Rhodococcus sp. Ac-P as well as zymophyte liquid and application thereof

Abstract

The invention provides rhodococcus sp. Ac-P as well as a zymophyte solution and application thereof, and belongs to the technical field of microorganisms. The collection number of the Rhodococcus qingshengii Ac-P disclosed by the invention is CGMCC (China General Microbiological Culture Collection Center) No.32243, and the collection number of the Rhodococcus qingshengii Ac-P is CGMCC No.32243. The Rhodococcus fence celebration Ac-P disclosed by the invention can effectively degrade main malodorous gases NH3 and H2S generated in the composting fermentation process of livestock and poultry manure, and also has a relatively high inhibition rate on urease activity. According to the Rhodococcus fence celebrating Ac-P and the zymophyte liquid thereof, an effective microbial solution is provided for odor emission reduction in the livestock and poultry manure composting process, the technical vacancy of the link is filled, and the whole livestock and poultry manure treatment technical system can be perfected; the development of harmless treatment and resource utilization technologies of the livestock and poultry manure is promoted.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a Rhodococcus faecalis Ac-P, its fermentation broth, and its applications. Background Technology

[0002] Composting is an effective way to harmlessly treat solid waste from livestock and poultry farming, with low processing costs and the ability to meet the requirements for waste harmlessness. However, the biggest problem with the application of this technology is the nuisance caused by odor during the composting process. The main components of compost odor include ammonia, hydrogen sulfide, and volatile organic compounds (VOCs), with ammonia having the highest concentration among the odor components and hydrogen sulfide having the highest odor potential.

[0003] Odor release during conventional composting fermentation can be divided into two stages: concentrated release and disordered release. The concentrated release stage mainly occurs during the turning of the compost pile and for 1-2 hours after turning, while the remaining time is the disordered release stage. Data on nitrogen loss due to frequent turning reveals that ammonia release is substantial during the concentrated release stage. Odor release is relatively low during the disordered release stage. Therefore, odor reduction during composting should focus on the concentrated release stage while also addressing the disordered release stage. However, current odor control measures primarily focus on source reduction and end-of-pipe reduction, with a lack of equipment and processes for process-level emission reduction.

[0004] From the perspective of the deodorization process, deodorization technologies can be broadly divided into two categories: one is ex-situ control, which involves removing malodorous substances directly through physical, chemical, and biological means after the malodor has been released; the other is in-situ control, which involves removing malodor by directly degrading malodorous substances in feces or inhibiting odor-producing microorganisms in feces through chemical reagents, enzymes, and functional bacteria. In-situ control technology is currently the main technology for controlling malodor in livestock and poultry manure.

[0005] Currently, the control or treatment of odorous gases in intensive farms is basically limited to exhaust ventilation and air circulation to dilute the level of odorous gases. The control of these odorous gases, from preventing the source to treatment, is basically "non-existent". This is also different from the treatment of livestock and poultry wastewater and manure, and is one of the current problems in animal husbandry. Summary of the Invention

[0006] To solve the above-mentioned technical problems, the present invention provides a Rhodococcus qingshengii Ac-P, the preservation number of which is CGMCC No. 32243.

[0007] The present invention also provides a fermentation broth containing the aforementioned *Rhodococcus fanningeri* Ac-P. Preferably, the viable cell count of the fermentation broth is ≥1×10⁻⁶. 8CFU / mL.

[0008] The present invention also provides a method for preparing the fermentation broth, wherein *Rhodococcus fannings* Ac-P is inoculated into a liquid culture medium and cultured at a constant temperature of 25-40°C with shaking.

[0009] Preferably, the liquid culture medium is #48 liquid culture medium; the inoculum amount of Rhodococcus faecalis Ac-P is 3%-10% (v / v); and the culture rotation speed is 150-200 r / min.

[0010] The present invention also provides the application of the aforementioned Rhodococcus faecalis Ac-P or the aforementioned fermentation liquid in the preparation of deodorizing functional liquid fertilizer and in the inhibition of urease activity.

[0011] This invention also provides a method for preparing deodorizing liquid fertilizer, wherein cow dung, water, and Rhodococcus faecalis Ac-P fermentation liquid are mixed at a mass-volume ratio of 1g:10-20mL:0.5-1mL as raw materials, and after aeration for 3-5 days, the solid and liquid are separated, and the liquid part is the deodorizing liquid fertilizer.

[0012] The present invention also provides a deodorizing functional liquid fertilizer prepared by the aforementioned preparation method.

[0013] The present invention also provides the application of the aforementioned deodorizing liquid fertilizer in the treatment of odorous gases in livestock and poultry farming, wherein the odorous gases include NH3 and H2S.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The *Rhodococcus fanningsense* Ac-P strain of this invention can effectively degrade the main odorous gases NH3 and H2S produced during the composting and fermentation of livestock and poultry manure. Experiments show that it has a good effect on reducing the release of ammonia and hydrogen sulfide during livestock and poultry manure fermentation, effectively improving the odor problem during composting. Furthermore, *Rhodococcus fanningsense* Ac-P also helps regulate the pH during livestock and poultry manure fermentation, providing a more suitable environment for microbial growth and fermentation, and promoting the smooth progress of the fermentation process.

[0016] Rhodococcus fanningsense Ac-P also exhibits a high inhibition rate against urease activity. Urease catalyzes the decomposition of urea in livestock and poultry manure to produce ammonia. Rhodococcus fanningsense Ac-P inhibits urease activity, which can reduce ammonia production at the source, further reducing the odor during the fermentation process of livestock and poultry manure. It also helps to reduce nitrogen loss and improve the nutrient retention rate of fertilizer.

[0017] The Rhodococcus fanningsacchari Ac-P and its fermentation liquid of this invention provide an effective microbial solution for odor reduction during the composting process of livestock and poultry manure, filling a technological gap in this process, helping to improve the entire technical system of livestock and poultry manure treatment, and promoting the development of harmless treatment and resource utilization technologies for livestock and poultry manure.

[0018] Biological Preservation Instructions

[0019] Biological material (strain): Ac-P, classified as Rhodococcus qingshengii, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 32243, and deposited on October 17, 2024. Attached Figure Description

[0020] Figure 1 The colony morphology of Rhodococcus faecalis Ac-P is shown.

[0021] Figure 2 Phylogenetic tree of Rhodococcus faecalis Ac-P;

[0022] Figure 3 The growth curve of Rhodococcus faecalis Ac-P is shown.

[0023] Figure 4 The pH change curve of Rhodococcus faecalis Ac-P;

[0024] Figure 5 The effect of different culture temperatures on the viable Ac-P count of Rhodococcus fannings.

[0025] Figure 6 The effects of different microbial agents on the concentration efficiency of NH3 degradation during cow manure fermentation;

[0026] Figure 7 The effects of different microbial agents on the concentration efficiency of H2S degradation during cow manure fermentation;

[0027] Figure 8 The inhibition rate of urease activity by different bacterial agents. Detailed Implementation

[0028] This invention provides a *Rhodococcus qingshengii* Ac-P strain, with accession number CGMCC No. 32243. This *Rhodococcus qingshengii* Ac-P strain exhibits the ability to degrade NH3 and H2S during the composting and fermentation of livestock and poultry manure, and also has the function of inhibiting urease activity.

[0029] The present invention also provides a fermentation broth containing the aforementioned Rhodococcus fanningsacacioides Ac-P, preferably wherein the viable count of the fermentation broth is ≥1×10⁻⁶. 8 CFU / mL.

[0030] The present invention also provides a method for preparing the fermentation broth, wherein *Rhodococcus fannings* Ac-P is inoculated into a liquid culture medium and cultured at a constant temperature of 25-40°C with shaking, preferably at a constant temperature of 35°C.

[0031] The preferred liquid culture medium of this invention is #48 liquid culture medium, with the following formula: 1.0g yeast extract, 1.0g beef extract, 2.0g nitrogen supplement (NE amine A), 10g glucose, and 1L distilled water; the preferred inoculum amount of Rhodococcus faecalis Ac-P is 3%-10% (v / v), more preferably 5% (v / v); the preferred culture speed is 150-200 r / min, more preferably 180 r / min. Preferably, the fermentation broth is also concentrated to achieve a viable count ≥1×10⁻⁶. 8 The standard for CFU / mL.

[0032] The present invention also provides the application of the aforementioned Rhodococcus faecalis Ac-P or the aforementioned fermentation broth in the fermentation of livestock and poultry manure and in the inhibition of urease activity.

[0033] This invention also provides a method for preparing a deodorizing liquid fertilizer. The method involves mixing cow dung with water and *Rhodococcus faecalis* Ac-P fermentation liquid at a mass-to-volume ratio of 1g:10-20mL:0.5-1mL as raw materials. After aeration for 3-5 days, the solid and liquid components are separated, and the liquid portion is the deodorizing liquid fertilizer. Preferably, cow dung is mixed with water and *Rhodococcus faecalis* Ac-P fermentation liquid at a mass-to-volume ratio of 1g:15mL:0.5mL; preferably, aeration is performed for 4 days.

[0034] This invention also provides a deodorizing functional liquid fertilizer prepared by the aforementioned method and its application in the treatment of odorous gases in livestock and poultry farming. Preferably, the odorous gases include NH3 and H2S. As one possible implementation, the deodorizing functional liquid fertilizer can be directly sprayed onto livestock and poultry housing, or added during the fermentation process of livestock and poultry manure.

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] Isolation, screening and identification of strain Ac-P

[0038] Weigh 5g of riverbed mud and add it to 100mL of #48 medium (1.0g yeast extract, 1.0g beef extract, 2.0g nitrogen supplement, 10g glucose, 1L distilled water, autoclaved at 121℃ for 20min). Shake at 37℃ and 120r / min for 2 days, then allow to stand. Take 10mL of the supernatant and inoculate it into #48 liquid medium, shaking for 5 days. Spread the best-growing culture onto LB agar plates and incubate at 37℃. After the colonies reach a suitable size, isolate the strain through repeated streak plating, naming it Ac-P. Finally, freeze-store the strain at -70℃.

[0039] The isolated strain Ac-P was subcultured, plated on LB agar plates, and incubated in a constant temperature incubator. After being sealed with film, it was sent to Beijing Liuhe BGI Genomics Co., Ltd. for microbial identification.

[0040] The colony morphology of strain Ac-P is as follows Figure 1 As shown, the colonies are light pink, round, with neat edges, moist, viscous, and opaque.

[0041] Strains Ac-P were sequenced using 16S rDNA, and the sequencing results were compared with NCBI-BLAST. The identification result was: Ac-P is Rhodococcus qingshengii, belonging to the phylum Actinobacteria. The phylogenetic tree is as follows. Figure 2 As shown.

[0042] Example 2

[0043] Determination of Ac-P growth curve of Rhodococcus fannings

[0044] Take 100 μL of bacterial culture from 100 mL of the test bacteria culture medium and inoculate it into another 10 mL of #48 liquid medium. Prepare 12 test tubes to repeat the operation. Place all 10 mL of liquid medium containing the inoculated bacterial culture into a shaker at 37℃ and 120 rpm. Start the UV-Vis spectrophotometer, adjust the wavelength to 600 nm, and preheat for 20 min. Using distilled water as a reference, zero the spectrophotometer. Every 3 hours, use a pipette to draw an appropriate amount of bacterial culture into a cuvette and measure its OD value using the spectrophotometer. Plot the OD value on the ordinate and time on the abscissa to obtain the growth curve of the test bacteria.

[0045] like Figure 3As shown, from 0 to 10 hours, the optical density value is low and increases slowly, indicating that *Rhodococcus fanningsense* Ac-P is adapting to the new environment and its cellular metabolic activity is gradually becoming more active, but the increase in cell number is not significant. From 10 to 30 hours, *Rhodococcus fanningsense* Ac-P enters the logarithmic growth phase, proliferating exponentially and rapidly increasing the cell number. From 30 to 45 hours, the optical density value reaches a relatively high plateau, and the curve becomes flatter, indicating that *Rhodococcus fanningsense* Ac-P is in a stable phase, where the growth rate and death rate reach equilibrium, and the cell number remains relatively stable. After 45 hours, the optical density value begins to decrease slowly, and *Rhodococcus fanningsense* Ac-P enters the death phase.

[0046] Example 3

[0047] Determination of Ac-PpH Changes in Rhodococcus fanqingsheng

[0048] In Example 2, while determining the growth curve of the test bacteria, its pH was measured using a pH meter, the data was recorded, and finally the pH change of the test bacteria was obtained.

[0049] like Figure 4 As shown, the pH value of the bacterial solution gradually increases with the extension of the culture time, reaching about 7 at 40h (when the number of viable bacteria is the highest), and then decreases.

[0050] Example 4

[0051] Study on the optimal growth temperature of Rhodococcus faecalis Ac-P

[0052] The liquid culture medium corresponding to the test bacteria was dispensed into Erlenmeyer flasks, sterilized, and inoculated at an inoculation rate of 5% (v / v). The flasks were then incubated in constant temperature shaking incubators at 20℃, 25℃, 30℃, 35℃, and 40℃, with a rotation speed of 180 r / min. The OD (600 nm) of the culture medium was measured 42 h after inoculation.

[0053] like Figure 5 As shown, the viable count of Rhodococcus faecalis Ac-P reached its highest value at 35℃, reaching 6.8 × 10⁻⁶. 5 CFU / mL.

[0054] Example 5

[0055] A deodorizing liquid fertilizer, prepared as follows:

[0056] Mix cow dung with water and Rhodococcus faecalis Ac-P fermentation broth (live bacteria count ≥ 1 × 10⁻⁶). 8 The CFU / mL solution was mixed at a mass-volume ratio of 1g:15mL:0.5mL as raw material. After aeration for 4 days, the solid and liquid were separated, and the liquid part was the deodorizing liquid fertilizer.

[0057] Example 6

[0058] An experiment using Rhodococcus fanqingshengi Ac-P to remove odorous gases NH3 and H2S from cow dung.

[0059] 1. Experimental Design

[0060] The deodorization test was conducted in different treatment groups, with each group tested three times, as shown in Table 1. Each group received 50g of cow dung and 50mL of sterile distilled water, and each treatment group received 50mL of bacterial solution. The bacterial solution was measured using OD... 600 Rhodococcus faecalis Ac-P culture medium and photosynthetic bacteria culture medium with a value of 1.

[0061] The photosynthetic bacteria was *Rhodopseudomonas palustris*, purchased from Wuhan Fengtian Biotechnology Co., Ltd.

[0062] Table 1. Test groups for Ac-P removal of odor gases NH3 and H2S

[0063]

[0064]

[0065] 2. Data Collection

[0066] Construction of the ammonia concentration standard curve: Prepare the following solutions according to the requirements for determining the ammonia standard curve: 0.005 mol / L sulfuric acid absorption solution (diluted 10 times before use), 50.00 g / L salicylic acid solution, 10.00 g / L sodium nitrosoferricyanide solution, 0.05 mol / L sodium hypochlorite solution, ammonia standard stock solution, and ammonia standard working solution. Prepare a standard series of bottles using 10 10 mL volumetric flasks. Add 0.50 mL of salicylic acid solution to each bottle, then add 0.10 mL of sodium nitrosoferricyanide solution and 0.10 mL of sodium hypochlorite solution, mix well, and let stand at room temperature for 1 hour. Using a 1 cm cuvette, measure the absorbance of each solution at a wavelength of 697.5 nm, using water as a reference. Plot the standard curve with ammonia content (μg) on ​​the x-axis and absorbance on the y-axis.

[0067] After one day of treatment in each treatment group, an atmospheric sampler was connected to a bubble absorption tube containing 10 mL of absorbent solution, and 5 L of gas was sampled at a flow rate of 0.5 L / min. After sampling, the samples were stored at room temperature and analyzed within 24 hours. The sample solution was transferred to a 10 mL volumetric flask, and the absorbance was measured following the procedure for preparing a standard curve. The ammonia concentration in each treatment group of the deodorization experiment was determined and the data were recorded.

[0068] The H2S concentration of each treatment group in the deodorization test was measured using a pump-suction hydrogen sulfide detector, and the data were recorded.

[0069] 3. Test Results

[0070] like Figure 6 As shown, the treatment group with the best NH3 degradation effect was treatment group 1 (Rhodococcus faecalis Ac-P treatment group), with a treatment efficiency of 53.96%. Figure 7 As shown, the treatment group with the best H2S degradation effect was also treatment group 1 (Rhodococcus fanningeri Ac-P treatment group), with a treatment efficiency of 49.12%. This indicates that Rhodococcus fanningeri Ac-P can effectively degrade NH3 and H2S produced during the composting and fermentation of cow manure.

[0071] Example 7

[0072] Tests on the inhibition of urease activity by Ac-P of Rhodococcus fanningeri

[0073] The urease activity inhibition test was divided into three different treatment groups: treatment group 1 was the treatment group with added Rhodococcus fannings Ac-P, treatment group 2 was the treatment group with added photosynthetic bacteria, and treatment group 3 was the treatment group with added Rhodococcus fannings Ac-P and photosynthetic bacteria. Each group was tested three times, as shown in Table 2.

[0074] The photosynthetic bacteria was *Rhodopseudomonas palustris*, purchased from Wuhan Fengtian Biotechnology Co., Ltd.

[0075] Table 2. Experimental groups for Ac-P inhibition of urease activity.

[0076]

[0077]

[0078] Each group was subjected to a urease activity inhibition test according to the following analytical method.

[0079] First, the first row of the 96-well plate was designated as the blank group, the second row as the control group, and the rest as the experimental group. In each well of the 96-well plate, 25 μL of urease solution (0.35 μg / mL) was added. For the blank and control groups, 25 μL of phosphate buffer solution was added to each well. For the experimental groups, 25 μL of different treatment groups and 25 μL of urease inhibitor phosphate buffer solution were added to each well. The plates were incubated at 37°C for 50 min. For the blank group, 50 μL of phosphate buffer solution was added to each well. For the control and experimental groups, 50 μL of 25 mmol / L urea solution was added to each well. After incubation at 37°C for 30 min, 50 μL of reagent A (127 mmol / L phenol and 0.168 mmol / L sodium nitroprusside) and 50 μL of reagent B (125 mmol / L NaOH and 11.3 mmol / L NaClO) were added sequentially. The plates were incubated at 37°C for 35 min, and the absorbance A was measured at 620 nm. The inhibition rate was calculated using the following formula.

[0080] Inhibition rate (%) = [1 - (A)] 测定 -A 空白 ) / A 对照 -A 空白 ]×100

[0081] Test results: such as Figure 8 As shown, treatment group 1 (Rhodococcus faecalis Ac-P treatment group) showed the highest inhibition rate of urease activity, reaching 35.60%.

[0082] 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 Rhodococcus qingshengii Ac-P, characterized in that: The preservation number of the Rhodococcus fanqingshengii Ac-P is CGMCC No.32243.

2. A fermentation liquid containing the Rhodococcus fanqingshengii Ac-P according to claim 1.

3. The fermentation liquid according to claim 2, characterized in that: The number of viable bacteria in the fermentation solution is ≥ 1×10 8 CFU / mL.

4. A method for preparing the fermentation liquid according to claim 2 or 3, characterized in that: The liquid culture medium was inoculated with Rhodococcus fanqingshengii Ac-P and cultured at a constant temperature of 25-40°C with shaking.

5. The preparation method according to claim 4, characterized in that: The liquid culture medium is #48 liquid culture medium; the inoculation amount of the Rhodococcus fanqingshengii Ac-P is 3%-10%; and the culture rotation speed is 150-200r / min.

6. Use of the Rhodococcus fanqingshengii Ac-P described in claim 1 or the fermentation liquid described in any one of claims 2 to 3 in the preparation of deodorizing functional liquid fertilizer.

7. A method for preparing a deodorizing functional liquid fertilizer, characterized in that: Cow dung is mixed with water and the fermentation liquid of Rhodococcus fanqingshengii Ac-P as described in any one of claims 2-3 at a mass volume ratio of 1g:10-20mL:0.5-1mL as raw materials, and after aeration for 3-5 days, the solid and liquid are separated, and the liquid part is the deodorizing functional liquid fertilizer.

8. The deodorizing functional liquid fertilizer obtained by the preparation method according to claim 7.

9. Use of the deodorizing functional liquid fertilizer according to claim 8 in treating malodorous gases in livestock and poultry farming, wherein the malodorous gases include NH3 and H2S.

10. Use of the Rhodococcus fanqingshengii Ac-P described in claim 1 or the fermentation broth described in any one of claims 2 to 3 in inhibiting urease activity.

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