A colorless bacterium and its application in degradation of chloroaniline

Abstract

The invention relates to the technical field of sewage treatment, and proposes a colorless bacterium and its application in degrading chloroaniline. The colorless bacterium is a colorless bacterium (Achromobacter sp.) HZ-O-033, and the deposit number is CGMCC No.30975. The colorless bacterium (Achromobacter sp.) HZ-O-033 strain screened by the present invention has the function of degrading m-chloroaniline and o-chloroaniline. For sewage containing 500 mg / L m-chloroaniline mixture, the degradation rate reaches more than 99% in 72 hours, and has good application prospects in the degradation of chloroaniline pollution.

Description

Technical Field

[0001] The invention relates to the technical field of sewage treatment, in particular to a colorless bacterium and application thereof in degrading chloroaniline. Background Art

[0002] Chloroaniline compounds such as m-chloroaniline and o-chloroaniline are widely present in wastewater from textile printing and dyeing, pharmaceutical and petrochemical industries. They are a type of pollutant with a large pollution area, strong toxicity and difficult biodegradation. They can exist in the environment for a long time and are easy to bioaccumulate, causing serious harm to the environment and human health. The traditional physical and chemical methods for treating m-chloroaniline and o-chloroaniline are costly and prone to secondary pollution. The biological treatment method has the advantages of low operating cost and no secondary pollution, and is the main method for treating wastewater containing m-chloroaniline and o-chloroaniline.

[0003] At present, researchers at home and abroad have made some progress in the microbial degradation of chloroaniline compounds. For example, the invention patent with patent number CN114806968A discloses Pseudoxanthomonas that can degrade chlorobenzene, chlorophenol and / or chloroaniline. However, it takes more than 10 days for this strain to degrade 100 mg / L of p-chloroaniline, and the degradation efficiency is low. Therefore, screening a strain that can efficiently degrade chloroaniline compounds is an urgent problem that needs to be solved. Summary of the invention

[0004] In view of this, the present invention provides a colorless bacterium and application thereof in degrading chloroaniline, wherein the bacterium is a strain capable of efficiently degrading chloroaniline compounds.

[0005] The technical solution of the present invention is implemented as follows: On the one hand, the present invention provides a colorless bacterium, which is Achromobacter sp. HZ-O-033, with a preservation number of CGMCC No.30975.

[0006] On the basis of the above technical solution, preferably, the nucleotide sequence of 16S rRNA of the Achromobacter sp. HZ-O-033 is as shown in SEQ ID No.1.

[0007] Application of a colorless bacterium in the degradation of chloroaniline.

[0008] On the basis of the above technical solution, preferably, the chloroaniline is selected from one or both of m-chloroaniline and o-chloroaniline.

[0009] On the basis of the above technical scheme, preferably, Achromobacter sp. HZ-O-033 is added into the wastewater containing chloroaniline to degrade the chloroaniline.

[0010] On the basis of the above technical scheme, preferably, the inoculation amount of the Achromobacter sp. HZ-O-033 in the sewage is 5-15 mL / L, and the strain concentration is 1×10 6 cell / mL.

[0011] The invention discloses an application of a colorless bacterium in preparing a chloroaniline-degrading bacterial agent.

[0012] On the basis of the above technical solution, preferably, the chloroaniline is selected from one or both of m-chloroaniline and o-chloroaniline.

[0013] The achromobacterium of the present invention and its application in degrading chloroaniline have the following beneficial effects compared with the prior art:

[0014] The Achromobacter sp. HZ-O-033 strain screened by the present invention has the function of degrading m-chloroaniline and o-chloroaniline. For sewage containing 500 mg / L m-chloroaniline mixture, the degradation rate reaches more than 99% within 72 hours, and has good application prospects in the degradation of chloroaniline pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is a scanning electron micrograph of Achromobacter sp. HZ-O-033;

[0017] Figure 2 This is the phylogenetic tree analysis diagram of Achromobacter sp. HZ-O-033 strain;

[0018] Figure 3 The chromatogram of the sample is shown in the following example. DETAILED DESCRIPTION

[0019] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Example 1 Strain enrichment, separation, purification and identification

[0021] (1) Strain enrichment

[0022] The activated sludge from a biochemical pool of a pharmaceutical factory in Hubei was used as the mud for screening and enrichment. After the bottom mud was centrifuged, the supernatant was removed, and the precipitate was added to the enrichment medium at 10% (w / v), and the enrichment was carried out on a shaking table at 30°C and 150r / min. The content of the m / o-chloroaniline mixed solution in the culture medium was detected every day. Once the degradation rate of the m / o-chloroaniline mixed solution in the culture medium reached more than 95%, it was inoculated into a new culture medium again at a ratio of 10%, and the content of the m / o-chloroaniline mixed solution was increased at the same time. After acclimation for 3 to 4 weeks according to this method, the m / o-chloroaniline degradation efficiency of the sludge was improved, and the m / o-chloroaniline degrading bacteria enrichment solution was obtained at this time, and the enriched bacterial solution could be separated and purified by plate dilution coating and streaking method.

[0023] Enrichment medium formula: m / o-chloroaniline mixture 0.1g-1.5g, K 2 HPO 4 0.5g, KH 2 PO 4 0.52g, NaCl 0.2g, MgSO 4 7H 2 O 0.2g, MnSO 4 ·H 2 O 0.15 g, CaCl 2 0.1 g, (NH 4 ) 2 SO 4 0.1-0.3g, add distilled water to 1L, pH 7.0-7.2.

[0024] Preparation method of m / o-chloroaniline mixture: m-chloroaniline and o-chloroaniline are mixed in a mass ratio of 1:1.

[0025] Strain isolation and purification: Take 1 mL of the mixed m / o-chloroaniline-degrading bacteria enrichment solution and perform gradient dilution. The dilution solution uses sterilized purification medium. The enrichment is diluted to 10 -1 -10 -7 7 gradients, 100 μL of each gradient was spread on the solid purification medium, 5 parallels were made for each gradient, and cultured at 30°C for 48 hours, and different colonies grown on the plate were picked for streak purification. After 6-7 rounds of streak purification, a strain was finally purified and named HZ-O-033.

[0026] Purification medium (liquid): m / o-chloroaniline mixture 0.1g-0.5g, K 2 HPO 4 0.5g, KH 2PO 4 0.52g, NaCl 0.2g, MgSO 4 7H 2 O 0.2g, MnSO 4 ·H 2 O 0.15 g, CaCl 2 0.1 g, (NH 4 ) 2 SO 4 0.1g, add distilled water to 1L, pH 7.0-7.2.

[0027] Purification medium (solid): Add 1.5%-2% (w / v) agar powder to the liquid purification medium.

[0028] (2) Strain identification

[0029] Strain morphology: The strain HZ-O-033 was streaked on beef extract peptone solid medium and cultured at 30°C under aerobic conditions for 24-48 hours. The colony diameter of HZ-O-033 was 0.2-0.5mm, and the colony was white, raised, smooth and dull, round, with smooth edges, transparent, odorless and tasteless. The microscopic morphology of the strain was observed by scanning electron microscopy, and it was found that the bacteria was a bacillus, 1-1.5μm long and 0.3-0.5μm wide. Figure 1 shown.

[0030] Molecular biological identification: The genome of strain HZ-O-033 was extracted and 16S rDNA sequencing was performed. The obtained sequence is shown in SEQ ID No.1.

[0031] The sequencing results were compared with the known sequences in the Genbank database. As shown in the figure, strain HZ-O-033 has the highest sequence homology with Achromobacter sp. Figure 2 Phylogenetic tree analysis identified it as Achromobacter sp.

[0032] In general, the strain HZ-O-033 obtained by the present invention is a colorless bacillus (Achromobacter sp.). Therefore, the strain HZ-O-033 was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) on June 17, 2024, with the deposit number: CGMCC No. 30975, and the protection center identified the strain HZ-O-033 as active on June 17, 2024.

[0033] Example 2 Screening of culture medium for expansion of strain HZ-O-033

[0034] (1) Strain activation: Prepare the three culture media in Table 1, take 3 mL of each culture medium into a glass tube for sterilization. Pick a single colony on the solid enrichment medium and add it to each of the above culture media. Incubate at 30°C, 150 rpm in a shaker for 48 h. The concentrations of the strains in the three activated culture media are 1×10 6 cell / mL (medium No. 1), 1×10 5 cell / mL (medium No. 2), 1×10 7 cells / mL (medium No. 3) and activation is complete.

[0035] (2) Strain expansion: The three activated strains were inoculated into the corresponding culture medium in Table 1 at 1% (v / v) and cultured aerobically at 30°C and 150 rpm in a shaker for 48 h. The bacterial concentration (OD 600 ) and m / o-chloroaniline concentrations. The results are shown in Table 2.

[0036] Table 1 Culture medium formula

[0037]

[0038] Note: The mass ratio of m / o-chloroaniline in the m / o-chloroaniline mixture is 1:1.

[0039] Table 2 Degradation efficiency of m / o-chloroaniline by bacteria cultured in different media

[0040]

[0041]

[0042] From the table above, we can see that the degradation efficiency of m / o-chloroaniline by the culture medium No. 1 is the highest, and the degradation rate of the m / o-chloroaniline mixed solution reaches 99.73% in 24 hours; the degradation efficiency of the culture medium No. 3 is second, and the degradation rate can reach 99.73% in 48 hours; while the degradation rate of the culture medium No. 2 can only reach 31.86% in 48 hours. From the perspective of bacterial concentration, the bacteria in the culture medium No. 3 grow faster, followed by No. 1, and No. 2 is the slowest. Considering the degradation efficiency and bacterial growth concentration, the subsequent experiments will choose the culture medium No. 1.

[0043] Example 3 Ability of strain HZ-O-033 to degrade m-chloroaniline and o-chloroaniline

[0044] In order to verify whether strain HZ-O-033 has the ability to degrade both m-chloroaniline and o-chloroaniline, the m- / o-chloroaniline mixture in culture medium No. 1 was replaced with m-chloroaniline and o-chloroaniline to check whether strain HZ-O-033 can degrade them alone.

[0045] Experimental method: HZ-O-032 strain (1×10 6 The m-chloroaniline, o-chloroaniline and the m- / o-chloroaniline mixture were inoculated into a culture medium containing only m-chloroaniline, o-chloroaniline and the m- / o-chloroaniline mixture (the culture medium was the above-mentioned culture medium No. 1), and the culture medium was treated at 30° C. and 150 r / min. The m-chloroaniline, o-chloroaniline and the m- / o-chloroaniline mixture were tested regularly. The results are shown in Table 3.

[0046] Table 3 Degradation ability of strain HZ-O-033 on m-chloroaniline and / or o-chloroaniline

[0047]

[0048] As can be seen from the above table, strain HZ-O-033 has the ability to degrade m-chloroaniline and o-chloroaniline respectively. The degradation rate of m-chloroaniline and / or o-chloroaniline at 139-154 mg / L in 48 h is more than 99%. Among them, the degradation rate of o-chloroaniline by strain HZ-O-033 is faster than that of m-chloroaniline, which may be related to the structure of o-chloroaniline. o-chloroaniline is easier to open the ring, making it degrade faster than m-chloroaniline.

[0049] Example 4 Effect of initial m / chloroaniline concentration on strains

[0050] Experimental method: The activated HZ-O-033 strain (1×10 6 The 1% (v / v) 2-chloroaniline mixture was inoculated into the simulated wastewater containing different concentrations of m-chloroaniline and m-chloroaniline mixtures at 30°C and 150 r / min. The content of the m-chloroaniline mixture was tested regularly. The results are shown in Table 4.

[0051] Simulated wastewater preparation method: Use the effluent from the biochemical pool of a pharmaceutical factory, and adjust the concentration of the m / chloroaniline mixed solution in the effluent to 20 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 500 mg / L respectively. The mass ratio of m / o-chloroaniline in the mixed solution is 1:1.

[0052] Table 4 Effect of initial m / chloroaniline concentration on the degradation ability of strain HZ-O-033

[0053]

[0054] As shown in Table 4: Different initial concentrations of m / chloroaniline mixed solutions have little effect on the degradation ability of strain HZ-O-033. When the initial concentration is 20-100 mg / L, the degradation rate of strain HZ-O-033 can reach more than 98% within 48 hours, and the degradation rate can reach more than 99% within 72 hours. When the initial concentration of m / chloroaniline mixed solution is 200-500 mg / L, the degradation is relatively slow in the first 48 hours, and it is immediately degraded at 72 hours. It may be that the concentration is high, and the strain needs more time for ring-opening cleavage to degrade m / chloroaniline into an available carbon source for growth metabolism.

[0055] Example 5 Effect of different inoculation amounts on the degradation of m / chloroaniline by strain HZ-O-033

[0056] The activated strain HZ-O-033 (1×10 6 The cells were inoculated into simulated wastewater with an initial m / o-chloroaniline concentration of 100 mg / L at an inoculum size of 0.5% (v / v), 1% (v / v), 1.5% (v / v), 2% (v / v) and 3% (v / v), respectively, and cultured in a shaker (30°C, 150 r / min). Samples were taken at 48 h to detect the m / o-chloroaniline concentration. The results are shown in Table 5.

[0057] Table 5 Effect of inoculation size on the degradation of m / chloroaniline by strain HZ-O-033

[0058]

[0059] As can be seen from Table 5, when the inoculation amount of strain HZ-O-033 is 0.5% to 1.5%, the 24h degradation rate can reach more than 99%. When the inoculation amount is ≥2%, the degradation of m / chloroaniline by strain HZ-O-033 will be inhibited.

[0060] Example 6 Effect of salinity on the degradation of m / chloroaniline by strain HZ-O-033

[0061] Experimental method: The activated strain HZ-O-033 was added at 1% (v / v) (1×10 6 cell / mL) were inoculated into simulated wastewater containing m / chloroaniline mixtures of different salinities, and treated at 30°C and 150r / min. The content of m / chloroaniline mixtures was tested regularly. The results are shown in Table 6.

[0062] Preparation method of simulated wastewater with different salinities: using the effluent from the biochemical pool of a pharmaceutical factory, the salt concentration in the effluent was adjusted to 0.02%, 1%, 2% and 3% respectively.

[0063] Table 6 Effect of salinity on the degradation ability of strain HZ-O-033

[0064]

[0065] Since high-salt wastewater often appears in chemical wastewater and petrochemical wastewater, it is necessary to examine the tolerance of strain HZ-O-033 to salinity. As shown in Table 5, strain HZ-O-033 can tolerate a salinity of 0.02%-2%. When the salinity exceeds 3%, the ability of strain HZ-O-033 to degrade m / o-chloroaniline is inhibited. When the salinity is 2%, the degradation rate of m / o-chloroaniline in 72 hours is 96.65%, and when the salinity is 0.02%-1%, the degradation rate of m / o-chloroaniline in 72 hours exceeds 99%.

[0066] In summary, the optimal salinity for strain HZ-O-033 is 0-2%.

[0067] Example 7 Strain Application Examples

[0068] (1) Application of strains in pharmaceutical wastewater

[0069] The system in the biochemical system of wastewater produced by a pharmaceutical company often fluctuates, and the ammonia nitrogen in the effluent often exceeds the standard. Through GCMS testing of the effluent, it was found that the effluent contained m-chloroaniline, which can inhibit the activity of nitrifying bacteria in the biochemical system, thereby causing the ammonia nitrogen in the effluent to exceed the standard. The GCMS test results are shown in Figure 3 .

[0070] The test found that the concentration of meta-chloroaniline in the effluent was 87.04 mg / L. In order to solve the problem of meta-chloroaniline, strain HZ-O-033 was added to the effluent. The strain HZ-O-033 was cultured at 0.1-0.5% (v / v) (1×10 6 cell / mL) was added to the biochemical pool for 3-5 days. After one week, the concentration of chloroaniline in the water was detected to be 1.27 mg / L.

[0071] After one month of stable operation, the concentration of meta-chloroaniline in the effluent was maintained at 0.1-0.5 mg / L. During this period, the ammonia nitrogen in the effluent of the biochemical system was maintained within the effluent standard and did not exceed the standard. This shows that the strain HZ-O-033 can effectively degrade meta-chloroaniline in wastewater.

[0072] (2) Application of strains in high-salinity chemical wastewater

[0073] The salinity of the effluent from a chemical plant reached 2.05%, which contained m-chloroaniline and o-chloroaniline. The index parameters are as follows:

[0074]

[0075] The meta-chloroaniline / ortho-chloroaniline in the effluent is the main source of COD in the effluent, and the COD in the effluent often exceeds the standard. Therefore, it is necessary to degrade the meta-chloroaniline / ortho-chloroaniline in the effluent and reduce the COD to meet the effluent requirements.

[0076] The strain HZ-O-033 was added to the biochemical system at 0.01%-0.5% for 1 month. The effluent COD met the requirements and the effluent indicators were as follows:

[0077]

[0078] After using the bacterial agent, the concentration of intermediate / o-chloroaniline in the effluent was maintained within 2 mg / L, and the COD was within 50 mg / L, meeting the requirement of effluent <COD 100 mg / L.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A colorless bacterium, characterized in that: The achromobacter is achromobacter ( Achromobacter sp. )HZ-O-033, the deposit number is CGMCC No.30975.

2. The use of a colorless bacterium in the degradation of chloroaniline as claimed in claim 1, characterized in that: The chloroaniline is selected from one or both of m-chloroaniline and o-chloroaniline.

3. The use according to claim 2, characterized in that: Achromobacter Achromobacter sp. )HZ-O-033 is added into wastewater containing chloroaniline to degrade chloroaniline.

4. The use according to claim 2, characterized in that: The Achromobacterium Achromobacter sp. ) The inoculation volume of HZ-O-033 in sewage is 5-15 mL / L, and the bacterial concentration is 1×10 6 cell / mL.

5. The use of a colorless bacterium as claimed in claim 1 in the preparation of a chloroaniline-degrading bacterial agent, characterized in that: The chloroaniline is selected from one or both of m-chloroaniline and o-chloroaniline.

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