in GDH activity of strain HUBU1 at different culture time points, which is provided in the embodiments of this application.

[0039] Figure 7 The graph shows the changes in GS activity of strain HUBU1 at different culture time points, which is provided in the embodiments of this application.

[0040] Figure 8 The graph shows the changes in GOGAT activity of strain HUBU1 at different culture time points, as provided in the embodiments of this application. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0042] The materials used in the following embodiments are not limited to those listed below and may be replaced by other similar materials. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0043] In this application, unless the context clearly indicates otherwise, the terms “including,” “comprising,” “containing,” “having,” etc., shall be understood as open-ended and mean “including but not limited to.”

[0044] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying common rounding techniques.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before providing a detailed description of this application, the following terms and definitions are provided to better understand this application:

[0046] 1. Ammonia assimilation: refers to the metabolic process by which microorganisms convert inorganic nitrogen (such as ammonium nitrogen, nitrate nitrogen, etc.) into their own organic nitrogen (such as amino acids, proteins). In this application, it specifically refers to the conversion of ammonium nitrogen (NH4+) into organic nitrogen. 4+ The process of converting nitrogen into organic nitrogen.

[0047] 2. Ammonium nitrogen: refers to nitrogen in the form of ammonium ions (NH4+). 4+ Nitrogen in the form of ) is one of the inorganic nitrogen sources that microorganisms preferentially utilize, and it is also the main form of nitrogen that is easily lost through volatilization during the treatment of organic waste.

[0048] 2. Nitrate nitrogen and nitrite nitrogen: These refer to nitrogen in the form of nitrate ions (NO3, N2O3, 2N ... 3- ) and nitrite (NO) 2- Nitrogen in the form of ammonium nitrogen must be reduced to ammonium nitrogen by microorganisms before it can enter the assimilation pathway.

[0049] 3. Glutamine synthase (GS): In this application, it refers to the enzyme that catalyzes the reaction of ammonium ions with glutamate to produce glutamine. It is one of the key enzymes in the ammonia assimilation pathway and has a high affinity for low concentrations of ammonium.

[0050] 4. Glutamate dehydrogenase (GDH): In this application, it refers to the enzyme that catalyzes the direct formation of glutamate from α-ketoglutarate and ammonium ions, and plays a major role in ammonia assimilation at high ammonium concentrations.

[0051] 5. Glutamate synthase (GOGAT): In this application, it refers to the enzyme that works synergistically with GS to catalyze the reaction of glutamine and α-ketoglutarate to produce two molecules of glutamate, forming the GS-GOGAT cycle.

[0052] 6.OD 600 : refers to the optical density value of the bacterial solution at a wavelength of 600 nm. In this application, it is used to characterize the relative concentration of bacteria in the culture medium.

[0053] The following are specific examples:

[0054] Example 1: Isolation, Purification and Identification of Strains

[0055] 1.1 Sample Source and Pretreatment

[0056] The tested insect was the diamondback moth (Plutella xylostella), collected from the field at Huazhong Agricultural University. The insects were reared in an artificial climate chamber under the following conditions: temperature 25±1 ℃, relative humidity 60%–70%, and photoperiod of 16 h light / 8 h dark. Fresh cabbage leaves were provided daily. Nine newly hatched first-instar larvae were selected and divided into three biological replicates, each containing three individuals (n=3). The larvae were starved for 24 hours in sterile petri dishes to reduce interference from exogenous intestinal microorganisms. In a laminar flow hood, the larvae were immersed in 75% ethanol for 1 min, followed by washing their body surface three times with sterile water to eliminate interference from surface microorganisms.

[0057] 1.2 Culture medium preparation

[0058] The main components include the preparation of the following culture media:

[0059] LB liquid medium: 25 g LB broth powder, 1000 mL distilled water, adjust pH to 7.0-7.5, autoclave at 121 ℃ for 15 min.

[0060] LB solid medium: 25 g LB broth powder, 15 g agar powder, 1000 mL distilled water, adjust pH to 7.0-7.5, autoclave at 121 ℃ for 15 min, pour into plates after sterilization for later use.

[0061] Ammonia assimilation solid medium: 1.4 g potassium dihydrogen phosphate, 2.1 g sodium dihydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 10 g glucose, 1 g ammonium chloride, 15 g agar powder, pH 7.0-7.4, 1000 mL distilled water, autoclaved at 121 ℃ for 15 min, pour into plates for later use.

[0062] Ammonium nitrogen liquid culture medium: 1.4 g potassium dihydrogen phosphate, 2.1 g sodium dihydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 10 g glucose, 1 g ammonium chloride, pH 7.0~7.4, 1000 mL distilled water, autoclaved at 121 ℃ for 15 min.

[0063] Nitrate nitrogen liquid culture medium: replace ammonium chloride with 1 g of sodium nitrate, and the remaining components are the same as those for ammonium nitrogen liquid culture medium.

[0064] Nitrite nitrogen liquid culture medium: replace ammonium chloride with 1 g of sodium nitrite, and the remaining components are the same as those for ammonium nitrogen liquid culture medium.

[0065] 1.3 Screening Methods

[0066] The pretreated insect samples were placed in 1.5 mL sterile centrifuge tubes, and 900 μL of sterile phosphate buffer was added. The mixture was then thoroughly homogenized using a sterile grinding rod to obtain a bacterial suspension. The suspension was then diluted with sterile water for 10 minutes. -2 10 -3 10 -4 Serial dilutions were performed, with 100 μL of each dilution spread onto the surface of ammonia assimilation solid medium plates, with three replicates for each dilution. The plates were incubated upside down at 30 °C for 1–5 days, and colony growth was observed. Based on colony appearance time, size, color, edge morphology, and surface characteristics, colonies growing well on the ammonia assimilation medium were selected and purified three times consecutively using the streak plating method to obtain single-colony pure cultures. The obtained candidate strains were inoculated into LB liquid medium and cultured at 30 °C with shaking at 180 r / min until OD500 reached. 600 =1, serving as seed liquid for subsequent nitrogen source utilization and ammonia assimilation performance testing.

[0067] Several bacterial strains derived from diamondback moth were screened to identify those that could stably grow on media with ammonium as the sole inorganic nitrogen source. Further screening revealed that strain HUBU1 exhibited rapid growth and good stability on both ammonia assimilation plates and ammonium nitrogen liquid media, and was therefore selected as the target functional strain.

[0068] 1.4 Colony morphology characteristics

[0069] The purified strain HUBU1 was inoculated onto LB solid medium and ammonia assimilation solid medium, respectively, and incubated at 30 ℃ for 3-5 days. Colony morphology was observed. The results are as follows: Figure 1 As shown in the figures, A represents the colony morphology of the target strain HUBU1 on LB solid medium, and B represents the colony morphology of the target strain HUBU1 on ammonia assimilation solid medium. The figures show that on ammonia assimilation solid medium, colonies grow slowly, are small, round, opaque, grayish-white, relatively dry, and adhere well. On LB solid medium, colonies grow quickly, are round, opaque, pale yellow, and have a relatively dry surface. The bacteria are Gram-positive after staining.

[0070] 1.5 16S rRNA gene amplification and sequence analysis

[0071] Genomic DNA was extracted from strain HUBU1 using the bacterial genomic DNA extraction kit (DP302) from Tiangen Biotech (Beijing) Co., Ltd. PCR amplification of the 16S rRNA gene was performed using universal primers 27F and 1492R. The universal primer sequences are as follows:

[0072] Forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO:2);

[0073] Reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO:3);

[0074] The PCR reaction system (25 μL) consisted of: 22 μL of Gold Mix (Green), 1 μL of 27F primer (10 μM), 1 μL of 1492R primer (10 μM), and 1 μL of template DNA.

[0075] PCR reaction program: 95 ℃ pre-denaturation for 5 min; 95 ℃ denaturation for 30 s, 56 ℃ annealing for 15 s, 72 ℃ extension for 30 s, for a total of 30 cycles; 72 ℃ final extension for 5 min.

[0076] The amplification products were detected by 1% agarose gel electrophoresis for 25 min. DNA dye was added, and the bands were observed under UV light. PCR products with clear bands were selected and sent to Sangon Biotech Co., Ltd. for Sanger sequencing. The obtained sequence was 1235 bp in length, and its nucleotide sequence is shown in SEQ ID NO:1.

[0077] 16S rDNA sequence of HUBU1 strain (SEQ ID NO:1):

[0078]

[0079] The 16S rRNA gene sequence (SEQ ID NO:1) obtained by sequencing was compared with the NCBI database using BLAST. The results showed that the target strain had the highest homology (96.95%) with Actinomadura nitritigenes NBRC 15918 (accession number NR112736.1). Therefore, the strain was named Actinomadurasp. HUBU1.

[0080] 1.6 Phylogenetic Analysis

[0081] A phylogenetic tree was constructed using MEGA 12 software based on the neighbor-joining method, with 1000 replicates for bootstrapping testing. *Spirillospora albida* was used as the outgroup for definitive determination. The phylogenetic tree results are as follows: Figure 2 As shown, strain HUBU1 clusters with related standard strains of the genus Actinomadura into the same branch.

[0082] 1.7 Preservation of Strains

[0083] The strains selected above were deposited at the China Center for Type Culture Collection on April 24, 2026, and were classified as Actinomadura sp. HUBU1, with accession number CCTCC NO:M 2026802, and the deposit address is No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0084] Example 2: Morphological characteristics and growth characteristics of strain HUBU1 under different nitrogen sources

[0085] 2.1 Determination of growth curves under different nitrogen sources

[0086] Strains HUBU1 were cultured in LB liquid medium at 30 °C with shaking at 180 rpm until the logarithmic growth phase (OD200). 600 =1), and inoculated into ammonium nitrogen liquid medium, nitrate nitrogen liquid medium, and nitrite nitrogen liquid medium at an inoculum rate of 1% (v / v), respectively, and cultured at 30 ℃ with shaking at 180 rpm. Samples were taken at 0, 12, 24, 36, 48, and 60 h to measure the OD of the culture medium. 600 Values ​​are used to plot growth curves.

[0087] Figure 3 This is a growth curve of strain HUBU1 under different nitrogen source conditions. From... Figure 3It can be seen that strain HUBU1 can grow in all three inorganic nitrogen source media over time, but the growth status differs significantly, generally showing the following order: NH4Cl (ammonium nitrogen) > NaNO3 (nitrate nitrogen) > NaNO2 (nitrite nitrogen). Under ammonium nitrogen conditions, OD... 600 The strain reached a plateau at approximately 36 hours. These results indicate that the strain exhibits a clear preference for ammonium nitrogen and possesses strong potential for ammonium nitrogen utilization. It also has some ability to utilize nitrate nitrogen, but because nitrate needs to be reduced before entering the assimilation pathway, its utilization efficiency is lower than that of ammonium nitrogen, resulting in moderate growth. When NaNO2 is used as the sole nitrogen source, the strain exhibits almost entirely limited growth. OD throughout the culture period... 600 The concentration remained relatively stable within the range of 0.05-0.08, with growth plateauing after 24 hours and showing almost no significant increase at 48 and 60 hours. The corresponding time point in the figure is marked as 'c', indicating that it was significantly lower than the other two groups. This suggests that the bacterium has a weak ability to utilize nitrite nitrogen. This may be because nitrite itself is somewhat toxic to cells, and its metabolic transformation process places higher demands on the bacterial cells, thus hindering rapid bacterial proliferation.

[0088] Example 3: Determination of ammonia nitrogen assimilation capacity of strain HUBU1

[0089] 3.1 Plotting the Ammonia Nitrogen Standard Curve

[0090] Prepare ammonium chloride standard solutions with concentration gradients of 0, 0.02, 0.05, 0.10, 0.50, 1.00, and 2.00 mg / L. Take 50 mL of each concentration standard solution into a 50 mL colorimetric tube, add 1.0 mL of potassium sodium tartrate solution (500 g / L), mix well, then add 1.5 mL of Nessler's reagent and mix well. After standing for 10 min, measure the absorbance at 420 nm using a 10 mm path length cuvette, with water as a reference. Plot a standard curve with the corrected absorbance (minus the absorbance of the zero-concentration blank tube) on the ordinate and the ammonia nitrogen concentration on the abscissa, as shown below. Figure 4 As shown in the figure. This standard curve is used for converting ammonia nitrogen concentration in samples.

[0091] 3.2 Ammonia nitrogen removal test

[0092] Strain strain HUBU1, in its logarithmic growth phase, was inoculated at a rate of 1% (v / v) into 100 mL of ammonium nitrogen liquid medium and cultured at 30 ℃ with shaking at 180 rpm. Samples were taken at 0, 12, 24, 36, 48, and 60 h, and the supernatant was collected by centrifugation. The ammonia nitrogen concentration in the supernatant was determined according to method 3.1. Three replicates were performed for each time point. The ammonia nitrogen removal rate was calculated using the following formula:

[0093] Removal rate (%) = (C0 - C) t) / C0×100%;

[0094] Where C0 is the initial ammonia nitrogen concentration, C t Let t be the remaining ammonia nitrogen concentration at time t.

[0095] 3.3 Results

[0096] The strain was inoculated into ammonium chloride liquid medium and cultured for 60 h. The ammonia nitrogen concentration and removal rate were then measured. Figure 5 This is a graph showing the change in ammonia nitrogen concentration during the cultivation of strain HUBU1. One-way ANOVA showed that the ammonia nitrogen concentration decreased significantly over time (F...). 5,10 =85.38, P < 0.001). Post-hoc multiple comparisons showed no significant difference between 0 h and 12 h (P = 0.5929), but both were significantly higher than at 24 h and subsequent time points (P < 0.05). The ammonia nitrogen concentration at 24 h was significantly higher than at 36, 48, and 60 h (P < 0.01). There was no significant difference between 36 h and 48 h (P = 0.7570), but both were significantly higher than at 60 h (P < 0.05). Figure 5 As shown, different letters indicate significant differences (P<0.05). After 60 h of cultivation, the ammonia nitrogen concentration decreased significantly from the initial 263.95±2.99 mg / L to 217.82±2.05 mg / L (paired t-test, t2=43.44, P<0.001), and the ammonia nitrogen removal rate was 17.5%.

[0097] The above results demonstrate that strain HUBU1 can effectively reduce the ammonia nitrogen concentration in the culture medium in a culture system where ammonium salt is the sole inorganic nitrogen source, exhibiting a clear ammonia assimilation capacity. This embodiment functionally proves that the strain of this application can be used as an ammonia assimilation functional bacterium in technical scenarios such as nitrogen retention in composting, nitrogen retention in organic solid waste, and nitrogen removal from ammonia-containing wastewater.

[0098] Example 4: Determination of the activity of key enzymes in ammonia assimilation of strain HUBU1

[0099] 4.1 Preparation of crude enzyme solution

[0100] The HUBU1 strain was cultured according to the method in Example 3, and bacterial cells were collected at 12, 24, 36, 48, and 60 h. The cells were centrifuged at 8000 r / min for 10 min at 4 °C, the supernatant was discarded, and the cells were washed twice with phosphate-buffered saline (pH=7.0). The cells were resuspended in an appropriate amount of buffer and disrupted by sonication (ice bath, 200 W, sonication for 3 s, 10 s interval, repeated 30 times). The supernatant was collected after centrifugation as the crude enzyme solution. Three biological replicates were set up for each time point.

[0101] 4.2 Glutamate dehydrogenase (GDH) activity assay

[0102] The GDH activity assay kit from Beijing Solarbio Science & Technology Co., Ltd. was used, and the procedure was followed according to the instructions. Assay principle: NADH oxidation method. Enzyme activity unit definition: Under standard reaction conditions, the amount of enzyme required to catalyze the oxidation of 1 μmol of NADH per minute is defined as 1 enzyme activity unit (U).

[0103] 4.3 Glutamine synthase (GS) activity assay

[0104] The GS activity assay kit from Beijing Solarbio Science & Technology Co., Ltd. was used, and the procedure was followed according to the instructions. Assay principle: γ-glutamyl hydroxamic acid colorimetric method. Enzyme activity unit definition: Under standard reaction conditions, the amount of enzyme required to generate 1 μmol of γ-glutamyl hydroxamic acid per minute is defined as 1 enzyme activity unit (U).

[0105] 4.4 Glutamate synthase (GOGAT) activity assay

[0106] The GOGAT activity assay kit from Beijing Solarbio Science & Technology Co., Ltd. was used, and the procedure was followed according to the instructions. Assay principle: NADH consumption method. Enzyme activity unit definition: Under standard reaction conditions, the amount of enzyme required to oxidize 1 μmol of NADH per minute is defined as 1 enzyme activity unit (U).

[0107] 4.5 Statistical Analysis

[0108] All data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA and Tukey's post-hoc test, with a significance level set at P < 0.05.

[0109] 4.6 Enzyme activity assay results

[0110] 4.6.1 Dynamic changes in GDH activity

[0111] Figure 6 This is a graph showing the changes in GDH activity of strain HUBU1 at different culture time points. One-way ANOVA showed highly significant differences in GDH activity at different time points (F4, ...). 10= 13.14, P < 0.001. The highest enzyme activities were observed at 24 h and 36 h, at 0.430 ± 0.031 U / mL and 0.439 ± 0.031 U / mL, respectively, with no significant difference between the two, but both significantly higher than those at 12 h, 48 h, and 60 h (P < 0.05). There was no significant difference in enzyme activity between 48 h (0.349 ± 0.044 U / mL) and 12 h (0.336 ± 0.027 U / mL), but both were significantly higher than those at 60 h (0.257 ± 0.036 U / mL) (P < 0.05). These results indicate that GDH maintained a high level during the mid-stage of cultivation (24–36 h), possibly rapidly assimilating ammonium to glutamate under high ammonium concentrations.

[0112] 4.6.2 Dynamic changes in GS activity

[0113] Figure 7 One-way ANOVA showed that there were significant differences in GS activity at different time points (F4, 10 = 3.94, P = 0.035). The enzyme activity at 48 h (0.523 ± 0.040 U / mL) was significantly higher than that at 12 h (0.419 ± 0.045 U / mL) (P = 0.018), while there were no significant differences among the other time points (P > 0.05). The results indicate that during the stationary growth period (after 48 h), ammonium concentration decreases, the high-affinity GS pathway is activated, and GS activity increases to capture low concentrations of ammonium.

[0114] 4.6.3 Dynamic changes in GOGAT activity

[0115] Figure 8 This is a graph showing the changes in GOGAT activity of strain HUBU1 at different culture time points. One-way ANOVA showed no significant difference in GOGAT activity at different time points (F4, ...). 10 = 3.09, P = 0.068). Tukey's test revealed a significant difference in enzyme activity between 12 h and 36 h (P = 0.023), while no significant differences were found between other time points (P > 0.05). The results indicate that GOGAT enzyme activity is relatively stable throughout the culture period. GOGAT, as an auxiliary or compensatory pathway for ammonia assimilation, converts glutamine and α-ketoglutarate into two molecules of glutamate, maintaining the smooth operation of the metabolic pathway. Its activity may be regulated by substrate (glutamine) concentration.

[0116] The above enzymatic results indicate that strain HUBU1 can dynamically and synergistically utilize the GDH and GS-GOGAT pathways to achieve ammonia assimilation at different culture stages: the GDH pathway has a certain advantage when the ammonium concentration is high, while the GS-GOGAT pathway is enhanced when the ammonium concentration decreases, thus achieving sustained ammonia utilization. This metabolic characteristic makes it suitable for biological treatment systems with dynamically changing ammonia concentrations, demonstrating good process adaptability.

[0117] Example 5: Preparation and Application of Microbial Inoculants

[0118] 5.1 Preparation of microbial agents

[0119] The purified Actinomadura sp. HUBU1 was inoculated into LB liquid medium and cultured at 30°C with shaking at 180 rpm until the logarithmic growth phase (OD200). 600 = 1). Collect bacterial cells by centrifugation of the culture medium, wash twice with sterile physiological saline, and then resuspend in a cryoprotectant containing 5% glycerol, 2% trehalose, 1% skim milk, and 0.2% polyvinylpyrrolidone, adjusting the bacterial concentration to 1.0 × 10⁻⁶. 9 ~1.0×10 10 The liquid inoculum can be prepared by adding cfu / mL. Alternatively, wheat bran (mixed in a 1:2 ratio) can be added and dried to prepare a solid inoculum.

[0120] 5.2 Application Method Examples

[0121] Inoculating the above-mentioned microbial agent at 1%–10% (v / v) into wastewater containing ammonium nitrogen, organic solid waste composting systems, or other ammonia nitrogen treatment systems, and treating them at 25–40 °C and pH=6.5–8.5 can effectively reduce the concentration of ammonium nitrogen in the system and reduce the loss of nitrogen in the form of ammonia gas volatilization.

[0122] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A strain of Actinomadura sp. HUBU1, which efficiently utilizes ammonium nitrogen, was deposited on April 24, 2026, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2026802, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. Actinomadura sp. HUBU1 according to claim 1, which was isolated from the intestine of diamondback moth (Plutella xylostella).

3. The Actinomadura sp. HUBU1 strain according to claim 1 or 2, wherein the glutamate dehydrogenase activity of the strain reaches its peak between 24 and 36 hours of culture, and the glutamine synthase activity is significantly higher at 48 hours of culture than at 12 hours of culture.

4. The Actinomadura sp. HUBU1 strain according to claim 1 or 2, wherein after being cultured in ammonium nitrogen liquid medium for 60 hours, the ammonia nitrogen removal rate is not less than 17%.

5. A microbial inoculant comprising Actinomadura sp. HUBU1 as described in any one of claims 1-4.

6. The microbial agent according to claim 5, used to remove ammonium nitrogen in a liquid environment.

7. The use of Actinomadura sp. HUBU1 as described in any one of claims 1-4 in the preparation of microbial preparations for assimilating ammonium nitrogen.

8. The application according to claim 7, wherein the microbial preparation is used to treat wastewater containing ammonia nitrogen or organic solid waste.

9. A method for reducing ammonia nitrogen loss during organic waste treatment, comprising: The Actinomadura sp. HUBU1 of any one of claims 1-4, or the microbial agent of claim 5 or 6, is inoculated into organic waste for treatment.

10. The method according to claim 9, wherein the inorganic nitrogen source in the culture medium used for culturing the Actinomadura sp. HUBU1 is ammonium nitrogen.

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