Sphingomonas yanoikuyae and application thereof in degrading indole compounds

Abstract

The application discloses a Sphingobium yanoikuyae FL021 and application thereof in degrading indole compounds. The Sphingobium yanoikuyae FL021 capable of efficiently degrading indole odor compounds under anoxic or facultative anaerobic conditions is obtained through screening and identification, and has been preserved in the China Center for Type Culture Collection on February 2, 2026, with a preservation number of CCTCC NO: M 2026334. It is found through verification that the degradation rates of the Sphingobium yanoikuyae FL021 to different indole compounds are all greater than 95%, the highest degradation rate can reach 99%, the Sphingobium yanoikuyae FL021 has strong tolerance to high-concentration indole compounds and strong environmental adaptability, and can be used as a microbial preparation to effectively degrade indole odor generated in the environment, such as livestock and poultry breeding manure, to improve the ecological environment and promote the green and healthy development of the livestock and poultry industry.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Sphingobium yanoikuyae FL021 and its application in the degradation of indole compounds. Background Technology

[0002] Indole, with the chemical formula C8H7N, is a nitrogen-containing heterocyclic aromatic compound formed by the parallel linkage of a benzene ring and a pyrrole ring. Its derivative, 3-methylindole (skatole), often co-occurs with indole in animal intestines and excrement, and is one of the main causes of the foul odor of feces. The continuous expansion of livestock and poultry farming in my country has resulted in the generation of large amounts of feces. High concentrations of indole and 3-methylindole, among other substances, not only produce unbearable odors but also possess certain biotoxicity, posing a serious threat to the ecological environment surrounding farms, the health of workers, and the sustainable development of animal husbandry. The continuous accumulation of these compounds in the environment not only exacerbates environmental pollution but also has long-term negative impacts on residents' lives and public health.

[0003] Currently, technologies for treating indole odor compounds are mainly divided into physicochemical methods and biological methods. While physicochemical methods such as ultrasound and ultraviolet light have some effect, their practical applications are limited by high treatment costs and high energy consumption, making them difficult to promote economically and sustainably in large-scale farms and similar settings. Therefore, biological deodorization technologies centered on microbial degradation, such as biofilters, are a more promising solution. Biodegradation pathways often involve microbial denitrification and aerobic bacteria, but they generally suffer from poor tolerance to high concentrations of indole, low degradation efficiency, and long microbial cultivation cycles, making it difficult to achieve high-efficiency degradation under anaerobic and high-concentration indole compound conditions.

[0004] Among known degrading microorganisms, several species of *Sphingobium* have attracted considerable attention due to their remarkable ability to degrade aromatic compounds, making them important candidate species for the bioremediation of recalcitrant organic pollutants such as polycyclic aromatic hydrocarbons, pesticides, and dyes in the environment. However, indole compounds are nitrogen-containing heterocyclic aromatic compounds with unique structures, possessing both benzene and pyrrole rings. This structural characteristic leads to indole being more toxic to microorganisms and significantly more difficult to biodegrade than conventional aromatic compounds such as phenol. Therefore, microorganisms capable of efficiently degrading common aromatic compounds may not be effective at degrading indole. Furthermore, currently, *Sphingobium* species primarily degrade aromatic compounds under ideal aerobic conditions, exhibiting low degradation efficiency and poor tolerance to high concentrations of compounds in common anaerobic environments such as wastewater or livestock manure. Therefore, there is an urgent need in this field for a strain that can efficiently degrade indole compounds and adapt to complex real-world environments, such as anaerobic and high-concentration environments. Summary of the Invention

[0005] This invention addresses a gap in existing technologies by providing a strain of *Sphingobium yanoikuyae* FL021 and its application in degrading indole compounds. Through screening and identification, this invention obtained *Sphingobium yanoikuyae* FL021, which can efficiently degrade indole odor compounds under anoxic or facultative anaerobic conditions. Verification showed that its degradation rate for different indole compounds was greater than 95%, with a maximum degradation rate reaching 99%. Furthermore, it exhibits strong tolerance to high concentrations of indole compounds and strong environmental adaptability. It can be used as a microbial agent to effectively degrade indole odors generated in environments such as livestock and poultry manure, improving the ecological environment and promoting the green and healthy development of animal husbandry.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a strain of *Sphingobium yanoikuyae* FL021, which was deposited at the China Center for Type Culture Collection (CCTCC) on February 2, 2026, with accession number CCTCC NO: M 2026334. The deposit address is: Wuhan University, Wuhan, China.

[0007] The present invention also provides a microbial agent comprising an effective dose of the above-mentioned Sphingobium yanoikuyae FL021 or its bacterial suspension or centrifuged cells or its fermentation product.

[0008] Furthermore, the microbial agent also contains an agriculturally or microbiologically acceptable carrier.

[0009] Furthermore, the bacterial agent is a liquid bacterial agent, a solid bacterial agent, or a lyophilized powder.

[0010] The present invention also provides the application of the above-mentioned Sphingobium yanoikuyae FL021 or the above-mentioned microbial inoculant in any of the following: A1) Application in the degradation of indole compounds; A2) Application in the preparation of products that degrade indole compounds.

[0011] Furthermore, the indole compounds include: indole, 1-methylindole, 2-methylindole, and / or 3-methylindole.

[0012] Furthermore, the *Sphingobium yanoikuyae* FL021 degrades indole compounds under facultative anaerobic or hypoxic conditions.

[0013] The present invention also provides a method for degrading indole compounds, the method comprising inoculating the above-mentioned Sphingobium yanoikuyae FL021 or the above-mentioned microbial agent into a substrate containing indole compounds, and treating it under conditions suitable for degradation.

[0014] Furthermore, the substrate includes wastewater, waste gas, or solid waste. Preferably, it is livestock and poultry breeding wastewater, manure, or waste gas containing indole-like odors.

[0015] Furthermore, the inoculum size of *Sphingobium yanoikuyae* FL021 is not less than 1% v / v.

[0016] Further, the *Sphingobium yanoikuyae* FL021 was inoculated into LB medium and incubated statically at 25-30°C for at least 48 hours. OD 600 If the value is greater than 5, after obtaining the FL021 seed culture, it is then inoculated into a substrate containing indole compounds at an inoculation rate of not less than 1% v / v.

[0017] Furthermore, the degradation is carried out at 25-30°C under facultative anaerobic or anoxic conditions. Preferably, it is carried out at 28°C under static conditions.

[0018] Furthermore, the initial concentration of the indole compound in the substrate is not higher than 400 mg / L.

[0019] Beneficial effects: This invention screened a strain of Sphingobium yanoikuyae FL021 from activated sludge of a sewage treatment plant, which can efficiently degrade indole odor compounds under anoxic or facultative anaerobic conditions. It was deposited at the China Center for Type Culture Collection on February 2, 2026, with accession number CCTCC NO: M 2026334. Studies have found that this strain can efficiently degrade various indole compounds, such as indole, 1-methylindole, 2-methylindole, and 3-methylindole, under anaerobic or facultative anaerobic conditions, with degradation rates all exceeding 95%, and the highest degradation rate reaching 99%. Furthermore, this strain exhibits strong tolerance to high concentrations of indole compounds, high degradation efficiency, a broad degradation spectrum, strong environmental adaptability, and ease of cultivation. It can be formulated into microbial preparations for the effective degradation of indole-containing odors generated in livestock and poultry manure and other environmental pollutants. This provides excellent microbial strain resources for low-cost, high-efficiency biological purification of livestock and poultry manure and indole-containing waste gases, improving the ecological environment and promoting the green and sustainable development of animal husbandry. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is the phylogenetic tree of Sphingobium yanoikuyae FL021 in Example 1 of the present invention.

[0022] Figure 2 This invention demonstrates the degradation effect of Sphingobium yanoikuyae FL021 on four indole compounds (indole, 1-methylindole, 2-methylindole, and 3-methylindole) in Example 2 of this invention.

[0023] Figure 3 This invention demonstrates the degradation effect of Sphingobium yanoikuyae FL021 on different types (indole, 1-methylindole, 2-methylindole, 3-methylindole) and different concentrations (100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L) of indole compounds in Example 2 of this invention. Detailed Implementation

[0024] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless specifically stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless specifically stated, the reagents and materials used in the following embodiments are commercially available.

[0025] Example 1: Screening and Identification of Strains 1. Screening of strains (1) Sample source: Fresh activated sludge samples were collected from the aeration tank of Jiangxia Wastewater Treatment Plant in Wuhan City, Hubei Province and placed in sterile sample bags for microbial enrichment.

[0026] (2) Microbial enrichment culture: Prepare a culture medium with indole as the sole carbon source, and enrich microorganisms with indole degradation ability through multiple rounds of selective culture, as follows.

[0027] (2.1) Weigh 50g of fresh sludge and place it in a 500mL Erlenmeyer flask. Add 150mL of sterilized liquid synthetic medium containing 100mg / L indole. The formulation of the synthetic medium is as follows (g / L): K2HPO4 (0.025), KH2PO4 (0.025), NH4Cl (0.10), KCl (0.1), FeCl3·6H2O (0.02), CaCl2 (0.05), indole (0.1), and 1 mL / L trace element solution, with a pH of 6.0-6.5. The trace element solution composition (g / L) is: MgSO4·7H2O (0.5), EDTA (1.0), ZnSO4 (0.2), MnCl2·4H2O (0.1), FeSO4·7H2O (0.5), CuSO4·5H2O (0.5), and CoCl2·6H2O (0.2).

[0028] (2.2) Place the Erlenmeyer flask in a constant temperature incubator at 28℃ and let it stand for 3 days to create an environment conducive to the growth of facultative anaerobic and hypoxic microorganisms.

[0029] (2.3) Multiple rounds of selective culture: After the first culture for 3 days, the supernatant in the Erlenmeyer flask was discarded, and an equal amount of the above synthetic medium containing 100 mg / L indole was added, and the culture was allowed to stand for another 3 days; after 3 days, the supernatant in the Erlenmeyer flask was discarded again, and an equal amount of the above synthetic medium containing 100 mg / L indole was added, and the culture was allowed to stand for another 2 days; after 2 days, the supernatant in the Erlenmeyer flask was discarded again, and an equal amount of the above synthetic medium containing 100 mg / L indole was added, and the culture was allowed to stand for another 2 days. The dominant bacterial groups that can efficiently utilize indole were enriched by repeated subculturing.

[0030] (2.4) Two days later, use a pipette to take 0.1 mL of the culture from the Erlenmeyer flask and inoculate it onto the melted and cooled solid synthetic medium. Place it in a constant temperature incubator at 28℃ and incubate for 3 days. The solid synthetic medium is based on the above liquid synthetic medium containing 100 mg / L indole, with 15 g / L agar powder added.

[0031] (2.5) After 3 days of culture, select single colonies with relatively fast growth rate from the culture dish, place them in liquid synthetic medium containing 100 mg / L indole, and incubate at 28°C for about 48 hours. After the culture becomes turbid, use it for subsequent bacterial 16S rDNA identification, and prepare the bacterial strain into glycerol tubes and store them at -80°C.

[0032] 2. Identification of strains The strain was identified molecularly using the 16S rRNA method. 2 mL of bacterial culture was transferred to an EP tube and centrifuged at 12000 rpm for 10 min. The supernatant was discarded, and DNA was extracted from the genome using a DNA extraction kit (purchased from Guangzhou Meige Biotechnology Co., Ltd.). The 16S rDNA of the microorganism was amplified and sequenced using universal bacterial primers 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' and 1492R: 5'-TACGGYTACCTTGTTAYGACTT-3'. The specific experimental procedure was performed by Guangzhou Meige Gene Technology Co., Ltd. The 16S rDNA sequence of this strain is shown in SEQ ID NO.1.

[0033] The sequencing results were compared with those from NCBI, and then phylogenetic analysis was performed based on the similarity to ultimately determine the species of the microorganism. The phylogenetic tree is shown below. Figure 1 As shown, after sequencing, identification, and phylogenetic analysis, the microorganism was identified as *Sphingobium yanoikuyae*, and named *Sphingobium yanoikuyae* FL021. This strain was deposited at the China Center for Type Culture Collection on February 2, 2026, with accession number CCTCCNO: M 2026334, address: Wuhan University, Wuhan, China.

[0034] Example 2: Determination of the degradation performance and tolerance of Sphingobium yanoikuyae FL021 to different indole compounds. 1. Determination of the degradation performance of Sphingobium yanoikuyae FL021 on compounds containing different indole compounds.

[0035] This embodiment evaluates the degradation performance of strain FL021 on indole and its three methyl derivatives (1-methylindole, 2-methylindole, and 3-methylindole) by measuring its degradation ability on indole compounds, as detailed below.

[0036] (1) First, prepare a liquid synthesis culture medium without indole compounds, with the formula as described in Example 1. Then, add 100 mg / L of indole, 1-methylindole, 2-methylindole, and 3-methylindole to obtain simulated wastewater containing four different types of indole compounds.

[0037] (2) The FL021 strain was inoculated into LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0) and incubated statically at 28°C for 48 h to allow its OD to adjust. 600If the value is greater than 5, FL021 seed solution is obtained and used for degradation experiments.

[0038] (3) Take 1 mL of the above FL021 seed culture and inoculate it into 100 mL of simulated wastewater containing different types of indole compounds. Place it in a static culture at 28℃ to simulate a facultative anaerobic or hypoxic environment. Take samples every 24 h from the start of inoculation and determine the content of indole compounds by high performance liquid chromatography (HPLC) and calculate the degradation rate. The HPLC determination method is as follows: chromatographic column: Agilent C18 column, detector type: Agilent UV light source, mobile phase: methanol:water = 6:4 (v / v), flow rate: 1 mL / min, column temperature: 30℃. Detection wavelength: 313 nm, peak time: 4.5-5.1 min. The degradation rate is calculated as: (initial concentration - residual concentration) / initial concentration.

[0039] The degradation kinetics of four indole compounds by strain FL021 are shown in the figure below. Figure 2 As shown in Table 1, the highest degradation rate was finally calculated.

[0040] Table 1. Maximum degradation rate of indole compounds by FL021 The results showed that *Sphingobium yanoikuyae* FL021 of this invention could effectively degrade four test compounds, achieving maximum degradation efficiency within 72-96 hours, demonstrating high degradation efficiency. The highest degradation rates of the four indole compounds by strain FL021 were all above 95%, with the highest degradation efficiency reaching 99% for 1-methylindole. These results indicate that strain FL021 possesses a broad-spectrum ability to degrade indole compounds, not only efficiently degrading the indole itself but also exhibiting high removal efficiency for its derivatives methylated at different positions (1-, 2-, 3-) on the benzene ring. This provides a valuable microbial resource for addressing complex indole-related odor pollution in real-world environments.

[0041] 2. Tolerance and degradation efficiency of Sphingobium yanoikuyae FL021 to different concentrations of indole compounds.

[0042] This embodiment further simulates wastewater by configuring indole compounds of different concentration gradients to determine the tolerance and degradation efficiency of strain FL021 to high concentrations of indole compounds, as detailed below.

[0043] (1) First, prepare a liquid synthetic culture medium without indole compounds, with the formula as described in Example 1. Then, add the above four indole substances respectively, and set their concentrations to 100 mg / L, 200 mg / L, 300 mg / L and 400 mg / L to obtain simulated wastewater containing four different types and four different concentrations of indole compounds.

[0044] (2) Prepare FL021 seed liquid using the same method as above.

[0045] (3) Take 1 mL of the above-cultured FL021 seed culture and inoculate it into 100 mL of simulated wastewater containing different types and concentrations of indole compounds. Place it in a static culture at 28℃ to simulate a facultative anaerobic or hypoxic environment. Use uninoculated simulated wastewater as a blank control. Take a sample at 96 h after inoculation and determine the residual amount of indole compounds by high performance liquid chromatography. Calculate the degradation rate using the same method as above.

[0046] The degradation rate of strain FL021 under different concentrations of indole compounds was determined as follows: Figure 3 As shown, the results indicate that strain FL021 exhibited highly efficient degradation ability when the concentration of indole compounds was ≤200 mg / L, with the degradation rate remaining above 85% after 96 h. When the concentration increased to 300 mg / L, the degradation process was inhibited, and the degradation rate decreased to below 43%. However, even under high concentration stress of 400 mg / L, strain FL021 still retained approximately 20% degradation ability for various indole compounds, indicating that FL021 has excellent tolerance to high concentrations of indole compounds. These results demonstrate that strain FL021 can effectively tolerate high concentrations of indole compounds, providing important support for its application in treating practical high-concentration livestock and poultry manure or industrial wastewater.

[0047] In summary, this invention provides a strain of *Sphingobium yanoikuyae* FL021 that can efficiently degrade indole odor compounds under anoxic or facultative anaerobic conditions. It exhibits broad-spectrum indole degradation capabilities, demonstrating excellent degradation ability for indole and its methylated derivatives, with maximum degradation rates exceeding 95%. Furthermore, it maintains metabolic activity and degradation function even under high concentrations of indole compounds, demonstrating strong tolerance and environmental adaptability. Therefore, it can be used as a microbial preparation to effectively degrade indole odors generated in environments such as livestock and poultry manure, thereby improving the ecological environment and promoting the green and healthy development of animal husbandry.

[0048] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A strain of Sphingobium yanoikuyae FL021 was deposited at the China Center for Type Culture Collection on February 2, 2026, with accession number CCTCC NO: M 2026334.

2. A microbial inoculant, characterized in that, The microbial agent contains an effective dose of Sphingobium yanoikuyae FL021 as described in claim 1, or a suspension thereof, centrifuged cells thereof, or a fermentation product thereof.

3. The use of Sphingobium yanoikuyae FL021 as described in claim 1 or the microbial agent as described in claim 2 in any of the following: A1) Application in the degradation of indole compounds; A2) Application in the preparation of products that degrade indole compounds.

4. The application according to claim 3, characterized in that, The indole compounds include: indole, 1-methylindole, 2-methylindole and / or 3-methylindole.

5. The application according to claim 3, characterized in that, The *Sphingobium yanoikuyae* FL021 degrades indole compounds under facultative anaerobic or hypoxic conditions.

6. A method for degrading indole compounds, characterized in that, The method includes inoculating a substrate containing indole compounds with either Sphingobium yanoikuyae FL021 as described in claim 1 or the microbial agent as described in claim 2, and then treating it under conditions suitable for degradation.

7. The method according to claim 6, characterized in that, The substrate may include wastewater, waste gas, or solid waste.

8. The method according to claim 6, characterized in that, The inoculation amount of *Sphingobium yanoikuyae* FL021 is not less than 1% v / v.

9. The method according to claim 6, characterized in that, The treatment is carried out at 25-30°C under facultative anaerobic or hypoxic conditions to degrade the material.

10. The method according to claim 6, characterized in that, The initial concentration of indole compounds in the substrate is no higher than 400 mg / L.

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