Halotolerant Chlorobenzene-Degrading Strains and Their Applications

Abstract

The present invention relates to bioengineering and is applied in the technical field of wastewater treatment by microorganisms. The present invention provides a salt-tolerant chlorobenzene-degrading strain, which is Ochrobactrum sp with a preservation number of CGMCC NO. 27159. The present invention can be used for the treatment of high-salinity chlorobenzene-containing sewage, and while degrading chlorobenzene, it can simultaneously reduce the total nitrogen and ammonia nitrogen.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology. Specifically, it is a salt-tolerant chlorobenzene-degrading strain and its application. Background Art

[0002] Chlorobenzene is a commonly used industrial organic solvent and an intermediate in many chemical reactions. It is often used in industries such as textiles, pharmaceuticals, leather, and steel. Due to the mutagenic, carcinogenic, and teratogenic effects of chlorine, as well as its characteristics of cumulative biological toxicity and wide pollution area, it is listed as one of the key pollutant blacklists.

[0003] The chemical properties of chlorobenzene are relatively stable. Traditional physical or chemical oxidation methods have a long treatment cycle and poor efficiency.

[0004] Using microorganisms to degrade chlorobenzene is the most economical and effective method. The screening and cultivation of chlorobenzene-degrading bacteria have been used, but the chlorobenzene-degrading bacteria currently used have problems to be solved: a small concentration range for degrading chlorobenzene, a long degradation time, and a small applicable environmental range.

[0005] Therefore, it is necessary to provide a salt-tolerant chlorobenzene-degrading strain and its application to solve the above problems. Summary of the Invention

[0006] The purpose of the present invention is to provide a salt-tolerant chlorobenzene-degrading strain and its application.

[0007] The present invention realizes the above purpose through the following technical solutions:

[0008] One of the technical solutions is:

[0009] A salt-tolerant chlorobenzene-degrading strain is Ochrobactrum sp, and the preservation number is CGMCC NO.27159.

[0010] Furthermore, the nucleotide sequence of 16S rDNA is as shown in SEQ ID No.1.

[0011] Furthermore, the bottom sludge in the anoxic tank of the printing and dyeing factory sewage treatment system is enriched by an enrichment medium. After centrifuging the sludge, the supernatant is removed, and the sludge precipitate is added to the enrichment medium at a ratio of 5-10% w / v. Enrichment is carried out at 35°C under anoxic conditions with DO < 1mg / L.

[0012] Furthermore, the change in the chlorobenzene content in the medium is detected every day. When the chlorobenzene content in the medium drops to 0mg / L, it is inoculated into a new medium again at a ratio of 5-10%, and at the same time, the chlorobenzene content is increased. After 1-2 months of domestication, the chlorobenzene degradation efficiency of the sludge is improved compared with the initial stage. At this time, a chlorobenzene-degrading bacteria enrichment solution is obtained, and the enriched bacterial solution is separated and purified by the plate dilution coating and streaking methods.

[0013] Further, the enriched bacterial liquid was diluted into 10 -1 -10 -7 seven gradients with a purified culture medium, and 100 μl was taken from each gradient and spread on the solid purified culture medium. Five parallels were made for each gradient, and anaerobic culture was carried out at 30 °C for 48 h. Different colonies grown on the plate were picked for streak purification; after 7-8 generations of streaking, the strain was finally purified and named strain HZ-O-030.

[0014] Further, strain HZ-O-030 was streaked on the nutrient agar solid medium and cultured at 30 °C under anaerobic conditions for 24-48 h; the colony diameter of HZ-O-030 was 0.5-1 mm, the colony was white, raised, with a smooth and dull surface, round, with a smooth edge, transparent, odorless and tasteless; by observing the microscopic morphology through a scanning electron microscope, strain HZ-O-030 was a bacillus, 1-2 μm in length and 0.4-0.5 μm in width.

[0015] The second technical solution is as follows:

[0016] An application of a salt-tolerant chlorobenzene-degrading strain, characterized in that it is applied to the treatment of chlorobenzene wastewater, and the steps include:

[0017] 1) Strain activation;

[0018] 2) Application:

[0019] 2-1) The activated strain HZ-O-030 was added to the chlorobenzene sewage at a ratio of 1% for treatment;

[0020] 2-2) The activated strain HZ-O-030 was added to the saline chlorobenzene sewage at a ratio of 1% for treatment;

[0021] 2-3) The activated strain HZ-O-030 was added to the chlorobenzene sewage with different pH values at a ratio of 1% for treatment.

[0022] Further, the LB medium includes 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, supplemented with distilled water to 1 L, and the pH is 7.0-7.2.

[0023] Further, the strain activation is specifically: inoculating the strain HZ-O-030 into the LB medium for activation, and culturing it under shaking at 30 °C, 100 r / min under anaerobic conditions for 48 h.

[0024] Compared with the prior art, the present invention can be used for the treatment of high-salinity chlorobenzene-containing sewage, and while degrading chlorobenzene, it can synchronously reduce the total nitrogen and ammonia nitrogen. Description of the Drawings

[0025] Figure 1 Scanning electron micrograph of the salt-tolerant chlorobenzene-degrading strain of the present invention;

[0026] Figure 2 Phylogenetic tree of the screened strain of the present invention and related strains;

[0027] Figure 3 Schematic diagram of the sewage treatment system of Example 2;

[0028] Microbial preservation for patent procedures:

[0029] Date of deposit: April 19, 2023

[0030] Depository: China General Microbiological Culture Collection Center

[0031] Address of depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences

[0032] Accession number: CGMCC No. 27159

[0033] Taxonomic name: Ochrobactrum sp Detailed description of the invention

[0034] Example 1:

[0035] This example demonstrates a strain that can rapidly degrade high-concentration chlorobenzene, is applicable to high-salinity chlorobenzene-containing sewage, and can simultaneously reduce total nitrogen and ammonia nitrogen while degrading chlorobenzene;

[0036] The steps include:

[0037] 1) Screening and identification of the strain:

[0038] 1-1) Enrichment of the strain:

[0039] Enrichment was carried out using the bottom sludge in the anoxic tank of a printing and dyeing factory sewage treatment system. After centrifuging the bottom sludge, the supernatant was removed, and the sludge precipitate was added to the enrichment medium at a ratio of 5-10% w / v. Enrichment was carried out at 35°C under anoxic conditions (DO < 1 mg / L);

[0040] The change in the chlorobenzene content in the medium was detected daily. When the chlorobenzene content in the medium dropped to 0 mg / L, it was inoculated into a new medium again at a ratio of 5-10%, and the chlorobenzene content was increased simultaneously;

[0041] After 1-2 months of domestication, the chlorobenzene degradation efficiency of the sludge increased compared to the initial stage. At this time, an enriched solution of chlorobenzene-degrading bacteria was obtained, and the enriched bacterial solution could be separated and purified by the plate dilution coating method and the streaking method;

[0042] The enrichment medium formula is as follows:

[0043] 50 - 1000 mg of chlorobenzene, NH 4 Cl 0.4 - 1 g, NaCl 15 g, Na 2 HPO 4 1.3 g, KH 2 PO 4 2 g, MgSO 4 ·7H 2 O 0.2 g, FeSO 4 ·7H 2 O 0.01 g, CaCl 2 ·2H 2 O 0.05 g, make up with distilled water to 1 L, pH 7.2 - 7.5;

[0044] 1 - 2) Isolation and purification of the strain:

[0045] Dilution coating method and streak purification: Dilute the enriched bacterial liquid with the liquid purification medium into 7 gradients of 10 -1 -10 -7 , take 100 μl from each gradient and coat them on the solid purification medium respectively, make 5 parallels for each gradient, culture them anoxically at 30 °C for 48 h, pick different colonies grown on the plate for streak purification; After 7 - 8 generations of streaking, finally 1 strain of bacteria was purified and named HZ - O - 030;

[0046] The liquid purification medium formula is: 500 - 1000 mg of chlorobenzene, NH 4 Cl 0.8 - 1 g, KH 2 PO 4 2 g, Na 2 HPO 4 1.3 g, CaCl 2 ·2H 2 O 0.05 g, MgSO 4 ·7H 2 O 0.2 g, FeSO 4 ·7H 2 O 0.01 g, make up with distilled water to 1 L, pH 7.2 - 7.5.

[0047] The solid purification medium can also be used. The solid purification medium is based on the liquid purification medium and added with 1.5% - 2% (w / v) agar powder;

[0048] 2) Identification of strain morphology and molecular biology

[0049] 2 - 1) Strain morphology:

[0050] The strain HZ-O-030 was streaked on a nutrient agar peptone solid medium and cultured at 30 °C under anaerobic conditions for 24 - 48 h. The diameter of the HZ-O-030 colony was 0.5 - 1 mm. The colony was white, raised, with a smooth and dull surface, circular, with a smooth edge, transparent, odorless and tasteless. By observing the microscopic morphology of the strain through a scanning electron microscope and referring to Figure 1 , it was found that the strain HZ-O-030 was a bacillus, 1 - 2 μm in length and 0.4 - 0.5 μm in width.

[0051] 2) Molecular biological identification of the strain

[0052] The genome of the strain HZ-O-030 was extracted and 16S rDNA sequencing was performed. The obtained sequence was designated as SEQ ID No.1 and was as follows:

[0053]

[0054] The sequencing results were compared with the known sequences in the Genbank database, and with reference to Figure 2 , the strain HZ-O-030 had the highest sequence homology with the genus Ochrobactrum and was identified as Ochrobactrum.

[0055] 3) Growth and functional characteristics of the strain

[0056] 3-1) Physiological and biochemical functions of strain HZ-O-030:

[0057] Activation of the strain: The strain HZ-O-030 was inoculated into LB medium for activation and cultured with shaking at 30 °C, 100 r / min under anoxic conditions for 48 h;

[0058] Identification of the physiological and biochemical functions of the strain: The activated strain was inoculated into the basal medium at 1% v / v and cultured with shaking at 30 °C, 100 r / min under anoxic conditions for 48 h - 72 h; During the culture period, samples were taken every 8 h to detect the OD 600 (bacterial concentration), chlorobenzene content, and ammonia nitrogen content;

[0059] Among them, the formula of LB medium is:

[0060] Tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, supplemented with distilled water to 1 L, pH 7.0 - 7.2;

[0061] The formula of the basal medium is:

[0062] Chlorobenzene 500 - 800 mg, NH 4 Cl 0.5 - 1 g, K 2 HPO 4 1.05 g, KH 2 PO 4 0.45 g, sodium acetate 1 - 2 g, supplemented with distilled water to 1 L, pH 7.0 - 7.2;

[0063] The physiological and biochemical functions are shown in Table 1:

[0064] Table 1

[0065]

[0066] HZ-O-030 entered the exponential phase at 8 h, and the OD 600 reached a maximum value of 2.479 at 40 h, and the degradation efficiency of chlorobenzene reached the highest at 8 h, which was 39.20 mg / L·h;

[0067] The degradation efficiency of chlorobenzene is above 20 mg / L·h within 24 h; with the continuous degradation of chlorobenzene, chlorobenzene is basically degraded at 40 h, and the bacterial concentration also reaches the highest value;

[0068] The degradation rate of chlorobenzene is the fastest at 0 - 8 h, the growth rate of bacterial concentration is the fastest at 8 - 16 h, and the degradation rate of ammonia nitrogen is the fastest at 8 - 16 h. The change trend of the degradation rate of ammonia nitrogen is consistent with the growth rate of bacterial concentration, but the growth rate of bacterial concentration lags slightly behind the degradation rate of chlorobenzene because chlorobenzene is first degraded into small molecule acids before being used by the strain for growth and metabolism;

[0069] The degradation rate of chlorobenzene by strain HZ - O - 030 reaches 91.67% at 24 h and 99.99% at 48 h;

[0070] In summary, strain HZ - O - 030 has a very good degradation effect on chlorobenzene and a very fast degradation rate;

[0071] 4) Treatment of chlorobenzene wastewater by the strain:

[0072] 4 - 1) Conventional treatment of chlorobenzene wastewater: The strain HZ - O - 030 after activation was added to chlorobenzene sewage at a ratio of 1% (v / v) respectively. The chlorobenzene concentrations in the sewage were 50 mg / L, 500 mg / L, 1000 mg / L, and 1500 mg / L. Treatment was carried out under anoxic conditions at 30℃ and 100 r / min, and the chlorobenzene content was regularly detected;

[0073] Among them, the chlorobenzene sewage was the sewage from the anoxic tank of the printing and dyeing factory sewage treatment system, and the chlorobenzene content was adjusted to 50 - 1500 mg / L respectively, with pH 7.0 - 7.2;

[0074] Treatment results: The change of chlorobenzene was detected every 12 h, and the results are shown in Table 2. It can be seen from the table that the degradation efficiency of strain HZ - O - 030 is very high when the chlorobenzene concentration is 50 - 1500 mg / L. Among them, when treating chlorobenzene in the concentration range of 50 - 1000 mg / L, the degradation rate can reach 98% and above at 48 h, and the degradation rate can reach above 87% at 24 h; the treatment concentration range of chlorobenzene sewage is relatively large and the efficiency is relatively high.

[0075] Table 2:

[0076]

[0077] 4 - 2) Treatment of chlorobenzene wastewater with different salinities:

[0078] The strain was activated, and the activated HZ-O-030 strain was added to the chlorobenzene wastewater at a ratio of 1% (v / v). The chlorobenzene content in the wastewater was 500 mg / L, and the salinity was adjusted to 0%, 1%, 3%, and 5% respectively. The treatment was carried out under anoxic conditions at 30 °C and 100 r / min, and the chlorobenzene content was detected regularly.

[0079] Treatment results:

[0080] The change in chlorobenzene concentration was detected every 12 h, and the results are shown in Table 3. It can be seen from the table that the strain can degrade chlorobenzene in wastewater with a salinity of 1% - 5%. When the salinity is 1% - 3%, the degradation rate of chlorobenzene is the highest, and the degradation rate can reach more than 95% in 48 h. And when the salinity is 1%, the degradation rate is faster than that of the wastewater with a salinity of 0%. When the salinity is 5%, the chlorobenzene degradation rate is 83.3%, showing a slight decrease. The HZ-O-030 strain can treat high-salinity wastewater.

[0081] Table 3

[0082]

[0083] 4-3) Treatment of chlorobenzene wastewater with different pH values:

[0084] The activated HZ-O-030 strain was added to the chlorobenzene wastewater at a ratio of 1% (v / v). The chlorobenzene in the wastewater was 500 mg / L, and the salinity was 1%. The pH values were set to 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, a total of 6 groups. The treatment was carried out under anoxic conditions at 30 °C and 100 r / min, and the chlorobenzene content was detected regularly.

[0085] Treatment results: The change in chlorobenzene concentration was detected every 12 h, and the results are shown in Table 4. The pH has little effect on the chlorobenzene-degrading bacteria. When the pH is between 6.0 and 9.0, the chlorobenzene degradation rate is above 95%. When the pH is 4.0 and 5.0, the chlorobenzene degradation efficiencies are 66.3% and 83.3% respectively. When the pH value is below 5.0, it has a certain impact on the degradation efficiency of the HZ-O-030 strain.

[0086] In summary, the strain HZ-O-030 can treat chlorobenzene wastewater with a chlorobenzene concentration of 50 - 1500 mg / L, a pH range of 5.0 - 9.0, and a salt concentration of 1% - 5%, and the chlorobenzene degradation efficiency can reach more than 95%.

[0087] Table 4

[0088]

[0089] Example 2:

[0090] The wastewater of a printing and dyeing chemical enterprise in Shaoxing contains high-salt and high-concentration chlorobenzene wastewater. The chlorobenzene content is 200 - 500 mg / L, and the salinity is 2% - 2.5%. See Table 5 for the influent water quality.

[0091] Table 5

[0092]

[0093] The conventional sewage treatment system used is shown in Figure 3 , which mainly consists of four parts: iron-carbon Fenton, A / O, sedimentation tank effluent, and post-physical and chemical treatment. Due to the use of a large amount of chemical agents, the sewage treatment cost is very high. At the same time, since the physical and chemical agents cannot completely degrade chlorobenzene, the effluent COD is often high, and chlorobenzene affects the microbial activity, resulting in the exceeding of other pollution indicators.

[0094] The strain HZ-O-030 was added to the sewage treatment system at a dosing ratio of 0.1‰. After the addition, the sewage treatment system operated stably for one month. See Table 6 for the effluent water quality parameters and operating costs of the system before and after the use of the microbial agent.

[0095] Table 6:

[0096]

[0097] In summary, after using the strain HZ-O-030, the system effluent is stable and meets the discharge standards; and by using the strain HZ-O-030, the use of physical and chemical agents is reduced, greatly reducing the cost.

[0098] The above are only some embodiments of the present invention. For those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A salt-tolerant chlorobenzene-degrading strain, Characterized in that: Ochrobactrum anthropi( Ochrobactrum sp. ), with the preservation number of CGMCC NO.27159, abbreviated as strain HZ-O-030.

2. Application of a salt-tolerant chlorobenzene-degrading strain, Characterized in that: Applying the salt-tolerant chlorobenzene-degrading strain described in claim 1 to the treatment of chlorobenzene wastewater, the steps include: 1) Activation of strain HZ-O-030; 2) Application: 2-1) Adding the activated strain HZ-O-030 to chlorobenzene sewage at a ratio of v / v of 1% for treatment; 2-2) Adding the activated strain HZ-O-030 to chlorobenzene sewage containing salt at a ratio of v / v of 1% for treatment.

3. According to the application of a salt-tolerant chlorobenzene-degrading strain described in claim 2, Characterized in that: The activation of the strain is specifically: inoculating the strain HZ-O-030 into an LB medium for activation, culturing with shaking at 30 °C, 100 r / min under anoxic conditions for 48 h.

4. According to the application of a salt-tolerant chlorobenzene-degrading strain described in claim 3, Characterized in that: The LB medium includes 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, supplemented with distilled water to 1 L, and the pH is 7.0 - 7.2.

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