Montesporium g10 and application thereof in degrading kitchen waste odor

Abstract

The application discloses a montagnatia G10 and application thereof in degrading kitchen waste odor, and separates a high-efficiency hydrogen sulfide removing montagnatia CCTCC NO:M2023718 from sludge in a garbage disposal station, wherein the montagnatia has strong environmental adaptability, simple nutrition requirement, can survive in kitchen waste and efficiently degrade hydrogen sulfide and VOCs, the hydrogen sulfide removal rate can reach more than 90%, the VOCs removal rate is 60% to 90%, and the ammonia gas removal rate is 50% to 80%.

Description

(I) Technical Field

[0001] This invention relates to a species of *Trichosporium montana* G10 and its application in degrading odor from kitchen waste. (II) Background Technology

[0002] Odor pollution falls under the category of air pollution and is one of the world's seven major environmental hazards. It has become a hot topic of environmental pollution and an increasingly prominent social problem in public life. Food waste is a major component of urban residents' daily lives and a primary source of organic matter in urban solid waste. Its easy decomposition, high water content, and high organic matter content generate large amounts of strongly pungent odorous gases during collection, transportation, and disposal, directly or indirectly impacting the surrounding environment and human health.

[0003] Currently, there are three main methods for deodorization: physical methods, chemical methods, and biological methods. Physical and chemical methods are suitable for situations with high concentrations of odor pollutants and have advantages such as high odor treatment efficiency, short treatment residence time, and fast start-up; however, they also have disadvantages such as being prone to causing secondary pollution, high equipment costs, and high operational requirements.

[0004] Compared to physical and chemical methods, biological deodorization does not require the addition of chemicals and can be carried out at normal temperatures (10℃~40℃) and pressures. It is also more cost-effective and simpler to operate. Furthermore, microbial degradation typically involves oxidation reactions, with the final products of odor being carbon dioxide, water, sulfates, and nitrates, thus avoiding secondary pollution. The microbial deodorization methods used in landfills, waste compression stations, and waste compactors are similar to those used in livestock farms, primarily employing live bacteria sprays.

[0005] Patent document CN112159775A discloses the application of a bacterial strain in biological deodorization, specifically using a mixture of *Sphingomonas zeylans* cells and carriers such as diatomaceous earth, wheat straw, or corn cobs at a mass ratio of 1:0.8 to 1.2 for deodorizing kitchen waste.

[0006] Patent document CN114672433A discloses a compound microbial deodorizing agent, comprising *Lactobacillus longiflorus*, *Lactobacillus zei*, *Lactobacillus plantarum*, and *Lactobacillus paracasei*, with a total viable count of 13-17 billion / mL for these microorganisms. This deodorizing agent not only deodorizes livestock pens and manure, but also contains non-toxic and non-polluting microorganisms, making it environmentally friendly, harmless to humans, livestock, plants, and soil. It effectively prevents the breeding of mosquitoes and flies in livestock pens, providing a better welfare-oriented farming environment for livestock.

[0007] Therefore, in order to solve the pollution caused by the treatment of kitchen waste and reduce the emission of malodorous gases, developing more efficient deodorizing strains from the microbial-rich natural world is a technical problem that needs to be solved by those skilled in the art. (III) Summary of the Invention

[0008] The purpose of this invention is to provide a new strain of fungi with highly efficient deodorization ability—Montenesporium G10—and its application in degrading the odor of kitchen waste, thus providing a new and highly efficient deodorizing strain for solving the pollution caused by kitchen waste and reducing the emission of malodorous gases.

[0009] The technical solution adopted in this invention is:

[0010] This invention provides a strain with highly efficient deodorizing ability—Trichosporon montevideense G10, deposited at the China Center for Type Culture Collection, accession number: CCTCC NO: M2023718, deposit date: May 10, 2023, deposit address: Wuhan University, Wuhan, China, postcode 430072.

[0011] The *Trichosporium montana* G10 strain obtained in this invention, suitable for biological deodorization, was cultured on a PDA solid plate at 30°C for 2-3 days. The colonies were relatively moist, large and protruding, opaque, milky white, with scattering streaks on the surface. Under an electron microscope, the surface was uneven and contained hyphae.

[0012] This invention also provides an application of the aforementioned Montecristosporum G10 in degrading the odor of kitchen waste. The application involves mixing a biological deodorizing agent with the kitchen waste to be treated and placing it at a constant temperature of 30°C to achieve the purpose of degrading the odor of the kitchen waste. The biological deodorizing agent is a fermentation broth obtained by fermenting Montecristosporum G10 or a bacterial suspension of wet cells in deionized water after centrifugation of the fermentation broth.

[0013] Preferably, the odor from the food waste includes hydrogen sulfide, volatile organic compounds (VOCs), or ammonia.

[0014] Preferably, the volumetric dosage of the biological deodorizing agent is 0.05-0.15 mL / g based on the weight of the kitchen waste to be treated, and more preferably 0.1 mL / g.

[0015] Furthermore, the number of viable bacteria in the fermentation broth or bacterial suspension is greater than 1×10⁻⁶. 8 CFU / mL, preferably 3.0–4.0 × 10⁻⁶. 8 CFU / mL.

[0016] Furthermore, the fermentation broth is prepared as follows: *Trichosporium montana* G10 is inoculated into PDA liquid medium and cultured at 28-35℃ and 100-200 rpm for 20-48 h (preferably 30℃ and 200 rpm for 24 h) to obtain the fermentation broth; the PDA liquid medium consists of: 200 g / L peeled potato, 20 g / L glucose, water as solvent, and natural pH.

[0017] Furthermore, before fermentation, the *Trichosporium montana* G10 is first activated by slant culture and then seed culture is carried out. The seed culture is then inoculated into PDA liquid medium at a volume concentration of 1-3% (preferably 1%). The seed culture is prepared according to the following steps: *Trichosporium montana* G10 is inoculated into PDA solid medium and cultured at 30°C for 60-72 hours. Single colonies are picked and inoculated into PDA liquid medium and cultured at 30°C and 200 rpm for 15-18 hours to obtain the seed culture. The PDA solid medium consists of: 200 g / L peeled potato, 20 g / L glucose, and 15-20 g / L agar, with water as the solvent and natural pH.

[0018] This invention also provides a biological deodorizing agent prepared from *Trichoderma montana* G10 for degrading the odor of kitchen waste. The biological deodorizing agent is a fermentation broth obtained by fermenting *Trichoderma montana* G10 or a bacterial suspension of wet cells in deionized water after centrifugation of the fermentation broth. The biological deodorizing agent is a kitchen waste odor remover, and the odor originates from hydrogen sulfide, VOCs, or ammonia. The kitchen waste includes kitchen waste from canteen waste dumping areas, waste transfer stations, etc.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in the following: The present invention isolates a strain of Montecristosporum CCTCC NO:M2023718, which is highly efficient at removing hydrogen sulfide, from the sludge of the waste treatment plant. It has strong environmental adaptability and simple nutritional requirements. It can survive in kitchen waste and efficiently degrade hydrogen sulfide and VOCs. The hydrogen sulfide removal rate can reach more than 90%, the VOCs removal rate is 60% to 90%, and the ammonia removal rate is 50% to 80%. (iv) Description of the attached drawings

[0020] Figure 1 The images show the front (A) and back (B) morphology of strain G10 colonies.

[0021] Figure 2 The images are SEM scans of strain G10. A shows the surface structure of the strain at 5000x magnification, and B shows the surface structure of the strain at 12000x magnification.

[0022] Figure 3 Phylogenetic tree of strain G10. (V) Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0024] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] PDA solid culture medium: 200 g / L peeled potato, 20 g / L glucose, 15-20 g / L agar, water as solvent, natural pH.

[0026] PDA liquid culture medium: 200 g / L peeled potato, 20 g / L glucose, water as solvent, natural pH.

[0027] Example 1: Screening of strain G10

[0028] (1) Sampling and enrichment: Weigh 5g of sludge (wet weight) from a waste treatment plant in Wuhan and inoculate it into a 250mL Erlenmeyer flask containing 50mL of PDA liquid culture medium. Incubate at 30℃ and 200rpm for 24h on a shaker.

[0029] (2) Dilution and coating: Take 1 mL of the acclimated bacterial culture from step (1) and perform serial dilution in 9 mL of physiological saline. Take 10 mL of each dilution and apply it to the plate. 3 -10 6 100 μL of the diluted solution was spread on PDA solid culture medium plates and incubated in a 30℃ constant temperature incubator for 60-72 h.

[0030] (3) Purification and preservation: Use an inoculation loop to pick single colonies with obvious morphological differences from those in step (2) and inoculate them onto PDA solid medium. Incubate at 30°C for 60 hours. After single colonies have grown, inoculate them again onto PDA solid medium and incubate at 30°C for 60 hours to purify and ensure that pure single colonies are obtained. Record this as strain G10. Pick single colonies G10 and inoculate them into PDA liquid medium test tubes. Incubate at 30°C and 200 rpm for 15-18 hours. Then preserve them in glycerol at -80°C. Fill sterile glycerol preservation tubes with 30% glycerol at a 1:1 ratio.

[0031] Example 2: Identification of strain G10

[0032] 1. Colony morphology observation:

[0033] Using an inoculation loop, pick up strain G10 preserved in Example 1, streak it onto a PDA solid medium plate, and incubate at 30°C for 2-3 days. The colony morphology is shown in the figure. Figure 1 The colonies are relatively moist, large and protruding, opaque, milky white, and have radiating streaks on their surface.

[0034] 2. SEM (Scanning Electron Microscopy) observation:

[0035] Scanning electron microscope image of strain G10 is shown below. Figure 2 The cells are columnar in shape, with an uneven surface and hyphae.

[0036] 3. Molecular identification

[0037] Single colonies of strain G10 were picked and boiled in 10 μL sterile ddH2O PCR tubes for 10 min to serve as DNA templates. ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 were used as templates.

[0038] (5'-TCCTCCGCTTATTGATATGC-3') are the forward and reverse primers for PCR amplification.

[0039] The PCR amplification system was as follows (20 μL): 0.5 μL each of primers ITS1 and ITS4, 1 μL of DNA template, 8 μL of ddH2O, and 10 μL of 2*Rapid Taq Master Mix.

[0040] PCR reaction conditions: pre-denaturation 98℃ for 5 min, denaturation 98℃ for 30 s, annealing 57℃ for 30 s, extension 72℃ for 30 s, 30 cycles, 72℃ for 10 min, and storage at 4℃.

[0041] The obtained sequence (nucleotide sequence as shown in SEQ ID NO.1) was searched using BLAST on the NCBI website. rDNA sequences with high similarity to this sequence were selected and automatically analyzed using MEGA7 software Align by ClusterW. A neighbor-to-neighbor phylogenetic tree (NJ phylogenetic tree) was generated with 1000 repetitions. Figure 3 The results showed that strain G10 was most homologous to NR_073245.1Apiotrichummontevideense CBS 6721ITS region from TYPE material, with a homology of up to 98.64%.

[0042] SEQ ID NO.1:

[0043] attgatcgtg ccttccgtta ggggtgacgc ggaaggatca ttagtgattg ccttaattgg60cttaaaacta tatccatcta cacctgtgaa ctgtttgatt gaatcttcgg attcgatttt120tatacaaaca ttgtgtaatg aacgtcatta gatcataaca aaaaaaaact ttcaacaacg180gatctcttgg ctctcgcatc gatgaagaac gcagcgaaat gcgataagta atgtgaattg240cagaattcag tgaatcatcg aatctttgaa cgcaacttgc gctctctggt attccggaga300gcatgcctgt ttgagtgtca tgaaatctca accattaggg tttcttaatg gcttggattt360ggaggtttgc cagtctgact ggctcctctt aaaagagtta gcaagttgaa ctattgctat420ctggcgtaat aagtttcgct ggaatggtat tgtgaagcgt gcttctaatc gtctttcggg480acaatttttt tgactctggc ctcaaatcag gtaggactac ccgctgaact taagcatatc 540aaa543

[0044] 4. Biolog microbial physiological and biochemical identification

[0045] The metabolic activity of strain G10 to 95 different carbon sources was investigated using the Biolog (GENⅢ) automated microbial identification system. Strain G10 was inoculated into PDA liquid medium in test tubes and cultured at 30°C and 200 rpm for 15 h. After centrifugation, bacterial cells were collected and mixed with inoculum (FF-IF) using a sterile cotton swab to prepare a bacterial suspension. The suspension was adjusted to 75% T / FF using a turbidimeter. 100 μl of the bacterial suspension was added to each well of an 8-well Biolog FF microplate using an 8-well electric dispenser. The microplate was placed in a 30°C incubator, and the results were read using a Biolog reader after 24 h, 48 h, 72 h, 96 h, 168 h, and 240 h.

[0046] Metabolic fingerprint analysis using a Biolog reader revealed that strain G10 could utilize 67 carbon sources with relatively strong capabilities, but could not utilize or had weak utilization capabilities for the other 28 carbon sources. The Biolog system provided the identification results after 72 hours, as shown in Table 1.

[0047] Table 1. Utilization capacity of strain G10 of 95 carbon sources on Biolog FF plates

[0048]

[0049]

[0050] Notes:+,positive;-,negative;B,borderline

[0051] Based on morphological identification, molecular biological and Biolog microbial physiological and biochemical identification, strain G10 was preliminarily identified as *Trichosporon (Apiotrichum) montevideense*, and named *Trichosporon montevideense* G10. This strain has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2023718, and the deposit date is May 10, 2023.

[0052] Example 3: Preparation and application of biological deodorizing agents

[0053] Preparation of inoculum: The glycerol-preserved strain G10 was streaked onto PDA solid medium (same as in Example 1) and incubated at 30°C for 60-72 h. A single colony was picked and inoculated into a test tube containing 5 mL of PDA liquid medium and incubated at 30°C and 200 rpm for 15-18 h to obtain the seed culture. 1 mL of the seed culture was transferred to a 500 mL Erlenmeyer flask containing 100 mL of PDA liquid medium and incubated at 30°C and 200 rpm for 24 h to obtain the fermentation broth (with *Trichoderma montana* colony counts of 3.0–4.0 × 10⁻⁶). 8 CFU / mL is a biological deodorizing agent.

[0054] Experimental setup: Take kitchen waste from the swill storage area of ​​the school cafeteria. The sample should be uniform. Remove bones manually, crush and divide into 500mL shake flasks, 50±2g per flask. Add 5mL of bacterial agent to each shake flask, seal, and place in a constant temperature room at 30℃ for static fermentation. Add an equal amount of purified water to the control group. Measure the gas after 16 hours.

[0055] Hydrogen sulfide and VOCs were measured using the Yiyuntian PTM-600 portable multi-gas analyzer.

[0056] Ammonia determination method: Refer to standard HJ533-2009 Determination of ammonia in ambient air and exhaust gas - Nessler's reagent spectrophotometric method.

[0057] Removal rate calculation: (Control gas volume - Experimental group gas volume) / Control gas volume × 100%

[0058] The test results are shown in Table 2. The test results show that the bacterial solution has an excellent degradation effect on hydrogen sulfide, and also has a certain degree of degradation effect on ammonia and volatile organic compounds.

[0059] Table 2. Degradation effect of strain G10 on hydrogen sulfide, etc.

[0060]

[0061] Three parallel experiments showed that 16 hours after inoculation with the biological deodorizing agent, the degradation rate of ammonia in kitchen waste was 57.41%, the degradation rate of hydrogen sulfide was 95.45%, and the degradation rate of volatile organic compounds was 86.71%. The experimental results show that the biological deodorizing agent made from Monterey sporozoans G10 has a significant degradation effect on the odor generated by kitchen waste and has good application potential.

[0062] Example 4: Small-scale deodorization test using kitchen waste as substrate

[0063] Preparation of inoculum: The fermentation broth obtained in Example 3 was transferred at an inoculum concentration of 2% to a 2000mL Erlenmeyer flask containing 1000mL of LPDA liquid medium. The flask was incubated at 30℃ and 200rpm for 48h to obtain the fermentation broth (with *Trichoderma montana* colony counts of 3.0–4.0 × 10⁻⁶). 8 CFU / mL is a biological deodorizing agent.

[0064] Experimental setup: Fresh kitchen waste was crushed using a blender and divided into 3L garbage bins, each containing 1kg (wet weight) of fresh kitchen waste. Each experiment was conducted in triplicate. The prepared microbial agent was then added at a rate of 0.1mL / g, while the control group received an equal volume of purified water. The mixture was placed in a 30℃ constant-temperature laboratory, and the gas content changes were measured over 3 days.

[0065] Using the method in Example 3, the concentrations of hydrogen sulfide, volatile organic compounds, and ammonia were measured every 24 hours. The results are shown in Table 3.

[0066] The results showed that inoculation with Monterey Pleurotus erythrorhizon G10 effectively reduced the production of odorous gases. On the third day, ammonia emissions were reduced by 73.98%, hydrogen sulfide by 91.40%, and volatile organic compounds by 81.58%.

[0067] Table 3. Degradation effect of strain G10 on hydrogen sulfide, etc.

[0068]

[0069]

Claims

1. Trichosporon montevideense G10, deposited at China Center for Type Culture Collection, accession number: CCTCC NO: M2023718, deposit date: May 10, 2023, deposit address: Wuhan University, Wuhan, China, 430072, China.

2. The application of the *Trichosporium montana* G10 according to claim 1 in the degradation of odor from kitchen waste, characterized in that, The odor from the kitchen waste is hydrogen sulfide, VOCs, or ammonia.

3. The application as described in claim 2, characterized in that, The application is as follows: the biological deodorizing agent is mixed with the kitchen waste to be treated and placed at a constant temperature of 30°C to achieve the purpose of degrading the odor of the kitchen waste; the biological deodorizing agent is the fermentation broth obtained by fermenting and culturing Trichosporium montana G10 or the wet bacterial suspension of the fermentation broth in deionized water after centrifugation.

4. The application as described in claim 3, characterized in that, The fermentation broth or bacterial suspension contains a viable bacteria count greater than 1 × 10⁻⁶. 8 CFU / mL.

5. The application as described in claim 3, characterized in that, The volumetric dosage of the biological deodorizing agent is 0.05-0.15 mL / g based on the weight of the kitchen waste to be treated.

6. The application as described in claim 3, characterized in that, The fermentation broth was prepared as follows: Monterey sporozoans G10 was inoculated into PDA liquid medium and cultured at 28-35℃ and 100-200 rpm for 20-48 hours to obtain the fermentation broth.

7. The application as described in claim 6, characterized in that, Before fermentation, *Trichosporium montana* G10 is first activated by slant culture and then seed culture is carried out. The seed culture is then inoculated into PDA liquid medium at a volume concentration of 1-3%. The seed culture is prepared according to the following steps: *Trichosporium montana* G10 is inoculated into PDA solid medium and cultured at 30°C for 60-72 h. Single colonies are picked and inoculated into PDA liquid medium and cultured at 30°C and 200 rpm for 15-18 h to obtain the seed culture.

8. A biological deodorizing agent prepared from *Trichosporum cantorii* G10 according to claim 1, characterized in that, The biological deodorizing agent is a fermentation broth obtained by fermenting and culturing Trichosporium montana G10, or a bacterial suspension of wet bacterial cells in deionized water after centrifugation of the fermentation broth.

9. The biological deodorizing agent as described in claim 8, characterized in that, The biological deodorizing agent is an odor remover for kitchen waste, and the odor originates from hydrogen sulfide, VOCs, or ammonia.

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