Alcaligenes faecalis ALA-1 and application thereof in nitrous oxide emission reduction

Abstract

This invention relates to the field of microbial technology, specifically disclosing a strain of *Alcaligenes faecalis* ALA-1 and its application in nitrous oxide emission reduction. This invention provides a strain of *Alcaligenes faecalis* (… Alcaligenes faecalis ALA-1, with accession number CGMCC No. 35322, can efficiently convert N... 2 O is restored to N 2 When applied to aerobic composting processes, it can significantly reduce nitrogen emissions during the composting process. 2 O2 emissions will promote the green and low-carbon development of the organic solid waste composting industry.

Description

A strain of Alcaligenes faecalis ALA-1 and its application in nitrous oxide emission reduction Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a strain of Alcaligenes faecalis ALA-1 and its application in nitrous oxide emission reduction. Background Technology

[0002] Nitrous oxide (N2O) is a potent and persistent greenhouse gas, ranking as the third largest greenhouse gas. With increasing demand for food and the generation of organic waste, N2O emissions are projected to worsen further. It is estimated that approximately 8% of N2O generation is due to the management of livestock manure and municipal waste.

[0003] Although aerobic composting technology is widely used in organic waste management, the N2O emissions generated during the process have become a pressing obstacle, severely restricting the green and low-carbon development of the organic solid waste composting industry. Furthermore, in microbial nitrogen metabolism, N2O emissions depend on the combined effects of production and reduction. N2O production mainly includes heterotrophic denitrification, nitrifying bacterial denitrification, and anaerobic ammonia oxidation, while N2O reduction relies solely on two gene types, nosZ I and nosZ II, encoding nitrous oxide reductase (N2OR), driving the conversion of N2O to N2.

[0004] Numerous research teams are dedicated to finding microbial strains capable of effectively reducing N2O, aiming to reduce N2O emissions at the source. Currently, while some research exists on N2O reduction using microorganisms, it primarily focuses on screening common denitrifying bacteria such as Pseudomonas and Bacillus. Furthermore, these strains often exhibit insufficient N2O reduction efficiency and stability in the complex environments of actual composting (e.g., high temperature, high organic matter, complex nitrogen forms). There are few reports on the screening of strains specifically designed for highly efficient N2O reduction, especially those that can effectively function during the composting cooling period (i.e., the main N2O production stage). Some studies have found that *Alcaligenes faecalis* possesses unique metabolic capabilities in nitrogen conversion, demonstrating its ability to catalyze the oxidation of ammonia to nitrogen (N2) via hydroxylamine (NH2OH) under aerobic conditions. However, there are no reports on its N2O reduction function, and its N2O reduction capacity in practical composting applications remains to be considered. Summary of the Invention

[0005] One of the objectives of this invention is to provide a novel strain with highly efficient nitrous oxide (N2O) reduction function and its application.

[0006] This invention provides a strain of Alcaligenes faecalis ALA-1, with accession number CGMCC No. 35322.

[0007] This strain was deposited on July 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and was classified as Alcaligenes faecalis, with accession number CGMCC No. 35322.

[0008] This invention, through specific domestication and cultivation, screened and obtained a new strain of Alcaligenes faecalis, ALA-1, which can efficiently reduce N2O. It can grow using N2O as a nitrogen source and effectively reduce N2O, thereby reducing this greenhouse gas. When applied to aerobic composting, it can significantly reduce N2O emissions during the composting process, thus lowering greenhouse gas emissions from the organic solid waste composting industry.

[0009] The present invention also provides a microbial agent containing the above-mentioned Alcaligenes faecalis ALA-1.

[0010] The microbial agent of the present invention is a liquid fermentation broth or a solid freeze-dried powder.

[0011] The microbial agents of the present invention also include strains that have the same or different efficacy as Alcaligenes faecalis ALA-1.

[0012] The microbial agent of the present invention may also contain other bacteria with similar or different functions. It may be a solid microbial agent or a liquid microbial agent. Those skilled in the art can prepare the microbial agent according to methods known in the art.

[0013] The present invention also provides the application of the above-mentioned Alcaligenes faecalis ALA-1 or bacterial agent in nitrous oxide emission reduction, in aerobic composting, and in reducing N2O gas emissions during aerobic composting.

[0014] In the application of this invention, the raw materials for aerobic composting include animal manure and straw.

[0015] The present invention also provides a method for reducing N2O emissions during aerobic composting, which includes the step of inoculating the above-mentioned Alcaligenes faecalis ALA-1 or bacterial agent during the cooling period.

[0016] In the method of the present invention, the raw materials for aerobic composting include animal manure and straw, and the temperature during the cooling period is not higher than 40°C (preferably, the pH value is between 7.0 and 9.0).

[0017] And / or, inoculate with 0.1%-0.2% (mass fraction) of wet compost material.

[0018] Preferably, the C / N ratio of the raw materials for aerobic composting is 25:1 to 30:1, and the initial moisture content is 60% to 70%.

[0019] The Alcaligenes faecalis ALA-1 of the present invention can be used in aerobic composting processes, especially during the cooling period (temperature stable below 40°C, preferably 25-35°C; pH maintained at 7-9). By inoculating it (preferably at 0.1-0.2% (mass fraction) of wet compost material) into the compost material, its highly efficient nitrous oxide reductase activity encoded by the nosZ gene can reduce N2O generated during composting to N2, thereby significantly reducing N2O gas emissions during composting.

[0020] The beneficial effects of this invention are at least as follows:

[0021] This invention provides a novel strain of Alcaligenes faecalis ALA-1, which can efficiently reduce N2O to N2. When applied to aerobic composting processes, it can significantly reduce N2O emissions by more than 80%, promoting the green and low-carbon development of the organic solid waste composting industry. Attached Figure Description

[0022] Figure 1 shows the plate culture of the bacteria (left) and the microscopic observation after Gram staining (right).

[0023] Figure 2 shows the N2O removal effect of Alcaligenes faecalis ALA-1 and Pseudomonas stutzeri (ATCC17588) under pure culture conditions.

[0024] Figure 3 shows the daily and cumulative N2O emissions for different treatment groups during the composting process. Detailed Implementation

[0025] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0027] Example 1

[0028] I. Targeted screening methods for target strains are as follows:

[0029] 1. Sample collection: Collect samples during the cooling period of pig manure composting (usually when the composting temperature is stable below 40.0℃ and the pH value is between 7.0 and 9.0).

[0030] 2. Preparation of bacterial suspension: Weigh 10 g (wet weight) of sample, add 100 mL of sterile water, and place in a sterile Erlenmeyer flask. Shake on a constant temperature shaker (30±1℃, 150 rpm) for 30 minutes to fully disperse the microorganisms. After standing for 5 minutes, take the supernatant as the initial bacterial suspension. If necessary, perform 10-fold serial dilutions.

[0031] 3. Enrichment culture and domestication:

[0032] (1) Apparatus: A pressure-resistant and sealed 250ml serum bottle was used as the enrichment device.

[0033] (2) Culture medium: enrichment medium (L -1 ): 3.48 g K2HPO4, 0.195 g NH4Cl, 4 g succinic acid, 0.10 g glutamic acid, 0.04 g aspartic acid, 0.5 g NaCl, 0.2 g nitrotriacetic acid, 0.3 g MgSO4·7H2O, 0.015 g CaCl2·7H2O, 0.002 g FeSO3·7H2O, 0.1 mL trace element solution and 0.1 mL vitamin solution.

[0034] Trace element solution (L -1 ): 17.65 g EDTA(III), 109.5 g ZnSO4·7H2O, 50 g FeSO4·7H2O, 15.4 g MnSO4·H2O, 3.92 g CuSO4·5H2O, 2.48 g Co(NO3)2·6H2O, and 1.14 g H3BO3. Add H2SO4 until the solution is clear.

[0035] Vitamin solution (L) -1 ): 10.0g niacin, 5.0g thiamine hydrochloride and 0.10g biotin.

[0036] (3) Gas environment and domestication strategies:

[0037] Initially: High-purity N2O gas (concentration ≥ 80%) is introduced into the device until the initial partial pressure is 0.1 MPa, replacing the original gas in the device and serving as the sole nitrogen source.

[0038] Inoculation: Inoculate the bacterial suspension into the device containing enrichment medium at an inoculation rate of 10% (v / v).

[0039] Culture conditions: Place in a constant temperature shaker at 30±1℃ and shake at 150 rpm.

[0040] Medium replacement and pressure escalation: Fresh enrichment medium was introduced every 24 hours, and N2O was purged again. Each time the medium was replaced, the N2O partial pressure was gradually increased: starting at 0.1 MPa, increasing sequentially to 0.2 MPa, 0.3 MPa, 0.4 MPa, and finally reaching 0.5 MPa. This high-pressure selection process aimed to screen for strains that could tolerate high concentrations of N2O and grow effectively within it.

[0041] Acclimation period: The above enrichment and stress escalation process continues for 2-3 generations (about 7-10 days) to ensure the dominant growth of highly tolerant strains.

[0042] 4. Separation and purification:

[0043] (1) Take the enriched culture medium and perform a 10-fold serial dilution (e.g., 10... -1 Up to 10 -8 ).

[0044] (2) Using the spread plate method or the pour plate method, bacterial solutions of different dilutions are inoculated onto the screening and identification solid culture medium plates.

[0045] Screening and identification media include (L -1 ): KNO3 1.0g, sodium citrate 8.5g, bromothymol blue 0.05g, magnesium sulfate heptahydrate 1.0g, ferric chloride hexahydrate 0.05g, potassium dihydrogen phosphate 1.0g, calcium chloride dihydrate 0.2g, asparagine 1.0g.

[0046] Solid culture medium agar plates are made by adding 2.0% agar.

[0047] (3) Culture conditions: Invert the plate in a constant temperature incubator at 30±1℃ and culture for 48-72 hours. Introduce high-purity N2O gas (concentration ≥ 80%) until the initial partial pressure is 0.1 MPa.

[0048] (4) Selecting single colonies: Observe the single colonies growing on the plate. Based on the differences in morphology (such as size, shape, color, edge, transparency, etc.), select at least 30 single colonies with different morphologies for purification. Perform purification by streak plating at least 3 times until a pure culture is obtained. Temporarily number and store the purified strains on screening and identification medium slant (store at 4℃) or in glycerol tubes (store at -80℃).

[0049] II. Identification of the target strain

[0050] 1. Morphological observation: Gram staining of the purified strain revealed that it was a Gram-negative short bacillus (Figure 1).

[0051] 2. Metagenomic identification:

[0052] Total microbial DNA was extracted from pure cultures and pig manure compost samples using the Cetyltrimethyl ammonium bromide (CTAB) method. Metagenomic shotgun sequencing libraries were constructed using the NEB® Next Ultra™ DNA library preparation kit. During library construction, the extracted DNA was fragmented to 150 bp using a Covaris M220 ultrasonic disruptor (Covaris S2 System, MA, USA). PE libraries were then constructed through adapter selection, purification, and PCR amplification. Following PCR amplification, preliminary quantification was performed stepwise, including library dilution and detection for accurate quantification. The libraries were then sequenced on an Illumina Novaseq 6000 sequencing platform (Illumina Inc., San Diego, CA) using a 150 bp paired-end sequencing strategy.

[0053] Metagenomic assembly and identification were then performed to obtain the total abundance (TPM) of unigenes in pure culture and compost samples, which were then used for species annotation, and the actual relative abundance in the samples was predicted. Non-redundant protein sequences were compared with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database for functional annotation.

[0054] The results showed that the strains screened from pig manure and kitchen waste compost samples using the above culture method all had a similarity of more than 99% to the Alcaligenes faecalis model strain. At the same time, the abundance of the nosZ gene carried by Alcaligenes faecalis in the pure culture was greater than 200 TPM, and it also carried complete denitrification genes (nirK, nirS, norB, norC, nosZ).

[0055] Meanwhile, metagenomic sequencing analysis of enriched cultures and compost samples confirmed that the abundance of the nosZ gene of Alcaligenes faecalis in pure cultures was significantly higher than that in the original pig manure compost samples (background value of 50 TPM).

[0056] Finally, a strain of Alcaligenes faecalis was selected, named ALA-1, and biologically preserved, with the preservation number CGMCC No. 35322.

[0057] Example 2 Functional Verification Experiment

[0058] 1. Quantitative determination of N2O removal effect:

[0059] (1) Strains preparation: The strain ALA-1 obtained in Example 1 was activated and cultured in enriched medium (containing no ammonium or nitrate, with N2O as the sole nitrogen source) until the late logarithmic growth phase. Simultaneously, a known traditional denitrifying bacterium with N2O reducing ability, *Pseudomonas stutzeri* (ATCC 17588), was cultured as a control group. *Pseudomonas stutzeri* was activated and cultured in nutrient agar medium until the logarithmic growth phase.

[0060] (2) Test apparatus: Sterilized, pressure-resistant, and sealable serum bottles are used and connected to an N2O gas supply system.

[0061] (3) Experimental system:

[0062] Add a certain volume (e.g., 50 mL) of nitrogen-free basal medium (based on the enrichment medium described in Example 1, but with nitrogen sources such as NH4Cl, glutamic acid, and aspartic acid removed, and succinic acid used as the carbon source) to the bottle. Inoculate the bacterial culture to allow the initial OD to rise. 600 Reach 0.20. Seal the serum bottle and purge the air from the bottle three times using a vacuum pump. Inject high-purity N2O gas into the bottle until the initial partial pressure is 0.2 atm (approximately 0.02 MPa) or the set concentration (e.g., 1000 ppmv). Record the initial N2O concentration (C0).

[0063] (4) Culture and monitoring: The serum bottles were placed in a constant temperature shaker (150 rpm) at 30±1℃. At the set time points, 1 mL of headspace gas was drawn from the bottle using an airtight syringe, and the N2O concentration (Ct) was detected using a gas chromatograph.

[0064] (5) Data analysis: Calculate the N2O removal rate: Removal rate (%) = [(C0- Ct) / C0] × 100%. Plot the N2O removal rate over time (Figure 2).

[0065] (6) Key results are shown in Figure 2: N2O was rapidly removed in the first hour of the N2O removal test. After about 18 hours, the ALA strain was able to remove more than 90% of N2O, which was significantly higher than the 70% of the control group (Xanthomonas pseudoepiplo). The N2O removal rate of the ALA strain reached 98.5% ± 1.2% within 24 hours.

[0066] Example 3: Application Effect Verification (Composting Simulation Experiment)

[0067] This embodiment further demonstrates composting experiments on strain ALA-1 obtained in Example 1. Specifically, pig manure and corn stalks were used as compost materials in an aerobic composting experiment, maintaining an initial C / N ratio of 25:1 and an initial moisture content of approximately 65%. Furthermore, all composting reactors were connected to automatic ventilation systems to maintain continuous ventilation at a rate of 0.36 L / kg dry matter per minute. The compost was turned over every 7 days. During the compost cooling period, N2O emission reduction strain validation was conducted at a temperature of 35.5 ± 0.5°C. o C, the material moisture content was 62.4 ± 0.6%, and the pH was 7.8 ± 0.2. The experimental group (T2) consisted of ALA-1 strain culture solution (10 μL / kg) uniformly inoculated into the compost substrate at 0.2% (wet weight) during turning. 6 CFU / g). The N2O concentration in the reactor outlet gas was monitored periodically, and the cumulative N2O emissions were calculated. A blank control (CK) without inoculation and a positive control (T1) inoculated with Pseudomonas stutzeri strain (ATCC 17588) were set up, with the same inoculation amount as T2.

[0068] The specific results are shown in Figure 3. After inoculation (day 21, indicated by the arrow in the figure), the daily N2O emission flux of the ALA-1 treatment group (T2) rapidly decreased to levels significantly lower than the blank control group (CK) and the *Xanthomonas pseudoepiplo* treatment group (T1). By the end of composting (day 35), the cumulative N2O emissions of the T2 group were more than 80% lower than those of the CK group, and the N2O reduction effect was 30% higher than that of the T1 group, demonstrating a significant reduction effect (p<0.01).

[0069] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A strain of Alcaligenes faecalis ALA-1, characterized in that, The accession number is CGMCCNo.35322.

2. A microbial agent, characterized in that, Contains Alcaligenes faecalis ALA-1 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is either a liquid fermentation broth or a solid freeze-dried powder.

4. The microbial agent according to claim 2 or 3, characterized in that, The microbial agent also includes strains with the same or different efficacy as Alcaligenes faecalis ALA-1.

5. The application of Alcaligenes faecalis ALA-1 as described in claim 1 or the microbial agent as described in any one of claims 2-4 in aerobic composting.

6. The application of Alcaligenes faecalis ALA-1 as described in claim 1 or the microbial agent as described in any one of claims 2-4 in reducing N2O gas emissions during aerobic composting.

7. The application according to claim 5 or 6, characterized in that, The raw materials for the aerobic composting include animal manure and straw.

8. A method for reducing N2O gas emissions during aerobic composting, characterized in that, The step includes inoculating the Alcaligenes faecalis ALA-1 as described in claim 1 or the bacterial agent as described in any one of claims 2-4 during the cooling period.

9. The method according to claim 8, characterized in that, The raw materials for the aerobic composting include animal manure and straw, with the temperature during the cooling period not exceeding 40°C; and / or, the inoculation amount is 0.1%-0.2% w / w wet compost material.

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