Microbacterium sp. and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a microbacterium and application thereof. A specific technical scheme is as follows: a microbacterium (Microbacterium sp.) LD-D, which is preserved in the China General Microbiological Culture Collection Center on August 4, 2023, and has a preservation number of CGMCC NO. 28103. The application provides a new microbacterium, which has a strong adaptability in indole and coking wastewater, can efficiently degrade indole, and can effectively treat coking wastewater.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of microbacterium and its applications. Background Technology

[0002] Indole is a compound formed by the symmetric linkage of pyrrole and benzene, possessing a strong fecal odor and primarily originating from coal tar. High concentrations of indole and its derivatives in the environment are highly toxic to plants, animals, and microorganisms, causing hemolysis and chronic arthritis in humans and animals, leading to anemia in animals, and inhibiting cytochrome formation in plants. However, indole also has a strong inhibitory effect on most microorganisms, making it difficult to degrade with existing conventional removal methods.

[0003] Coking wastewater mainly originates from the primary cooling of coke oven gas, production water used in the coking process, and steam condensate. It contains high concentrations of inorganic and organic pollutants, such as ammonia, sulfides, phenolic resins, polycyclic aromatic hydrocarbons (PAHs), and nitrogen-containing heterocyclic compounds. The main treatment methods for coking wastewater include physical, chemical, and biological methods. Physical and chemical methods are complex, expensive, and prone to causing secondary pollution. Biological methods offer advantages such as environmental friendliness, low cost, simple technology, and ease of maintenance, and are currently the focus of research.

[0004] No microorganisms have yet been found that can simultaneously degrade indole and treat coking wastewater. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of microbacterium and its applications.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this invention is: a strain of Microbacterium sp. LD-D, which was deposited on August 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.28103.

[0007] Accordingly, the application of the microbacteria in the degradation of indole.

[0008] Accordingly, the application of the microbacteria in wastewater treatment.

[0009] Preferably, the wastewater is coking wastewater.

[0010] Preferably, the wastewater contains any one or more pollutants selected from ammonia, sulfides, phenolic resins, polycyclic aromatic hydrocarbons, and nitrogen-containing heterocyclic compounds.

[0011] Preferably, the wastewater contains indole and / or indole analogs or indole derivatives.

[0012] Preferably, the wastewater contains pyridine and / or quinoline.

[0013] Accordingly, the application of the microbacteria in COD degradation.

[0014] Preferably, the temperature range of the application is 20 to 40°C.

[0015] Preferably, the pH range of the application is 6 to 10.

[0016] The present invention has the following beneficial effects: The present invention provides a new microbacterium that has a strong adaptability to indole and coking wastewater, can efficiently degrade pyridine, indole, quinoline and similar compounds, and can effectively treat coking wastewater. Attached Figure Description

[0017] Figure 1 This is a colony morphology diagram of strain LD-D;

[0018] Figure 2 This is a comparison diagram showing the growth and indole degradation of strain LD-D in indole wastewater. Detailed Implementation

[0019] This invention provides a strain of Microbacterium sp. LD-D, which was deposited on August 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.28103 and address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.

[0020] The microbacteria can efficiently degrade indole, effectively treat indole wastewater and coking wastewater, and reduce COD in the wastewater.

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. All obtained data are average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0022] Example 1: Screening and Identification of Microorganisms

[0023] 1. Microbial Screening, Enrichment, and Isolation. Water and mud samples were taken from the Chongqing Coking Plant and transported in a low-temperature storage box. After shaking and mixing, 10 mL of water sample and 5 g of mud sample were added to 90 mL of inorganic salt medium with indole as the sole carbon and nitrogen source, respectively. The samples were incubated at 30℃ and 150 rpm for 7 days in a constant-temperature shaking incubator. 10 mL of the culture medium was then added to a fresh 90 mL of inorganic salt medium and incubated at 30℃ and 150 rpm. The inorganic salt medium was replaced every 7 days, and the indole concentration was gradually increased from 30 mg / L to 90 mg / L, 180 mg / L, 240 mg / L, and 300 mg / L, while maintaining other culture conditions. After increasing the indole concentration to 300 mg / L and incubating for 7 days, an enriched culture medium of indole-degrading strains was obtained.

[0024] Inorganic salt culture medium: MgSO4·7H2O 0.2g, K2HPO4 4.26g, KH2PO4 2.65g, CaCl2 0.02g, 1mL trace element solution, indole of the required concentration, 1000mL distilled water, pH=7.

[0025] Trace element solution: KI 0.005g, MnSO4·4H2O 0.2g, CuSO4·2H2O 0.02g, ZnSO4·7H2O 0.2g, Na2MoO4·2H2O 0.25g, H3BO3 0.008g, FeCl·6H2O 0.1g, add water to 100mL.

[0026] The enriched culture medium was serially diluted to 10⁻⁶ ppm. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 In a sterile environment, 80 μL of each dilution was pipetteed and spread onto LB agar plates. The plates were then inverted and incubated at 30°C for 24–48 h. Single colonies were picked and streaked onto new plates for purification. This process was repeated three times, and the resulting single colonies were stored in LB slant agar.

[0027] Add 30 mL of Gao's No. 1 liquid culture medium to a 50 mL Erlenmeyer flask, pick a single bacterium and incubate it for 2–3 days. Under aseptic conditions, take samples to measure OD. 600 Take OD 600=1 bacterial suspension was inoculated at a 5% inoculum volume (v / v) into a medium containing 245.08 mg / L indole inorganic salts. The indole concentration was determined by measuring the absorbance of the culture medium after cell removal at 270 nm using a UV-2800 UV-Vis spectrophotometer, and the degradation rate was calculated. A strain with good indole degradation effect was selected, with a degradation rate of 97.68%, and this strain was named: LD-D.

[0028] Gao's No. 1 liquid medium: 20g soluble starch, 1g KNO3, 0.5g K2HPO4, 0.5g MgSO4•7H2O, 0.5g NaCl, 0.01g FeSO4•7H2O, pH=7.4~7.6, diluted to 1L with deionized water. For the corresponding solid medium, add an additional 20g / L of agar.

[0029] 2. Identification. The colony morphology of strain LD-D is as follows: Figure 1 As shown, the colony morphology is: round with neat edges, Gram-negative, can survive at temperatures of 20-40℃, with an optimal growth temperature of 30℃, can adapt to pH 6-10, and has an optimal pH of 7.

[0030] The enzyme activities and physiological and biochemical indicators of strain LD-D were identified, including oxidase activity, amylase activity, lipase activity (oil hydrolysis experiment), urease activity (urea experiment), sugar fermentation test, methyl red test, and VP test. The results are shown in Table 1. The specific detection methods are as follows:

[0031] Oxidase activity: In a sterile environment, moisten the oxidase test strip with sterile water, then use a disposable inoculation needle to pick up LD-D colonies and smear them onto the test strip. Observe the color change; blue indicates a positive result, and no color change within 2 minutes indicates a negative result.

[0032] Amylase activity: Prepare solid starch culture medium (1% peptone, 0.5% beef extract, 0.2% soluble starch, 2% agar, 0.5% NaCl), pick LD-D colonies and streak them on a plate. Invert the plate and incubate it in a 37℃ constant temperature incubator for 24 hours. Then, add a small amount of Lugol's iodine solution. The appearance of a colorless transparent zone around the colony indicates that the amylase is positive.

[0033] Oil hydrolysis experiment: Prepare a solid oil culture medium (1% peptone, 0.5% beef extract, 0.5% NaCl, 1% peanut oil, 2% agar, 1 mL of 1.6% neutral red aqueous solution, pH=7.2. Adjust the pH before adding neutral red; stir constantly during dispensing to ensure the oil is evenly distributed in the medium). Pick LD-D colonies and streak them onto a plate. Invert the plate and incubate it in a 37℃ incubator for 24 hours. Observe the colony color; the appearance of red spots indicates a positive result, otherwise it is a negative result.

[0034] Urease activity identification: Prepare urea slant medium (urea 2%, agar 1.5%, NaCl 0.5%, potassium dihydrogen phosphate 0.2%, peptone 0.1%, phenol red 0.0012%, pH 6.8) in test tubes. Inoculate LD-D colonies into the slant medium and incubate at 35℃ for 48h. The medium turns red, indicating a positive result; otherwise, it is negative.

[0035] Sugar fermentation experiment: Prepare a liquid culture medium for sugar fermentation (1% peptone, 0.5% NaCl, 1% bromocresol purple ethanol solution, 1% sugar solution). Place an inverted Durbin tube into the culture medium. In a sterile environment, inoculate LD-D into the culture medium and incubate at 37°C for 48 hours. The culture medium turns yellow, indicating a positive result for acid production. The presence of air bubbles in the Durbin tube indicates a positive result (proving that fermentation can produce gas). Otherwise, it is a negative result.

[0036] Methyl red test: Prepare the test medium (0.5% peptone, 0.5% dipotassium hydrogen phosphate, 0.5% glucose). Take LD-D seed culture and inoculate it into glucose peptone water medium at a 10% inoculation rate under sterile conditions and operation. After incubation at 37℃ for 48 hours, add 2 drops of methyl red reagent. If the culture medium turns red, it is methyl red positive; otherwise, it is negative.

[0037] VP test: Prepare glucose peptone aqueous medium (0.5% peptone, 0.5% dipotassium hydrogen phosphate, 0.5% glucose). Take LD-D seed culture and inoculate it into glucose peptone aqueous medium at a 10% inoculum under sterile conditions. After incubation at 37℃ for 48h, add 5 drops of 40% KOH, then add an equal volume of 5% α-naphthol solution, shake vigorously, and then incubate at 37℃ for 15min to accelerate the reaction rate. If the culture turns red, the reaction is positive; otherwise, it is negative.

[0038] Table 1 Comparison of Physiological and Biochemical Characteristics

[0039]

[0040] Molecular identification: Total DNA was extracted from the purified LD-D strain using a Sangon Biotech bacterial DNA extraction kit. Using this DNA as a template, PCR amplification was performed using universal 16S rRNA primers. The DNA was then purified and recovered via gel electrophoresis. The PCR product was sequenced by Sangon Biotech, yielding a 484 bp sequence, as shown in SEQ ID NO:1. BLAST analysis was performed to compare the sequence with sequences already registered in the GenBank / EMBL / DDBJ databases. The closest homologous strain to LD-D was Microbacterium sp., with 100% sequence coverage and 100% similarity.

[0041] Example 2: Demonstration of the effect of microbial LD-D in the degradation of pyridine, indole, and quinoline

[0042] Strain LD-D was inoculated into Gao's No. 1 medium and cultured at 30℃ for 2 days until the late logarithmic phase. After centrifugation at 5000 rpm for 10 min, the culture was washed three times with sterile water, and the OD was adjusted. 600 =1.0, to prepare bacterial suspensions. The bacterial suspensions were inoculated into inorganic salt media containing indole, pyridine, and quinoline at an inoculation volume of 5% (v / v). The media were incubated at 30°C on a shaker at 150 rpm. Samples were taken every 3 hours to measure the OD of the samples. 600 The concentrations of pyridine, indole, and quinoline, and the degradation results after a certain period of cultivation are shown in Table 2. The growth of LD-D and the degradation of indole over time are shown in Table 2. Figure 2 As shown.

[0043] Table 2 Comparison of the effects of LD-D on the degradation of nitrogen heterocyclic compounds

[0044]

[0045] Example 3: Demonstration of the effect of microbial LD-D in degrading COD in coking wastewater

[0046] Add 100 mL of coking wastewater with different COD values ​​to a 250 mL Erlenmeyer flask, and then add OD values ​​cultured in an indole-free inorganic salt medium. 600 A 10% (v / v) LD-D bacterial suspension with a concentration of 1 was incubated in a shaker at 30℃ and 150 rpm for 7 days. The COD concentration in the coking wastewater was measured, and the degradation rate was calculated. Each group was repeated three times, and the average value was taken. A corresponding control group was set up for each group. The control group used an equal volume of indole-free inorganic salt medium instead of the LD-D bacterial suspension, while the other conditions were the same. The results are shown in Table 3.

[0047] Table 3. COD Comparison Table of LD-D Degradation in Coking Wastewater

[0048]

[0049] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Microbacterium sp. LD-D, characterized by: The microbacterium was deposited on August 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.28103.

2. The application of the microbacterium LD-D described in claim 1 in the degradation of indole.

3. The application of the microbacterium LD-D described in claim 1 in the degradation of COD in coking wastewater.

4. The application according to claim 3, characterized in that: The coking wastewater contains pyridine and / or quinoline.

5. The application according to any one of claims 2 to 4, characterized in that: The temperature range for this application is 20–40°C.

6. The application according to any one of claims 2 to 4, characterized in that: The pH range for this application is 6–10.

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