Rhodococcus wenchengii ac-p and a fermented broth and application thereof

Abstract

The application provides Rhodococcus qingshengii Ac-P and a fermentation bacterial liquid and application thereof, and belongs to the technical field of microorganisms.The Rhodococcus qingshengii Ac-P has a preservation number of CGMCC No.32243.The Rhodococcus qingshengii Ac-P can effectively degrade the main foul-smelling gases NH3 and H2S generated in the fermentation process of livestock and poultry manure composting, and has a high inhibition rate on urease activity.The Rhodococcus qingshengii Ac-P and the fermentation bacterial liquid thereof provide an effective microbial solution for the odor emission reduction in the livestock and poultry manure composting process, fill the technical vacancy of this link, help to perfect the technical system of the whole livestock and poultry manure treatment, and promote the development of the harmless treatment and resource utilization technology of livestock and poultry manure.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a Rhodococcus qingshengii Ac-P, and a fermentation broth and application thereof. BACKGROUND

[0002] Composting is an effective way for harmless treatment of solid waste from livestock and poultry breeding, and has low treatment cost, and can meet the harmless requirement of waste. However, odor nuisance is the biggest problem in the application of composting technology. The main components of compost odor include ammonia, hydrogen sulfide and volatile organic compounds (VOC), among which ammonia has the highest concentration in each odor component, and hydrogen sulfide has the highest odor potential.

[0003] The odor release in the conventional composting fermentation process can be divided into two stages, i.e. concentrated release and disordered release. The concentrated release stage mainly occurs in the composting turning process and 1-2 hours after turning, and the other period is the disordered release stage. From the data of nitrogen loss caused by turning frequency, it can be found that in the concentrated release stage, the ammonia release amount is huge. In the disordered release stage, the odor release amount of composting is relatively small. Therefore, the process of reducing odor in composting should focus on the concentrated release stage, and the disordered release stage should also be considered. However, at present, the related deodorization measures mainly focus on source reduction and end reduction, and the equipment and process of process reduction are relatively lacking.

[0004] From the deodorization process, the deodorization technology can be roughly divided into two categories: one is ex-situ control, that is, the odor has been released, and physical, chemical, biological and other means are used to directly remove the released odor; the other is in-situ control, that is, through chemical reagents, enzymes, functional bacteria and other means to directly degrade the odor in the feces or inhibit the odor-producing microorganisms in the feces to remove the odor. Generally, it can be divided into in vivo control and in vitro control, and the in-situ control technology is the main technology for controlling the odor of livestock and poultry manure at present.

[0005] At present, the regulation or treatment of odor gas in intensive livestock farms is basically limited to exhaust circulating air, which can dilute the level of odor gas. The regulation of these odor gases from source prevention to treatment is basically "none", which is different from the treatment of livestock and poultry wastewater, manure and other wastes, and is one of the current problems in animal husbandry. SUMMARY

[0006] In order to solve the above technical problems, the present application provides a Rhodococcus qingshengii Ac-P, and the preservation number of the Rhodococcus qingshengii Ac-P is CGMCC No. 32243.

[0007] The present application also provides a fermentation broth containing the Rhodococcus qingshengii Ac-P. Preferably, the viable bacterial count of the fermentation broth is ≥1×10 8CFU / mL.

[0008] The application also provides a preparation method of the fermentation bacterial liquid, inoculating Rhodococcus qingshengii Ac-P in a liquid medium and culturing at a constant temperature of 25-40 DEG C with constant shaking.

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

[0010] The application also provides application of the Rhodococcus qingshengii Ac-P or the fermentation bacterial liquid in preparation of deodorization functional liquid fertilizer and in inhibition of urease activity.

[0011] The application also provides a preparation method of deodorization functional liquid fertilizer, mixing cow dung, water and fermentation bacterial liquid of Rhodococcus qingshengii Ac-P according to a mass-volume ratio of 1g:10-20mL:0.5-1mL as raw materials, aerating for 3-5 days, and then performing solid-liquid separation, so that the liquid part is the deodorization functional liquid fertilizer.

[0012] The application also provides the deodorization functional liquid fertilizer prepared by the preparation method.

[0013] The application also provides application of the deodorization functional liquid fertilizer in treatment of foul gas in livestock and poultry breeding, wherein the foul gas includes NH3 and H2S.

[0014] Compared with the prior art, the application has the following beneficial effects:

[0015] The Rhodococcus qingshengii Ac-P can effectively degrade the main foul gases NH3 and H2S generated in the composting fermentation process of livestock and poultry manure. Tests show that the Rhodococcus qingshengii Ac-P has good effects in reducing the release of ammonia and hydrogen sulfide in the fermentation process of livestock and poultry manure, and can effectively improve the odor problem in the composting process. In addition, the Rhodococcus qingshengii Ac-P also helps to adjust the pH value in the fermentation process of livestock and poultry manure, provides a more suitable environment for the growth of microorganisms and the fermentation process, and promotes the smooth progress of the fermentation process.

[0016] The Rhodococcus qingshengii Ac-P also has a high inhibition rate on urease activity. Urease can catalyze the decomposition of urea to produce ammonia in livestock and poultry manure, and the Rhodococcus qingshengii Ac-P can inhibit the urease activity, thereby reducing the generation of ammonia from the source, further reducing the foul odor degree in the fermentation process of livestock and poultry manure, and also helping to reduce the loss of nitrogen and improve the nutrient retention rate of the fertilizer.

[0017] The Rhodococcus qingshengii Ac-P and the fermentation bacterial liquid thereof provide an effective microbial solution for odor emission reduction in the livestock and poultry manure composting process, fill the technical gap in this link, help to perfect the whole livestock and poultry manure treatment technical system, and promote the development of harmless treatment and resource utilization technology of livestock and poultry manure.

[0018] Biological preservation instructions

[0019] Biological material (strain): Ac-P, classified as Rhodococcus qingshengii, the preservation unit is China General Microbiological Culture Collection Center (CGMCC), the address of the preservation unit is No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing Chaoyang District, North Chenxi Road No. 1, the preservation number is CGMCC No. 32243, and the preservation date is October 17, 2024. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a colony morphology of the Rhodococcus qingshengii Ac-P;

[0021] Figure 2 It is an evolutionary tree of the Rhodococcus qingshengii Ac-P;

[0022] Figure 3 It is a growth curve of the Rhodococcus qingshengii Ac-P;

[0023] Figure 4 It is a pH change curve of the Rhodococcus qingshengii Ac-P;

[0024] Figure 5 It is an influence of different culture temperatures on the viable count of the Rhodococcus qingshengii Ac-P;

[0025] Figure 6 It is a concentration efficiency of different bacterial agent treatments on degradation of NH3 in the fermentation process of cow manure;

[0026] Figure 7 It is a concentration efficiency of different bacterial agent treatments on degradation of H2S in the fermentation process of cow manure;

[0027] Figure 8 It is an inhibition rate of different bacterial agent treatments on urease activity. DETAILED DESCRIPTION

[0028] The application provides a Rhodococcus qingshengii Ac-P, and the preservation number is CGMCC No. 32243. The Rhodococcus qingshengii Ac-P has the ability of degrading NH3 and H2S in the livestock and poultry manure composting fermentation process, and also has the effect of inhibiting urease activity.

[0029] The application also provides a fermentation broth containing the Rhodococcus qingshenii Ac-P, preferably, the viable cell count of the fermentation broth is greater than or equal to 1x10 8 CFU / mL.

[0030] The application also provides a preparation method of the fermentation broth, inoculating the Rhodococcus qingshenii Ac-P in a liquid medium, and culturing at a constant temperature of 25-40 DEG C, preferably, the culture temperature is a constant temperature of 35 DEG C.

[0031] The application preferably uses #48 liquid medium as the liquid medium, and the formula is: yeast extract 1.0 g, beef extract 1.0 g, nitrogen source supplement (NE amine A) 2.0 g, glucose 10 g, and distilled water 1 L; preferably, the inoculation amount of the Rhodococcus qingshenii Ac-P is 3%-10% (v / v), and further preferably 5% (v / v); preferably, the rotation speed of the culture is 150-200 r / min, and further preferably 180 r / min. Preferably, the fermentation broth is concentrated to reach the standard of the viable cell count being greater than or equal to 1x10 8 CFU / mL.

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

[0033] The application also provides a preparation method of a deodorization functional liquid fertilizer, and the preparation method uses cow dung, water and the fermentation broth of the Rhodococcus qingshenii Ac-P as raw materials, and the mass / volume ratio is 1 g:10-20 mL:0.5-1 mL; after aeration for 3-5 days, solid-liquid separation is performed, and the liquid part is the deodorization functional liquid fertilizer; preferably, the mass / volume ratio of the cow dung, the water and the fermentation broth of the Rhodococcus qingshenii Ac-P is 1 g:15 mL:0.5 mL; and preferably, the aeration is performed for 4 days.

[0034] The application also provides the deodorization functional liquid fertilizer prepared by the preparation method and application of the deodorization functional liquid fertilizer in treatment of foul gas in livestock and poultry breeding. Preferably, the foul gas includes NH3 and H2S. As an implementable mode, the deodorization functional liquid fertilizer can be directly sprayed in a livestock and poultry breeding house, or deodorization is added in the fermentation process of livestock and poultry manure.

[0035] The technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the application.

[0036] Example 1

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

[0038] Take 5g of river sediment and add it to 100mL of #48 medium (yeast extract 1.0g, beef extract 1.0g, nitrogen source supplement 2.0g, glucose 10g, distilled water 1L, autoclave 121℃, sterilization 20min) and shake in a constant temperature shaker at 37℃, 120r / min for 2d, then let stand. Take 10mL of supernatant and inoculate into #48 liquid medium and shake culture for 5d. The best growing culture is spread on LB plates and cultured in a constant temperature incubator at 37℃. After several times of plate streaking, a strain is obtained, named Ac-P, and finally stored in a -70℃ refrigerator.

[0039] The isolated strain Ac-P is subcultured, spread on LB plates, and placed in a constant temperature incubator for culture. After wrapping with sealing film, it is sent to Beijing Lihe Huada Gene Technology Co., Ltd. for microbial identification.

[0040] The colony morphology of strain Ac-P is shown in Figure 1 , which is a light pink round colony with neat edges, wet, viscous and opaque.

[0041] The strain Ac-P is sequenced by 16s rDNA, and the sequencing results are compared by NCBI-BLAST. The identification result is: Ac-P is Rhodococcus qingshengii, belonging to Actinobacteria, and the phylogenetic tree is shown in Figure 2 .

[0042] Example 2

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

[0044] Take 100μL of bacterial liquid from 100mL of culture liquid of the test bacteria and inoculate into another 10mL of #48 liquid medium. Prepare 12 test tubes for repeated operation. Place all the inoculated 10mL liquid medium in a constant temperature incubator at 37℃, 120r / min. Start the UV-visible spectrophotometer, adjust the wavelength to 600nm, and preheat for 20min. Use distilled water as reference, zero the spectrophotometer, and every 3h, take an appropriate amount of bacterial liquid in a cuvette with a pipette, and measure the OD value with a spectrophotometer. Take the OD value as the vertical coordinate and the time as the horizontal coordinate, and draw a smooth curve, which is the growth curve of the test bacteria.

[0045] As shown in 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] Test of removing NH3 and H2S from cow dung by Rhodobacter sphaeroides Ac-P

[0059] 1. Test design

[0060] The deodorization test was divided into different treatment groups, each group was repeated 3 times, as shown in Table 1, the amount of added cow dung in each group was 50 g, 50 mL of sterile distilled water was added, and 50 mL of bacterial liquid was added to each treatment group. The bacterial liquid was taken from the Rhodobacter sphaeroides Ac-P culture solution with an OD value of 1 and the photosynthetic bacterial culture solution. 600

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

[0062] Table 1 Test grouping of Ac-P removing NH3 and H2S

[0063]

[0064]

[0065] 2. Data collection

[0066] Preparation of ammonia concentration standard curve: According to the need to determine the ammonia standard curve, the following solutions were prepared: 0.005 mol / L sulfuric acid absorbent (diluted 10 times before use), 50.00 g / L salicylic acid solution, 10.00 g / L sodium nitroprusside solution, 0.05 mol / L sodium hypochlorite solution, ammonia standard stock solution, and ammonia standard working solution. Take 10 10 mL volumetric flasks, prepare standard series bottles. Add 0.50 mL of salicylic acid solution to each bottle, then add 0.10 mL of sodium nitroprusside solution and 0.10 mL of sodium hypochlorite solution, mix well, and let stand at room temperature for 1 h. With a 1 cm cuvette, at a wavelength of 697.5 nm, with water as the reference, measure the absorbance of each solution. With ammonia content (μg) as the horizontal coordinate and absorbance as the vertical coordinate, draw the standard curve.

[0067] After 1 day of treatment in each test group, use an air sampler to connect a gas bubble absorption tube containing 10 mL of absorption solution, at a flow rate of 0.5 L / min, and collect 5 L of gas. After sampling, the sample is stored at room temperature and analyzed within 24 h. Transfer the sample solution into a 10 mL volumetric flask, and then follow the operation steps for preparing the standard curve to measure the absorbance of the sample. Measure the ammonia gas concentration of each treatment group in the deodorization test and record the data.

[0068] Use a pump suction type hydrogen sulfide detector to measure the H2S concentration of each treatment group in the deodorization test and record the data.

[0069] 3. Test results ​

[0070] As shown in Figure 6 Table 2, the treatment group 1 (Rhodobacter sphaeroides Ac-P treatment group) had the best effect on degrading NH3 concentration, with a treatment efficiency of 53.96%. As shown in Figure 7 Table 2, the treatment group 1 (Rhodobacter sphaeroides Ac-P treatment group) also had the best effect on degrading H2S concentration, with a treatment efficiency of 49.12%. It was shown that Rhodobacter sphaeroides Ac-P could effectively degrade NH3 and H2S produced in the fermentation process of cow manure compost.

[0071] Example 7

[0072] Test of Rhodobacter sphaeroides Ac-P on urease activity inhibition

[0073] The urease activity inhibition test was divided into three different treatment groups. The treatment group 1 was the treatment group adding Rhodobacter sphaeroides Ac-P, the treatment group 2 was the treatment group adding photosynthetic bacteria, and the treatment group 3 was the treatment group adding Rhodobacter sphaeroides Ac-P and photosynthetic bacteria mixed bacteria. Each test was repeated three times, as shown in Table 2.

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

[0075] Table 2: Grouping of Ac-P urease activity inhibition test

[0076]

[0077]

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

[0079] First, the first row of the 96-well plate was set as the blank group, and the second row was set as the control group, and the rest was set as the test group. In the 96-well plate, 25 μL of urease solution (0.35 μg / mL) was added to each well, and 25 μL of phosphate buffer solution was added to each well of the blank group and the control group; 25 μL of different treatment groups and 25 μL of urease inhibitor phosphate buffer solution were added to each well of the test group, and the plate was incubated in a 37°C incubator for 50 min. 50 μL of phosphate buffer solution was added to each well of the blank group, and 50 μL of 25 mmol / L urea solution was added to each well of the control group and the experimental group. After incubation at 37°C for 30 min, 50 μL of A reagent (127 mmol / L phenol and 0.168 mmol / L sodium nitroprusside) and 50 μL of B reagent (125 mmol / L NaOH and 11.3 mmol / L NaClO) were added in turn, and the color was developed at 37°C for 35 min. 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 qinghaensis (R. qinghaensis) Ac-P, characterized in that, Rhodococcus qingshengii ) Ac-P, characterized in that, The preservation number of the Rhodococcus qingshengii Ac-P is CGMCC No.32243. 2.A fermented bacterial solution containing the Rhodococcus qingshengii Ac-P of claim 1.

3. The fermentation broth of claim 2, wherein, The viable cell count of the fermentation bacterial liquid is ≥1×10 8 CFU / mL.

4. A method for producing the fermentation broth according to claim 2 or 3, characterized by, Inoculate the Rhodococcus qingshengii Ac-P in a liquid medium, and incubate at 25-40℃ with constant shaking.

5. The production method according to claim 4, characterized by, The formula of the liquid medium is: yeast extract 1.0g, beef extract 1.0g, nitrogen source supplement 2.0g, glucose 10g, distilled water 1L; the inoculation amount of the Rhodococcus qingshengii Ac-P is 3%-10%; the rotation speed of the culture is 150-200r / min. 6.The application of the Rhodococcus qingshengii Ac-P of claim 1 or the fermented bacterial solution of any one of claims 2-3 in the preparation of a deodorizing functional liquid fertilizer.

7. A method for preparing a deodorizing functional liquid fertilizer, characterized by, Mix cow dung, water and the fermented bacterial solution of the Rhodococcus qingshengii Ac-P of any one of claims 2-3 according to the mass-volume ratio of 1g:10-20mL:0.5-1mL as raw materials, aerate for 3-5 days, and then separate the solid and liquid, and the liquid part is the deodorizing functional liquid fertilizer. 8.The deodorizing functional liquid fertilizer prepared by the preparation method of claim 7. 9.The application of the deodorizing functional liquid fertilizer of claim 8 in the treatment of foul gas in livestock and poultry breeding, wherein the foul gas includes NH3 and H2S. 10.The application of the Rhodococcus qingshengii Ac-P of claim 1 or the fermented bacterial solution of any one of claims 2-3 in the inhibition of urease activity.

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