2, 5-dichloroaniline degrading bacterium and application thereof

Abstract

The invention discloses a 2, 5-dichloroaniline degrading bacterium and application of the 2, 5-dichloroaniline degrading bacterium, the bacterial strain is stenotrophomonas acidaminiphila BA2, the preservation number is CGMCC No.29164, and the bacterial strain can grow by taking 2, 5-dichloroaniline as a sole carbon source and a sole nitrogen source under anaerobic and aerobic conditions. The 2, 5-dichloroaniline degrading bacteria are applied to a biological pretreatment process and an AO process, wastewater sequentially passes through the biological pretreatment process, the oxidation process, the AO process, the biological aerated filter process and the denitrification process, 2, 5-dichloroaniline and most of other pollutants in the wastewater are degraded through a biochemical section, and meanwhile, the ammonia and nitrogen removal effect is achieved; the method realizes low-cost up-to-standard discharge of wastewater, and solves the technical problems that 2, 5-dichloroaniline wastewater treatment enterprises are high in investment and operation cost, can generate secondary pollution and are unstable in effluent index.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a 2,5-dichloroaniline-degrading bacterium and application thereof. Background Art

[0002] 2,5-Dichloroaniline is an important chemical intermediate in dye, pigment, pesticide and organic synthesis industries, and has a wide range of uses. 2,5-Dichloroaniline wastewater pollutants are highly toxic and difficult to degrade, which will pollute the ecological environment. At present, the main methods for treating organic compound wastewater at home and abroad are: physical and chemical method (Fenton oxidation, iron-carbon micro-electrolysis, activated carbon adsorption) and biological method. The main characteristics of the physical method are: Fenton oxidation and iron-carbon micro-electrolysis can improve the biodegradability of wastewater, but during the reaction, iron ions will gradually hydrolyze to produce Fe(OH)3, which has a flocculation effect and will produce iron mud, causing secondary pollution; the activated carbon adsorption method needs to be desorbed after the activated carbon adsorption is saturated, and the desorbed substances need to be reprocessed. Biological method is the most widely used wastewater treatment method, but there are few public reports on the treatment of 2,5-dichloroaniline wastewater. Therefore, it is necessary to develop an efficient, clean and economical method for treating 2,5-dichloroaniline production wastewater based on biological treatment, which can effectively remove COD and TN in the wastewater and meet the discharge standards, and reduce enterprise investment and operating costs. Summary of the invention

[0003] The purpose of the present invention is to provide a 2,5-dichloroaniline degrading bacterium and application thereof, mainly to solve the technical problems of high investment and operation costs, secondary pollution and unstable effluent index in 2,5-dichloroaniline wastewater treatment enterprises.

[0004] To achieve the above object, the present invention provides the following technical solutions: A 2,5-dichloroaniline-degrading bacterium, characterized in that the strain is Stenotrophomonas acidaminiphila BA2, with a preservation number of CGMCC No.29164, and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on November 28, 2023.

[0005] The present invention also provides application of the 2,5-dichloroaniline-degrading bacteria in wastewater treatment.

[0006] Furthermore, the concentration of 2,5-dichloroaniline in the wastewater is 2000-2500 mg / L.

[0007] Furthermore, the wastewater treatment comprises the following steps: Step 1: Adjust the pH of the wastewater to 7-8 and filter it. The wastewater indicators are as follows: COD 6500-7500 mg / L, 2,5-dichloroaniline 2000-2500 mg / L, TN 1000-1500 mg / L, NH4 + -N400-500mg / L, TDS 2000-5000 mg / L, pH 8.5-9.0; Step 2, passing the filtered wastewater into a biological pretreatment unit, wherein the wastewater residence time in the wastewater biological pretreatment process section is 24-48 hours, the operating temperature is 20-30°C, and the dissolved oxygen is 2-4 mg / L; Step 3: The wastewater treated by the biological pretreatment unit is passed into the oxidation treatment unit, and electrochemical catalytic oxidation is performed to maintain a current density of 33.3-66.6 mA / cm 2 , wastewater retention time is 60-180min; Step 4, the wastewater treated by the oxidation treatment unit is passed into the AO treatment unit, the wastewater retention time of the A process section is 24-48h, the operating temperature is 20-30°C; the wastewater retention time of the O process section is 24-48h, the operating temperature is 20-30°C, and the dissolved oxygen is 2-4mg / L; Step 5, passing the wastewater treated by the AO treatment unit into the aerated biological filter unit, with an operating temperature of 20-30°C, dissolved oxygen of 2-4 mg / L, and a wastewater retention time of 8-12 hours; Step 6, the wastewater treated by the aerated biological filter unit is passed into the denitrification unit, the operating temperature is 20-30°C, the dissolved oxygen is less than 0.5mg / L, and the wastewater retention time is 4-8h.

[0008] Furthermore, in step 4, process section A is a 1-6 mesh granular activated carbon filler immobilized 2,5-dichloroaniline degrading bacteria biofilm treatment form, and the granular activated carbon filling amount is 30% (V / V) of the tank volume; process section O of the wastewater is a polyurethane sponge filler immobilized 2,5-dichloroaniline degrading bacteria biofilm treatment form, and the polyurethane sponge filler filling amount is 10% (V / V) of the tank volume.

[0009] Furthermore, in step 6, the denitrification unit is an autotrophic denitrification filter or a heterotrophic denitrification filter, the electron donor used in the heterotrophic denitrification filter is one of methanol, sodium acetate, and glucose or a combination of any two or more thereof, and the electron donor used in the autotrophic denitrification filter is pyrite particles.

[0010] Furthermore, the electron donor dosage is the molar amount of NO3 in the effluent of the aerated biological filter treatment unit. - -4-10 times the amount of N.

[0011] The treatment process of the present invention firstly reduces 2,5-dichloroaniline and COD in wastewater by using a biological pretreatment process with 2,5-dichloroaniline-degrading bacteria as the main degradation agent (adding 5% (V / V) 2,5-dichloroaniline-degrading bacteria (bacterial liquid density 10 9 cfu / mL), 50% (V / V) nutrient solution (nutrient solution formula: glucose 2g, amino acid 1g, KH2PO40.6g, K2HPO40.4g, MgSO40.2g, NaCl 0.2g, NaOH 0.35g, tap water 1L)), at which time 2,5-dichloroaniline N is basically converted into NH4 + -N, and then use the oxidation process to modify some of the difficult-to-biodegrade organic matter into easily biodegradable organic matter, and then use the biofilm AO treatment process to completely degrade the organic matter modified by the oxidation process (add 5% 2,5-dichloroaniline degrading bacteria (V / V) in the wastewater A process section (bacterial liquid density 10 9 cfu / mL), 50% (V / V) filtered wastewater, 45% (V / V) nutrient solution; 5% 2,5-dichloroaniline degrading bacteria (V / V) was added to the wastewater O process section (bacterial solution density 10 9 cfu / mL), 20% (V / V) filtered wastewater, 75% (V / V) nutrient solution. Among them, the wastewater A process section is filled with 1-6 mesh granular activated carbon, the granular activated carbon filling amount is 30% (V / V) of the tank volume, and the wastewater O process section is filled with polyurethane sponge filler, the polyurethane sponge filler (RAS special biological filler of Shandong Banghao Environmental Protection Technology Co., Ltd., the shape specification is 22±1mm, the expansion coefficient after water absorption is 1.2, and the filling is calculated according to the volume after water absorption and expansion) The filling amount is 10% (V / V) of the tank volume, and then the NH4 + -N is converted to NO3 - -N, and finally NO3 - -N is converted into N2 and finally meets the emission standards. This method is mainly based on biological process treatment, supplemented by oxidation process modification. Compared with the existing treatment process, the treatment cost is greatly reduced, no secondary pollution is generated, and the effluent indicators are stable.

[0012] Meanwhile, experiments have shown that the biological pretreatment process using the 2,5-dichloroaniline-degrading bacteria screened by the present invention as the main degradation agent is significantly better than the activated sludge aerobic pretreatment process in removing 2,5-dichloroaniline from wastewater, removing COD and converting organic amines.

[0013] The 2,5-dichloroaniline-degrading bacteria used in the present invention are separated and screened from sludge samples of the New Concept Wastewater Treatment Plant in Yixing City, and the biological preservation description is as follows.

[0014] Latin name: Stenotrophomonas acidaminiphila; Chinese translation: Microacidophilic oligotrophic monocytogenes; Full name of the depository: Deposited in the General Microbiology Center of China Microbiological Culture Collection Administration; Abbreviation of depository unit: CGMCC; Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: November 28, 2023; Deposit number: CGMCC No.29164. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The degradation of 2,5-dichloroaniline in the wastewater of each process section in Examples 2 and 3 is shown.

[0016] Figure 2 The degradation of COD in the wastewater of each process section in Examples 2 and 3 is shown.

[0017] Figure 3 The degradation of various types of nitrogen in the wastewater of each process stage in Example 2 is shown.

[0018] Figure 4 The degradation of various types of nitrogen in the wastewater of each process stage in Example 3. DETAILED DESCRIPTION

[0019] The present invention is described below by specific examples, but the present invention is not limited thereto. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified. The following examples are not intended to limit the scope of the present invention, and any equivalent implementation or modification that does not depart from the present invention should be included in the scope of protection of this patent.

[0020] Example 1 Domestication, isolation and identification of strains Test medium and preparation (1) Liquid separation medium formula: KH2PO4 1.0 g, K2HPO4 1.0 g, FeSO4 0.01 g, MgSO4 0.05 g, NaCl 1.0 g, tap water 1 L, NaOH 0.35 g.

[0021] (2) Solid separation medium formula: KH2PO4 1.0 g, K2HPO4 1.0 g, FeSO4 0.01 g, MgSO4 0.05 g, NaCl 1.0 g, tap water 1 L, NaOH 0.35 g, 2,5-dichloroaniline 2 g, agar 15 g.

[0022] (3) Enrichment medium formula: fish meal peptone 10 g, beef extract 3 g, NaCl 5 g, tap water 1 L, NaOH 0.35 g.

[0023] Acclimation of strains Add 2 g of sludge sample from Yixing New Concept Wastewater Treatment Plant into a 250 mL conical flask containing 100 mL of enrichment culture medium, and culture it in a constant temperature culture shaker at 30°C and 150 r / min for 16 to 24 h. Let the suspension stand after shaking, and the supernatant is the sample for inoculation.

[0024] Take three 500mL conical flasks containing 200mL of liquid separation medium, number them Y1, Y2, and Y3, add 0.1mL of 2,5-dichloroaniline to each, add 2mL of enriched supernatant to Y1, and culture in a constant temperature incubator at 30℃ and 150r / min for 24h, take 2mL of bacterial suspension from Y1 to Y2, and culture in a constant temperature incubator at 150r / min for 24h, take 2mL of bacterial suspension from Y2 to Y3, and culture for 24h as above, in order to remove a large number of non-target bacteria. Isolation of target strains Take 5 500mL conical flasks containing 200mL of liquid separation medium, numbered A1, A2, A3, A4, and A5, add 0.25mL, 0.55mL, 1mL, 1.25mL, and 2mL of 2,5-dichloroaniline as the sole carbon source and nitrogen source to A1-A5, respectively, and add 2mL of the bacterial suspension in Y3, respectively, and culture in a constant temperature culture shaker at 30℃ and 150r / min for 48h for screening and domestication. After domestication, perform microscopic examination on the bacterial suspensions in A1-A5, select bacterial suspensions with moderate bacterial density and bacterial abundance, and bacterial suspensions with moderate bacterial density and relatively single bacterial abundance, dilute them appropriately and apply them on solid separation medium, culture them at 30℃ for 72h, pick a single colony for plate streaking separation, repeat 4 to 5 times for purification, and obtain the target strain.

[0025] Identification of strains Bacterial DNA was extracted using a DNA extraction kit (Sangon) according to conventional extraction procedures, and then PCR target fragment amplification was performed. A pair of universal primers designed to amplify the 16s rDNA sequence was: 27F5'-AGTTTGATCMTGGCTCAG-3'; 1492R 5 '-GGTTACCTTGTTACGACTT-3'; Using genomic DNA as a template, add Premix TapTM for PCR amplification. After the PCR product is detected by 1% agarose gel electrophoresis, it is purified with a DNA purification recovery kit, connected to the pGM-T vector, transformed into E. coli DH5α competent cells, and applied to LB solid medium containing ampicillin. Culture at 37°C for 12 hours, pick the colony into liquid LB medium, shake and culture at 37°C and 180rpm overnight, and extract the plasmid with a plasmid extraction kit for determination. Among them, the PCR reaction conditions are: 94°C pre-denaturation for 5 minutes; then 30 cycles of 94°C denaturation for 1 minute; 55°C annealing for 1 minute; and then 72°C extension for 5 minutes. The PCR amplification product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Its sequence is shown in SEQ ID NO:1: The strain was identified by 16S rDNA, and the results were compared for homology using the Genebank Blast program. The homology between the strain and Stenotrophomonas acidaminiphila was 100%, and the strain was identified as Stenotrophomonas acidaminiphila and named Stenotrophomonas acidaminiphila BA2.

[0026] Example 2 Table 1 Wastewater indicators Index wastewater name COD (mg / L) pH NH4+-N (mg / L) TN (mg / L) 2,5-Dichloroaniline (mg / L) TDS (mg / L) Wastewater 7700 8.66 444 2970 2398 1105 The wastewater indicators are shown in Table 1. The wastewater was filtered after adjusting the pH to 7-8 with sulfuric acid. Wastewater biological pretreatment, AO unit activation, adding 5% (V / V) 2,5-dichloroaniline degrading bacteria (bacterial liquid density 10 9 cfu / mL), 50% (V / V) nutrient solution (nutrient solution formula: glucose 2g, amino acid 1g, KH2PO40.6g, K2HPO40.4g, MgSO40.2g, NaCl0.2g, NaOH 0.35g, tap water 1L); 5% (V / V) 2,5-dichloroaniline degrading bacteria (bacterial solution density 10 9 cfu / mL), 50% (V / V) filtered wastewater, 45% (V / V) nutrient solution; 5% (V / V) 2,5-dichloroaniline-degrading bacteria (bacterial solution density 10 9cfu / mL), 20% (V / V) filtered wastewater, 75% (V / V) nutrient solution. Among them, the wastewater A process section is filled with 1-6 mesh granular activated carbon, and the granular activated carbon filling amount is 30% (V / V) of the pool capacity, and the wastewater O process section is filled with polyurethane sponge filler, and the polyurethane sponge filler filling amount is 10% (V / V) of the pool capacity (Note: the above-mentioned bacterial agent, granular activated carbon, and polyurethane sponge filler added / filled volume percentage is for the pool capacity; the volume percentage of wastewater and nutrient solution added is for the remaining pool capacity after adding the bacterial agent and filling the filler, which is also the case in Example 3).

[0027] The biochemical section uses 2,5-dichloroaniline degrading bacteria as the main degradation body. The activation stage of the biochemical unit is that the biological pretreatment unit and the O treatment unit are aerated and activated for one week at an operating temperature of 30°C and a dissolved oxygen of 2mg / L. The A process section is statically activated at an operating temperature of 30°C for one week. After the biochemical system is started, the filtered wastewater is continuously treated. The filtered wastewater is passed into the biological pretreatment unit with a residence time of 48h, an operating temperature of 30°C, and a dissolved oxygen of 4mg / L; the effluent from the biological pretreatment unit is passed into the electrochemical catalytic oxidation treatment unit, maintaining a current density of 66.6mA / cm 2 , the wastewater retention time is 180min; the wastewater treated by the electrochemical catalytic oxidation treatment unit is passed into the A treatment process section, the operating temperature is 30℃, the wastewater retention time is 48h; the wastewater treated by the A treatment process section is passed into the O treatment process section, the operating temperature is 30℃, the wastewater retention time is 48h, and the dissolved oxygen is 4mg / L; the effluent of the AO treatment process is passed into the aerated biological filter (the aerated biological filter is a patented product of Jiangsu Yiyu Environmental Protection Technology Co., Ltd., the patent number is CN207581453U, a baffled aerated biological filter for ammonia removal and denitrification), the operating temperature is 30℃, the wastewater retention time is 12h, the dissolved oxygen is 4mg / L, and during the operation, NaHCO3 is added according to the ammonia nitrogen content of the effluent of the O process section: NaHCO3 (M / M) is 1:11. The effluent of the aerated biological filter enters the heterotrophic denitrification biological filter, and monohydrate glucose is used as the organic electron donor. The dosage is NO3 - -6 times the amount of -N (M / M), wastewater retention time 8h.

[0028] The indicators of 2,5-dichloroaniline in each process stage are as follows Figure 1 As shown, COD index is as follows Figure 2 As shown, the N index is Figure 3 shown.

[0029] Example 3 The biochemical section uses activated sludge as the main degradation body, the wastewater biochemical unit is activated, and 20% (V / V) filtered wastewater and 80% (V / V) activated activated sludge are added to the aerobic pretreatment process section (the activated sludge comes from the New Concept Wastewater Plant in Yixing City. The activation method is to mix the activated sludge with a water content of 80% with water in a ratio of 1:2, air-dry for 24 hours, let it stand for 1 hour, discard the supernatant, add an equal amount of water (containing 2g glucose, 0.107g urea, and 0.044g potassium dihydrogen phosphate per liter), air-dry for 48 hours, discard the supernatant, and repeat the above steps 2-3 times until the activated sludge has good sedimentation properties and the sludge is basically converted into flocculent sludge, and the aerobic sludge activation is completed). 50% (V / V) filtered wastewater and 50% (V / V) activated activated sludge are added to the anaerobic process section (the activation method of activated sludge is the same as above). In the aerobic process section, add 20% (V / V) filtered wastewater and 80% (V / V) activated activated sludge (the activation method of activated sludge is the same as above). In the activation stage of the biochemical unit, the aerobic pretreatment unit and the O treatment unit are aerated activated for one week at an operating temperature of 30°C and dissolved oxygen of 2mg / L, and the A process section is statically activated at an operating temperature of 30°C for one week. After the biochemical system is started, the filtered wastewater is continuously treated. The filtered wastewater is passed into the aerobic pretreatment unit for a residence time of 48h, an operating temperature of 30°C, and a dissolved oxygen of 4mg / L; the effluent from the aerobic pretreatment unit is passed into the electrochemical catalytic oxidation treatment unit, maintaining a current density of 66.6mA / cm 2 , the wastewater retention time is 180min; the wastewater treated by the electrochemical catalytic oxidation treatment unit is passed into the A treatment process section, the operating temperature is 30℃, the wastewater retention time is 48h; the wastewater treated by the A treatment process section is passed into the O treatment process section, the operating temperature is 30℃, the wastewater retention time is 48h, and the dissolved oxygen is 4mg / L. The effluent from the AO treatment process is passed into the aerated biological filter (the aerated biological filter is a patented product of Jiangsu Yiyu Environmental Protection Technology Co., Ltd., the patent number is CN207581453U, a baffled aerated biological filter for ammonia removal and denitrification), the operating temperature is 30℃, the wastewater retention time is 12h, the dissolved oxygen is 4mg / L, and during the operation, NaHCO3 is added according to the ammonia nitrogen content of the effluent from the O process section: NaHCO3 (M / M) is 1:11. The effluent from the aerated biological filter enters the heterotrophic denitrification biological filter, and monohydrate glucose is used as the organic electron donor. The dosage is NO3 - -6 times the amount of -N (M / M), wastewater retention time 8h.

[0030] The indicators of 2,5-dichloroaniline in each process stage are as follows Figure 1 As shown, COD index is as follows Figure 2 As shown, the N index is Figure 4 shown.

[0031] Conclusion: Combining Figure 1-Figure 4It can be seen that the biological pretreatment process with 2,5-dichloroaniline degrading bacteria as the main degradation body has a removal rate of 89.12% and 65.42% for 2,5-dichloroaniline and COD in the wastewater, respectively, and this process section can basically convert organic nitrogen in the wastewater into ammonia nitrogen. Through the subsequent electrocatalytic, anaerobic, aerobic, aerated biological filter, and denitrification process sections, 2,5-dichloroaniline was not detected in the final effluent, COD was 221 mg / L, and TN was 15 mg / L, meeting the enterprise's emission requirements. The removal rates of 2,5-dichloroaniline and COD in the activated sludge biological pretreatment process section are lower than those of the biological pretreatment process with 2,5-dichloroaniline degrading bacteria as the main degradation body, and organic nitrogen can basically not be converted into ammonia nitrogen. The pretreatment effluent is subsequently treated through the same process section, and the effluent cannot meet the discharge standards. It can be seen that 2,5-dichloroaniline-degrading bacteria enhanced the degradation effect of 2,5-dichloroaniline and COD in the biological pretreatment process, especially the conversion of organic nitrogen into ammonia nitrogen, and reduced the pressure of nitrogen removal in the subsequent process.

Claims

1. A 2,5-dichloroaniline-degrading bacterium, characterized in that: The strain is Stenotrophomonas acidaminiphila BA2, with the deposit number CGMCC No.29164. It was deposited in the General Microbiology Center of China Culture Collection Administration on November 28, 2023.

2. Use of a 2,5-dichloroaniline-degrading bacterium as claimed in claim 1 in wastewater treatment.

3. The use of a 2,5-dichloroaniline-degrading bacterium in wastewater treatment according to claim 2, characterized in that: The concentration of 2,5-dichloroaniline in the wastewater is 2000-2500 mg / L.

4. The use of a 2,5-dichloroaniline-degrading bacterium in wastewater treatment according to claim 2, characterized in that: The wastewater treatment comprises the following steps: Step 1: Adjust the pH of the wastewater to 7-8 and filter it. The wastewater indicators are as follows: COD 6500-7500 mg / L, 2,5-dichloroaniline 2000-2500 mg / L, TN 1000-1500 mg / L, NH4 + -N 400-500 mg / L, TDS 2000-5000 mg / L, pH 8.5-9.0; Step 2, passing the filtered wastewater into a biological pretreatment unit, wherein the wastewater residence time in the wastewater biological pretreatment process section is 24-48 hours, the operating temperature is 20-30°C, and the dissolved oxygen is 2-4 mg / L; Step 3: The wastewater treated by the biological pretreatment unit is passed into the oxidation treatment unit, and electrochemical catalytic oxidation is performed to maintain a current density of 33.3-66.6 mA / cm 2 , wastewater retention time is 60-180min; Step 4, the wastewater treated by the oxidation treatment unit is passed into the AO treatment unit, the wastewater retention time of the A process section is 24-48h, the operating temperature is 20-30°C; the wastewater retention time of the O process section is 24-48h, the operating temperature is 20-30°C, and the dissolved oxygen is 2-4mg / L; Step 5, passing the wastewater treated by the AO treatment unit into the aerated biological filter unit, with an operating temperature of 20-30°C, dissolved oxygen of 2-4 mg / L, and a wastewater retention time of 8-12 hours; Step 6, the wastewater treated by the aerated biological filter unit is passed into the denitrification unit, and an electron donor is additionally added, the operating temperature is 20-30°C, the dissolved oxygen is <0.5mg / L, and the wastewater retention time is 4-8h.

5. The use of a 2,5-dichloroaniline-degrading bacterium in wastewater treatment according to claim 4, characterized in that: In step 4, process section A is a treatment form of 1-6 mesh granular activated carbon filler immobilized 2,5-dichloroaniline-degrading bacteria biofilm method, and the filling amount of granular activated carbon is 30% (V / V) of the tank volume; process section O of wastewater is a treatment form of polyurethane sponge filler immobilized 2,5-dichloroaniline-degrading bacteria biofilm method, and the filling amount of polyurethane sponge filler is 10% (V / V) of the tank volume.

6. The use of a 2,5-dichloroaniline-degrading bacterium in wastewater treatment according to claim 4, characterized in that: In step 6, the denitrification unit is an autotrophic denitrification filter or a heterotrophic denitrification filter, the electron donor used in the heterotrophic denitrification filter is one of methanol, sodium acetate, and glucose or a combination of any two or more thereof, and the electron donor used in the autotrophic denitrification filter is pyrite particles.

7. The use of a 2,5-dichloroaniline-degrading bacterium in wastewater treatment according to claim 4, characterized in that: The electron donor dosage is NO3 in the effluent of the aerated biological filter treatment unit. - -4-10 times the amount of N.

Images