WO2021068416A1 - Brevibacillus nitrificans strain yj1 and application thereof - Google Patents

Abstract

Provided are an n-hexadecane degrading bacterial strain YJ1 and an application thereof. The degrading bacterial strain with n-hexadecane as a carbon source is identified as Brevibacillus nitrificans on the basis of the bacterial strain morphology, physiological characteristics, Gram staining reaction, 16SrDNA gene sequencing analysis, and phylogenetic analysis, and is named YJ1. The bacterial strain was preserved in the General Microbiology Center of the China Microbial Culture Collection Management Committee in August 2019, and the preservation number is CGMCC NO.18418; the bacterial strain can use n-hexadecane as the sole carbon source and quickly degrade same: after culturing for 15 days in an environment with an initial concentration of n-hexadecane of 10 mg/L, the degradation rate can reach 66.7%. The optimal growth conditions for the bacterial strain are 30°C, pH=7, and inoculation amount 10%

Description

Brevibacillus nitrificans strain YJ1 and application thereof Technical field

The invention belongs to the technical field of bioengineering, and specifically provides a strain YJ1 of Brevibacillus nitrificans nitrificans and an application thereof.

Background technique

With the rapid development of global industrialization, petroleum products have become the main source of energy in industrial production and daily life. At the same time, due to the rapid increase in oil usage, long-term unscientific use, and leakage during transportation and exploitation, some of the hydrocarbons such as strong toxicity, high residue, difficult to degrade, carcinogenic, and teratogenic enter the soil and groundwater, which are harmful to the environment. Cause pollution and seriously endanger human health.

The various hydrocarbons contained in petroleum, from the simplest compounds to complex solid residues with dozens of carbon atoms, can be metabolized and degraded by microorganisms as long as the conditions are right, but the degree of difficulty and degradation rate are different. Generally speaking, _{linear compounds in the range of C 8} to C ₁₈ are easier to decompose, olefins are the easiest to decompose, alkanes are the second, aromatics are more difficult, and alicyclic hydrocarbons are the least sensitive to the effects of microorganisms. So far, only a few strains have been able to use them. . In petroleum hydrocarbons, alkanes are the main components, with a content of up to 50%-95%, and they are also the main components in petroleum pollutants. Alkanes in petroleum are generally divided into normal alkanes and isoalkanes. Common normal alkanes are C ₁ to C ₄₅ , while the carbon chain length of isoalkanes is usually less than 10. Short-chain alkanes, such as methane, are mostly gaseous and volatile, so they have little impact on the environment; medium- and long-chain alkanes, such as n-hexadecane, are relatively difficult to degrade in the environment and are likely to cause harm to the surrounding environment. The most common source of pollution in oil pollution. How to effectively reduce or eliminate petroleum hydrocarbon pollution has become a key issue of widespread concern. The current methods of controlling oil pollution mainly include physical remediation, chemical remediation and bioremediation. Compared with the other two remediation methods, bioremediation has the advantages of high efficiency, no secondary pollution, easy operation and low cost. An important method of oil pollution remediation.

At present, there are about 100 genera and more than 200 species of petroleum hydrocarbon degradation microorganisms, including bacteria, actinomycetes, molds, yeasts, and algae. Common petroleum-degrading microorganisms mainly include: Achromobacter, Pseudomonas, Flavobacterium, Archrobactar, Alcaligenes, Micrococcus (Mictococcus), Acinetobacter, Rhodococcus, Bacillus, Coryneforms, Nocardia, Microbacterium, Mycobacterium (Mycobacterium), Dietzia (Dietzia) and so on. However, there are few research fields on the degrading strains of n-hexadecane at home and abroad. Only the patent CN 106242072A relates to the application of non-determinus bacteria to degrade n-hexadecane. Therefore, finding a strain that can degrade n-hexadecane has become the basis. One of the technical problems in the field.

Summary of the invention

In view of the shortcomings of the above-mentioned technology, the present invention provides a n-hexadecane degrading bacteria, which can be used for the application in the bioremediation process of degrading n-hexadecane.

In order to achieve the above objectives, the present invention is achieved through the following technical solutions:

A strain of Brevibacillus nitrificans strain YJ1, its deposit number is CGMCC NO.18418.

The 16S rDNA sequence of Brevibacillus nitrificans strain YJ1 is shown in SEQ NO.1.

The colony of the strain YJ1 of Brevibacillus nitrificans strain YJ1 on BP agar medium was light yellow, raised, with neat edges, and moist surface; the bacterial cells were straight rod-shaped, and Gram staining was positive; the hemolysis of the strain was negative .

The bacterial agent for degrading n-hexadecane is characterized in that it comprises the strain YJ1 of Brevibacillus nitrificans.

The inoculum also includes auxiliary materials commonly used in the preparation of inoculants; preferably, the auxiliary materials commonly used in the preparation of inoculants are selected from: the culture medium of Brevibacillus nitrificans strain YJ1, or, other commonly used auxiliary materials for inoculants .

The culture medium of the Brevibacillus nitrificans strain YJ1 is selected from: beef extract peptone medium, and/or, n-hexadecane inorganic salt medium;

The beef extract peptone culture medium includes: peptone 10g/L, beef extract 5g/L, NaCl 10g/L, the rest is water, pH 7.2;

The medium containing n-hexadecane inorganic salt includes: n-hexadecane 2-20 mL/L, K ₂ HPO ₄ ·3H ₂ O 1.0 g/L, KH ₂ PO ₄ 1.0 g/L, MgSO ₄ ·7H ₂ O 0.5g/L, NH ₄ NO ₃ 1.0g/L, CaCl ₂ 0.02g/L, Fe ₂ (SO ₄ ) ₃ 0.02g/L, the rest is water, pH 7.0-7.2.

The concentration range of n-hexadecane in the medium mentioned above relates to the influence of the concentration of different n-hexadecane substrates in Experimental Example 2 on the growth of the strain, so there are multiple concentrations.

The above-mentioned culture media are all liquid culture media. If a solid culture medium is to be made, it is only necessary to add 1.2-2% agar powder with a mass-to-volume ratio on the basis of the original culture medium formula.

The Bacillus nitrificans Brevibacillus nitrificans strain YJ1, and/or the application of the bacterial agent in the fields of biodegradation and bioremediation.

The biodegradation and bioremediation refer to the degradation of n-hexadecane and/or the restoration of n-hexadecane pollution in the soil.

The method for degrading n-hexadecane is characterized in that it comprises treating n-hexadecane with Brevibacillus nitrificans strain YJ1 with the deposit number of CGMCC NO.18418.

The conditions for treating n-hexadecane are 30°C, pH=7, and inoculation amount 10%.

The present invention provides a strain capable of degrading n-hexadecane, which is characterized in that: the degrading strain is Brevibacillus nitrificans, the strain code is YJ1, and it was published on August 23, 2019 by the China Microbial Culture Collection and Management Committee. Deposited by the Center for Microbiology CGMCC, the biological deposit number is CGMCC NO.18418.

The bacterial colony of this strain YJ1 in beef extract peptone agar medium is light yellow, raised, with neat edges, moist surface, not easy to provoke; the bacterial cells are straight rod-shaped under the microscope, and Gram staining is positive; the strain is hemolyzed by blood agar plate culture In the sex test, there was no hydrolysis circle around the colony, and the hemolysis of the strain was negative.

The degrading strain can be applied to the bioremediation of n-hexadecane, and the compound is n-hexadecane.

For the degradation of n-hexadecane by the n-hexadecane degrading strain, the suitable degradation conditions are 30°C, pH=7, and 10% inoculum.

After the degrading strain YJ1 is activated, it is connected to an inorganic salt medium with an initial concentration of n-hexadecane of 10 mL/L, and the degradation effect can reach 66.7%.

The beef extract peptone culture medium (Beef extract peptone, BP) is: peptone 10g/L, beef extract 5g/L, NaCl 10g/L, distilled water is fixed to 1L, and the pH is adjusted to 7.2.

The inorganic salt culture medium is: K ₂ HPO ₄ ·3H ₂ O 1.0g/L, KH ₂ PO ₄ 1.0g/L, MgSO ₄ ·7H ₂ O 0.5g/L, NH ₄ NO ₃ 1.0g/L, CaCl ₂ 0.02g/L, Fe ₂ (SO ₄ ) ₃ 0.02g/L, distilled water, pH 7.0-7.2, sterilized at 121°C for 20 minutes.

The inorganic salt medium containing n-hexadecane is an inorganic salt medium with a n-hexadecane concentration of 2-20 mL/L.

Said agar medium only needs to add 1.2-2% agar powder on the basis of the original medium formula.

The strain has the ability to grow with n-hexadecane as the sole carbon source and energy source.

The 16S rDNA sequence of the strain is shown in SEQ NO.1.

Application of the strain in the bioremediation of n-hexadecane pollution.

The n-hexadecane degrading bacteria YJ1 of the present invention, the bacterial colony of the strain in the BP agar medium is light yellow, convex, the edges are neat, the surface is moist, and it is not easy to provoke; the bacterial body is in the shape of a straight rod under the microscope, and is gram Staining is positive; blood agar plate medium is negative for hemolysis; the results of phylogenetic tree analysis show that it has high sequence homology and sequence similarity with multiple strains of Brevibacillus sp. released by Genbank Reaching 79%, it was identified as Brevibacillus nitrificans by the General Microbiology Center (CGMCC) of the China Microbial Species Collection Management Committee.

The screening method of n-hexadecane degrading bacteria can be implemented in sequence according to the following steps:

(1) Take a 0-12cm soil sample from the oil depot of a relocated power parts factory and its surrounding oil contaminated surface as a bacterial source for natural enrichment culture. Add 10g of soil sample into a 250mL Erlenmeyer flask containing 90mL inorganic salt liquid medium containing n-hexadecane, place it on a constant temperature shaking incubator at 30℃ and 180r/min for 5 days, and take 5mL of the mixed solution into fresh Containing n-hexadecane in 95mL inorganic salt medium. Repeat the above process many times to complete the enrichment of n-hexadecane degrading strains.

The inorganic salt culture medium is: K ₂ HPO ₄ ·3H ₂ O 1.0g/L, KH ₂ PO ₄ 1.0g/L, MgSO ₄ ·7H ₂ O 0.5g/L, NH ₄ NO ₃ 1.0g/L, CaCl ₂ 0.02g/L, Fe ₂ (SO ₄ ) ₃ 0.02g/L, distilled water, pH 7.0-7.2, sterilized at 121°C for 20 minutes.

The said inorganic salt medium containing n-hexadecane is: the above-mentioned inorganic salt medium with a concentration of n-hexadecane of 2-20 mL/L.

Said agar medium only needs to add 1.2-2% agar powder on the basis of the original medium formula.

(2) Dilute the enriched culture solution described in (1) and spread it on an inorganic salt agar medium containing n-hexadecane. After the plate is stationary for 30 minutes, place it in a constant temperature incubator at 30°C for cultivation. After repeated observations, colonies with different morphologies were formed on the surface of the plate.

(3) Transfer the strains selected in (2) to the inorganic salt agar medium that does not contain n-hexadecane, and remove the strains that can grow on the inorganic salt agar medium that does not contain n-hexadecane to remove Autotrophs and agar-available bacteria. Repeat the above streaking and separation process until a single colony that can grow significantly on the n-hexadecane inorganic salt medium is selected, and the strain is numbered YJ1.

The colony of the YJ1 strain of the present invention in the BP medium is light yellow, raised, with neat edges, moist surface, and not easy to be provoked; the bacterial cells under the microscope are straight rod-shaped and Gram staining is positive; strain blood agar plate culture hemolytic test , No hydrolysis circle appeared around the colony, strain hemolysis was negative; contact enzyme test was positive, oxidase, gelatin, 3-methyl-D-glucose, citric acid, glycerol, sucrose test were negative.

The application of the strain YJ1 is characterized in that the n-hexadecane degrading strain can be used in the bioremediation of n-hexadecane pollution, and the compound is n-hexadecane.

The application of the strain YJ1 is characterized in that the suitable degradation conditions of the YJ1 strain for the degradation of n-hexadecane are 30° C., pH=7, and inoculation amount of 10%.

The application of the described n-hexadecane degrading strain YJ1 is characterized in that the activated strain YJ1 is connected to an inorganic salt medium with an initial concentration of 10 mL/L of n-hexadecane for cultivation, and the degradation effect can reach 66.7%.

The strain of the present invention has good degradation characteristics for n-hexadecane, and will not cause secondary pollution to the environment, and can be applied to degrade n-hexadecane and biodegradation and bioremediation of soil contaminated by n-hexadecane, etc. field.

The preservation information of Brevibacillus nitrificans strain YJ1 of the present invention is as follows:

Name: YJ1

Category name: Bacillus nitrificans

Latin name: Brevibacillus nitrificans

Deposit number: CGMCC No. 18418

Preservation institution: General Microbiology Center of China Microbial Species Preservation Management Committee;

Preservation address: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing;

Date of preservation: August 23, 2019.

Description of the drawings

Figure 1 (a) shows the growth of strain YJ1 on BP medium, and (b) shows the hemolytic test of strain YJ1.

Figure 2 shows the morphology of strain YJ1 under an optical microscope.

Figure 3 shows the growth curve of strain YJ1. Among them, the temperature is 30°C, and the rotation speed is 180r/min.

Figure 4 shows the effect of different growth conditions on the growth of YJ1. (A) is the pH, (b) is the inoculum, (c) is the culture temperature, and (d) is the substrate concentration.

Figure 5 shows the degradation of n-hexadecane by strain YJ1 in an inorganic salt medium containing n-hexadecane at a concentration of 10 mL/L.

Detailed ways

Hereinafter, the above-mentioned content of the present invention will be further described in detail through specific implementations in the form of examples, but it should not be understood that the scope of the above-mentioned subject of the present invention is limited to the following examples. All technologies implemented based on the foregoing content of the present invention belong to the scope of the present invention. Except for special instructions, the following embodiments are all completed by conventional existing technologies.

The first group of examples, Brevibacillus nitrificans strain YJ1 of the present invention

This group of examples provides a strain of Brevibacillus nitrificans strain YJ1, the preservation number of which is CGMCC NO.18418.

In some embodiments, the 16S rDNA sequence of Brevibacillus nitrificans strain YJ1 is shown in SEQ NO.1.

In a specific embodiment, the colony of the strain YJ1 of Brevibacillus nitrificans strain YJ1 is light yellow, convex, with neat edges, and a moist surface; the bacterial cells are straight rod-shaped and Gram staining is positive; the hemolysis of the strain is negative .

The second group of examples, the bacterial agent of the present invention

This group of embodiments provides a bacterial agent for degrading n-hexadecane, which is characterized by comprising the Brevibacillus nitrificans strain YJ1 provided by any one of the first group of embodiments.

In a further embodiment, the inoculum also includes auxiliary materials commonly used in the preparation of inoculants; preferably, the auxiliary materials commonly used in the preparation of inoculants are selected from: the culture medium of Brevibacillus nitrificans strain YJ1, Or, other commonly used excipients for bacterial agents.

In a specific embodiment, the culture medium of the Brevibacillus nitrificans strain YJ1 is selected from: beef extract peptone medium, or, an inorganic salt medium containing n-hexadecane; the beef extract peptone medium includes : Peptone 10g/L, beef extract 5g/L, NaCl 10g/L, pH=7.2; the inorganic salt medium containing n-hexadecane includes: n-hexadecane concentration of 2-20mL/L, K ₂ HPO ₄ ·3H ₂ O 1.0g/L, KH ₂ PO ₄ 1.0g/L, MgSO ₄ ·7H ₂ O 0.5g/L, NH ₄ NO ₃ 1.0g/L, CaCl ₂ 0.02g/L, Fe ₂ (SO ₄ ) ₃ 0.02g/L, distilled water, pH 7.0～7.2. If the experiment needs to prepare the corresponding agar medium, just add 1.2-2% agar powder on the basis of the original medium formula.

The inorganic salt medium containing n-hexadecane is a YJ1 strain selection medium, and the screening mechanism is: the YJ1 strain uses n-hexadecane added in the inorganic salt medium as a growth carbon source and energy source. BP medium is a widely used bacterial medium, a selection medium for non-strain YJ1. In the experiment process, BP medium was used as the enrichment culture after the YJ1 strain was selected.

The commonly used auxiliary materials of the other bacterial agents are selected from: carriers, excipients, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, Osmotic pressure regulators, stabilizers, glidants, flavors, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrating agents, penetration enhancers, pH regulators, buffers, enhancers Plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc.

The third group of examples, the application of the YJ1 strain of the present invention

This group of embodiments provides the application of the Brevibacillus nitrificans strain YJ1 of any one of the first group of embodiments, and/or the application of the bacterial agent of any one of the second group of embodiments in the field of biodegradation and bioremediation .

In some embodiments, the biodegradation or bioremediation refers to the degradation of n-hexadecane and/or the restoration of n-hexadecane pollution in the soil.

The fourth group of embodiments, the method for degrading n-hexadecane of the present invention

This group of embodiments provides a method for degrading n-hexadecane, which is characterized in that it comprises treating n-hexadecane with Brevibacillus nitrificans strain YJ1 with the deposit number of CGMCC NO.18418.

In a preferred embodiment, the conditions for treating n-hexadecane are 30° C., pH=7, and inoculation amount 10%.

The medium formula used in the following examples is:

The inorganic salt culture medium is: K ₂ HPO ₄ ·3H ₂ O 1.0g/L, KH ₂ PO ₄ 1.0g/L, MgSO ₄ ·7H ₂ O 0.5g/L, NH ₄ NO ₃ 1.0g/L, CaCl ₂ 0.02g/L, Fe ₂ (SO ₄ ) ₃ 0.02g/L, distilled water, pH 7.0-7.2, sterilized at 121°C for 20 minutes.

The inorganic salt medium containing n-hexadecane is an inorganic salt medium of n-hexadecane degrading bacteria with a n-hexadecane concentration of 2-20 mL/L.

The beef extract peptone medium (Beef extract peptone, BP) is: peptone 10g/L, beef extract 5g/L, NaCl 10g/L, distilled water to a constant volume of 1L, and adjust the pH to 7.2.

Said agar medium only needs to add 1.2-2% agar powder on the basis of the original medium formula.

The strain of the present invention used in the following examples is referred to as YJ1 strain, Brevibacillus nitrificans.

Experimental Example 1: Separation, Screening and Purification of Strains

Enrichment culture:

Take a 0-12cm soil sample from the oil depot of a relocated power parts factory and its surrounding oil contaminated surface. Add 10mL/L of n-hexadecane to the inorganic salt medium as the only carbon source and energy source, add 10g of soil sample into a 250mL Erlenmeyer flask of 90mL liquid medium containing n-hexadecane and place it in 30 Cultivate on a constant temperature shaking incubator at 180r/min for 5 days, and take 5 mL of the mixed solution and put it into a fresh 95 mL inorganic salt medium containing n-hexadecane. Repeat the above process many times to complete the enrichment of n-hexadecane degrading strains.

Strain screening: take out a certain amount of the last enriched culture solution and apply it to the inorganic salt agar medium containing n-hexadecane. After the plate is still for 30 minutes, it is placed in a 30℃ constant temperature incubator for cultivation. The medium period is more In the second observation, colonies with different morphologies were formed on the surface of the plate.

Strain isolation and purification: transfer the strains selected in the above-mentioned strain screening process to the inorganic salt agar medium without n-hexadecane to remove the strains that can grow on the inorganic salt agar medium without n-hexadecane, To remove autotrophic organisms and bacteria using agar. Repeat the above streaking and separation process until a colony that can grow significantly on the n-hexadecane inorganic salt medium is selected, and the strain number is YJ1.

The colony morphology of this strain YJ1 in the BP medium is shown in Figure 1(a), which is light yellow, raised, with neat edges, moist surface, and not easy to provoke; the bacteria are straight rod-shaped under the microscope, and Gram staining is positive (see Figure 2); See Figure 1(b) for the hemolytic test results of strain blood agar plate culture. There is no hydrolysis circle around the YJ1 colony, and the bacterial hemolysis is negative; the contact enzyme test is positive, oxidase, gelatin, 3-methyl- The D-glucose, citric acid, glycerol, and sucrose tests were negative.

Strain preservation: transfer the selected YJ1 strain to the corresponding slant medium 30°C incubator for 48h, and then store it in a 4°C refrigerator for a short period of time; or pick the purified single colony of YJ1 into the corresponding liquid medium and cultivate it to In the logarithmic phase, add sterile glycerin (final glycerol concentration of 30%) to the bacterial solution at a ratio of 1:1 (V/V), mix and dispense into pre-sterilized bacteria preservation tubes (1～2mL/ Tube), stored in the refrigerator at -80°C.

The inventor performed 16S rDNA sequencing on the strains, and analyzed the bacteria using 16S rDNA gene sequence: streaking the plate to obtain a single colony of degrading bacteria, picking a single colony for colony PCR amplification, primer 27F (5'-AGAGTT TGATCMTGG CTCAG-3 '), 1492R(5'-TACGGY TACCTT GTTACG ACTT-3'). PCR reaction conditions: pre-denaturation at 94°C for 5 min; entering the thermal cycle, denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min, totaling 30 cycles; finally, extension at 72°C for 5 min and holding at 10°C. Perform electrophoresis detection on a 1% agarose gel. Sequencing of the strain was completed by Shanghai Shenggong Biotechnology Co., Ltd.

The nucleotide sequence of strain YJ1 is shown in the sequence table. This sequence is the complete 16S rDNA sequence of the strain. The strain was identified as Brevibacillus nitrificans by CGMCC. Identification unit information: General Microbiology Center of China Microbial Species Collection Management Committee, the address is Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

Therefore, the inventor named its strain code YJ1, and carried out a biological deposit on it, and its biological deposit number is CGMCC NO.18418, and its classification is named Brevibacillus nitrificans.

Experimental example 2: Growth curve and growth conditions determination of strain YJ1

Determination of growth curve: Use an inoculating loop to pick a loop of the selected strains and dilute with an appropriate amount of 0.85% normal saline to OD ₆₀₀ =0.1 to prepare a bacterial suspension, and pipette 1 mL of the bacterial suspension into 99 mL of BP liquid medium at 30°C , Vibrate culture at 180r/min, take a sample every 2 hours, because the biomass of the bacterial solution in a fixed wavelength is proportional to the optical absorption value (OD), the OD ₆₀₀ value is measured by a spectrophotometer, expressed as OD ₆₀₀ For the growth of this strain, do 3 repetitions. The test determined the growth of the n-hexadecane degrading strain YJ1 in BP medium for 62 hours, and the results showed that 0-10 hours is the delay period of the strain, and 10 to 32 hours of culture is the logarithmic growth phase of the strain. , It enters the stable growth period between 32 and 42 hours, and enters the decay period from 44 hours, as shown in Figure 3.

The initial pH value to determine the growth of the strain: prepare the inorganic salt liquid medium with the n-hexadecane concentration of 10mL/L and different initial pH values, and then insert the bacteria that are cultivated to the logarithmic growth phase at 4% of the inoculum. The solution was cultured with shaking at 30°C and 180r/min for 7 days. During the culture period, the growth of the strain was observed every day. Finally, the OD ₆₀₀ value was measured with a spectrophotometer. There were 3 parallels in each group. The set pH is as follows: 5.0, 6.0, 7.0, 8.0, 9.0. The results are shown in Figure 4(a). The strain YJ1 can grow in an environment with a pH of 5.0 to 9.0, and the optimal growth pH is 7.0.

The influence of culture temperature on the growth of strains: prepare an inorganic salt liquid medium with a concentration of n-hexadecane of 10mL/L and pH=7.0, and then insert 4% of the inoculum into the bacterial solution cultured to the logarithmic growth phase. , Respectively, under different culture temperature, 180r/min conditions, shaking culture for 7 days, observe the growth of the strains every day during the culture, and finally use a spectrophotometer to determine the OD ₆₀₀ value, each group set 3 parallel. The set strain growth temperatures are: 20°C, 25°C, 30°C, 35°C, and 40°C. Results As shown in Figure 4(b), strain YJ1 can grow at a temperature of 20°C to 40°C, and the optimal growth temperature is 30°C.

The influence of the inoculum amount on the growth of the strain: prepare an inorganic salt liquid medium with a concentration of n-hexadecane of 10 mL/L and pH=7.0, and then insert the inoculum into the bacterial liquid cultured to the logarithmic growth phase according to different inoculum amounts. Under the conditions of 30°C and 180r/min, shake culture for 7 days, observe the growth of the strains every day during the culture period, and finally measure the OD ₆₀₀ value with a spectrophotometer. There are 3 parallels in each group. The set inoculation amounts are: 2%, 4%, 6%, 8%, and 10%. The results are shown in Figure 4(c). The strain YJ1 grew under the condition of inoculation amount of 2%-10%, and the optimal inoculation amount was 10%.

The influence of substrate concentration on the growth of strains: prepare inorganic salt liquid culture media with different n-hexadecane concentrations, adjust pH=7.0, and then insert 4% of the inoculum into the bacterial solution cultured to the logarithmic growth phase. Under the conditions of 30°C and 180r/min, shake culture for 7 days, observe the growth of the strains every day during the culture, and finally measure the OD ₆₀₀ value with a spectrophotometer, with 3 parallels in each group. The set n-hexadecane concentration is as follows: 2mL/L, 5mL/L, 10mL/L, 15mL/L, 20mL/L. The results are shown in Figure 4(d). The strain YJ1 can grow under the condition of n-hexadecane concentration of 2-20 mL/L, and its optimal substrate concentration is 10 mL/L.

Experimental example 3: Analysis of degradation ability of strain YJ1

According to the above test results, the optimal growth conditions for the strains were determined to be a temperature of 30°C and a pH of 7.0, and the screened bacteria were respectively inoculated into the corresponding BP liquid medium. 30℃, 180r/min constant temperature shaking culture for 24h. Count on the plate, and the bacteria count is about 1.0×10 ⁸ CFU/mL, ready for use.

Bacterial degradation experiment of n-hexadecane: Based on the liquid inorganic salt medium (pH=7) added with a concentration of 10mL/L n-hexadecane as the carbon source, the bacteria in the logarithmic growth phase were inoculated at 10% The amount was connected to the culture medium and cultured under constant temperature shaking at 30°C and 180 r/min. With no strain added as a control, the constant temperature shaking culture was carried out for 5 days, 10 days, and 15 days.

The concentration of n-hexadecane is determined by ultraviolet spectrophotometry, that is, the whole bottle is extracted from the culture solution, and the extract is extracted 3 times with a petroleum ether oscillating separatory funnel. The absorption value under the characteristic peak wavelength of alkane-petroleum ether: 256nm), calculate its degradation rate, sample and analyze the residual amount of n-hexadecane, each group has 3 parallels.

The selected strain YJ1 uses n-hexadecane as the sole carbon source to carry out metabolic activities and continuously consumes the carbon source in the medium. It is cultured in a medium with a n-hexadecane concentration of 10 mL/L until 5, 10, and 15 days. , Using ultraviolet spectrophotometry to determine the content of n-hexadecane in the solution after degradation, the results show (Figure 5), the strain YJ1 degraded n-hexadecane up to 66.7%.

Claims (10)

1. A strain of Brevibacillus nitrificans strain YJ1, its deposit number is CGMCC NO.18418.
2. The Brevibacillus nitrificans strain YJ1 of claim 1, wherein the 16S rDNA sequence is shown in SEQ NO.1.
3. The Brevibacillus nitrificans strain YJ1 according to claim 1 or 2, characterized in that the colony is light yellow, convex, the edges are neat, and the surface is moist; the bacteria are straight rod-shaped and Gram staining is positive; The hemolysis of the strain was negative.
4. The bacterial agent for degrading n-hexadecane is characterized by comprising the Brevibacillus nitrificans strain YJ1 of any one of claims 1-3.
5. The bacterial agent according to claim 4, which further comprises auxiliary materials commonly used for preparation of bacterial agents; preferably, the auxiliary materials commonly used for preparation of bacterial agents are selected from the group consisting of: the cultivation of Brevibacillus nitrificans strain YJ1 Base, or other commonly used excipients for bacterial agents.
6. The bacterial agent according to claim 4 or 5, wherein the culture medium of the Brevibacillus nitrificans strain YJ1 is selected from: beef extract peptone culture medium, and/or culture containing n-hexadecane inorganic salt base;

   The beef extract peptone culture medium includes: peptone 10g/L, beef extract 5g/L, NaCl 10g/L, the rest is water, pH 7.2;

   The medium containing n-hexadecane inorganic salt includes: n-hexadecane 2-20 mL/L, K ₂ HPO ₄ ·3H ₂ O 1.0 g/L, KH ₂ PO ₄ 1.0 g/L, MgSO ₄ ·7H ₂ O 0.5g/L, NH ₄ NO ₃ 1.0g/L, CaCl ₂ 0.02g/L, Fe ₂ (SO ₄ ) ₃ 0.02g/L, the rest is water, pH 7.0-7.2.
7. The application of the Bacillus nitrificans Brevibacillus nitrificans strain YJ1 according to any one of claims 1-3, and/or the application of the bacterial agent according to any one of claims 4-6 in the fields of biodegradation and bioremediation.
8. The application according to claim 7, wherein the biodegradation and bioremediation refer to degrading n-hexadecane and/or repairing n-hexadecane pollution in the soil.
9. The method for degrading n-hexadecane is characterized in that it comprises treating n-hexadecane with Brevibacillus nitrificans strain YJ1 with the deposit number of CGMCC NO.18418.
10. The method according to claim 9, characterized in that the optimal conditions for the treatment of n-hexadecane are 30°C, pH=7, and inoculation amount 10%.

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