Whole-process denitrifying pseudomonas and application thereof in textile printing and dyeing sewage

Abstract

The invention discloses a whole-process denitrifying pseudomonas and application thereof in textile printing and dyeing sewage, and relates to the field of environmental microorganism water environment governance, the key points of the technical scheme are as follows: the whole-process denitrifying pseudomonas is pseudomonas stutzeri S8 and is preserved in the China Center for Type Culture Collection (CCTCC), the preservation date is June 23, 2025, and the preservation number is CCTCC NO: M 20251451. The Pseudomonas stutzeri S8 can simultaneously perform whole-course denitrification and decolorization under the toxicity stress of hexavalent chromium ions, the removal rate of nitrate and nitrite in chromium-containing actual textile printing and dyeing sewage can be higher than 95% after standing culture is performed for 48 hours, the synchronous dye removal rate is higher than 82%, the tolerant nitrate and nitrite concentration is higher than 500 mg / L, and the application range is wide. The strain has the advantages that the concentration of hexavalent chromium ions tolerated by the strain is higher than 50mg / L, in addition, the strain also has good decoloring and denitrification effects in a decoloring and denitrification culture medium stressed by other five single dyes, the nitrate removal rate is higher than 98%, and the dye removal rate is 70%-100%.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental microorganism water environment treatment, more particularly, it relates to a full denitrification pseudomonas and application thereof in textile printing and dyeing wastewater. BACKGROUND

[0002] Textile printing and dyeing industry is a high energy consumption and high water consumption industry, and about 100-200 tons of water are consumed per ton of textile production. According to estimates, the annual discharge of textile printing and dyeing wastewater is as high as 2 billion tons, accounting for about 10% of the total annual industrial wastewater discharge. In the textile printing and dyeing industry, dyes are widely used, which leads to high content of dye molecules and their metabolites in the wastewater, and deep color. The discharge of a large amount of "colored" wastewater will seriously affect the aquatic plants and animals in natural water bodies and human health.

[0003] In addition, in the textile printing and dyeing process, urea is often used as an auxiliary agent to facilitate dye fixation, and hexavalent chromium salt is often used as an oxidizing agent. In the common textile printing and dyeing wastewater treatment process, organic nitrogen such as urea and dye metabolites in the influent is converted into nitrate nitrogen through microbial ammonification and nitrification, and is returned to the anoxic tank through the internal reflux pipeline to mix with the textile printing and dyeing raw water for denitrification. Therefore, in the biochemical process of textile printing and dyeing wastewater treatment, the simultaneous presence of internal reflux nitrate nitrogen, dyes and hexavalent chromium salt in the raw water is a common phenomenon.

[0004] Microbial decolorization and denitrification technology has been widely studied due to its low cost, high efficiency, safety and environmental protection. However, most of the strains reported for decolorization and denitrification of textile printing and dyeing wastewater only have single decolorization or denitrification function. Only the strain of Bacterium T3 (CN118652800A) is reported to have decolorization and short-term denitrification function. However, there is no report on a strain that has both decolorization and full denitrification ability in the presence of highly toxic hexavalent chromium ions.

[0005] In view of the problem that dyes, nitrate nitrogen and hexavalent chromium ions coexist in textile printing and dyeing wastewater, how to effectively decolorize and denitrify under the stress of hexavalent chromium ions has become the focus of attention. Therefore, screening a strain that is resistant to chromium and has both decolorization and full denitrification ability from natural environment and applying it to textile printing and dyeing wastewater will effectively reduce the color and total nitrogen of the wastewater, which is of great significance to ensure the stable operation of the biochemical system of textile printing and dyeing wastewater and the discharge of wastewater up to the standard. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application aims to provide a full denitrification pseudomonas and application thereof in textile printing and dyeing wastewater. The strain can tolerate the toxic effect of hexavalent chromium ions and achieve the effect of simultaneous decolorization and denitrification of textile printing and dyeing wastewater.

[0007] The above technical purpose of the present application is realized by the following technical scheme: a full denitrification Pseudomonas, which is Pseudomonas stutzeri S8, deposited in the China Center for Type Culture Collection (CCTCC) on June 23, 2025, and has a deposit number of CCTCC NO: M 20251451.

[0008] Further, the 16S rDNA gene sequence of the Pseudomonas stutzeri S8 is shown in SEQ ID NO. 1.

[0009] The application of the above-mentioned full denitrification Pseudomonas in textile printing and dyeing wastewater, the Pseudomonas stutzeri S8 can grow and metabolize in actual textile printing and dyeing wastewater containing hexavalent chromium, dyes and nitrate nitrogen or nitrite nitrogen, tolerate the toxicity of hexavalent chromium, and perform full denitrification and decolorization with organic carbon source and yeast extract as co-metabolism substances, so as to realize simultaneous decolorization and denitrification of textile printing and dyeing wastewater.

[0010] Further, the Pseudomonas stutzeri S8 seed liquid is added to actual textile printing and dyeing wastewater containing hexavalent chromium, dyes and nitrate nitrogen, and an appropriate amount of organic carbon source and yeast extract is added for static culture, so that the simultaneous removal of dyes and nitrate in textile printing and dyeing wastewater can be realized.

[0011] Further, the application is performed according to the following steps: the Pseudomonas stutzeri S8 is inoculated into LB liquid medium, and is cultured at 30℃ with 150rpm shaking bed oscillation for 24h; after the culture is completed, it is centrifuged at 8000xg for 5min, and then diluted with sterile water to OD600=1.5 to obtain a seed liquid for standby; the seed liquid is added to different concentrations of nitrate nitrogen, nitrite nitrogen and hexavalent chromium ion decolorization and denitrification culture medium, different dye concentration wastewater culture medium, different single dye decolorization and denitrification culture medium and an anoxic bioreactor at a proportion of 5%, the concentration of nitrate nitrogen and nitrite nitrogen is controlled between 100-500mg / L, the concentration of hexavalent chromium ion is controlled between 10-50mg / L, the concentration of dye wastewater is controlled between 20-100%, and the types of dyes are acid orange, orange yellow G, direct blue 1, congo red and scarlet B, respectively, and the culture is performed at 30℃.

[0012] In summary, the present application has the following beneficial effects: The Pseudomonas stutzeri S8 of the application can simultaneously perform full denitrification and decolorization under the toxicity stress of hexavalent chromium ions, and the removal rates of nitrate and nitrite in the actual textile printing and dyeing wastewater containing chromium are higher than 95% and the simultaneous dye removal rate is higher than 82% after static culture for 48 h, the tolerance of nitrate and nitrite concentration is higher than 500 mg / L, and the tolerance of hexavalent chromium ion concentration is higher than 50 mg / L, in addition, the bacteria also have good decolorization and denitrification effect in the decolorization and denitrification medium under the stress of the remaining five kinds of single dyes, and the removal rate of nitrate is higher than 98%, and the dye removal rate is between 70% and 100%. The problems that the traditional microorganisms of textile printing and dyeing wastewater need to be combined with multiple bacteria to simultaneously decolorize and denitrify and the existence of hexavalent chromium inhibits the growth and metabolism of the strain are solved; in addition, the overall removal efficiency and load of the wastewater biochemical process system are improved, the wastewater retention time is correspondingly shortened, the energy consumption cost is saved, and great economic benefits and environmental protection benefits are obtained. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is a morphological diagram of Pseudomonas stutzeri S8 strain; Figure 2 It is a synchronous decolorization and denitrification effect diagram of Pseudomonas stutzeri S8 under different concentrations of nitrate nitrogen; Figure 3 It is a synchronous decolorization and denitrification effect diagram of Pseudomonas stutzeri S8 under different concentrations of nitrite nitrogen; Figure 4 It is a synchronous decolorization and denitrification effect diagram of Pseudomonas stutzeri S8 under different concentrations of dyeing wastewater; Figure 5 It is a synchronous decolorization and denitrification effect diagram of Pseudomonas stutzeri S8 under different concentrations of hexavalent chromium ion stress; Figure 6 It is a synchronous decolorization and denitrification effect diagram of Pseudomonas stutzeri S8 under different kinds of dyes; Figure 7 It is a synchronous decolorization and denitrification effect diagram of Pseudomonas stutzeri S8 in a bioreactor on dyeing wastewater. DETAILED DESCRIPTION

[0014] The application will be described in detail below in combination with the drawings and examples.

[0015] The culture medium involved in the application is as follows: 1. LB liquid medium: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, and water as solvent; 2. Enrichment domestication medium: 1.5 g / L trisodium citrate, 1.5 g / L yeast extract, 1 g / L NaH2PO4, 1 g / L Na2HPO4, 0.05 g / L MgSO4, solvent is mixed textile printing and dyeing sewage (1:1 of dyeing raw water and printing and dyeing sewage station backflow nitrification liquid, after mixing, the sewage COD is 711 mg / L, NH3-N is 4.7 mg / L, NO2-N is 0.9 mg / L, NO3-N is 58 mg / L, TN is 88.5 mg / L, Cr 6+ 10.4 mg / L, absorbance at maximum absorption wavelength 580 nm is 1.22), and a proper amount of potassium nitrate and hexavalent chromium salt is additionally added to control the final nitrate nitrogen of the medium to be 100 mg / L, Cr 6+ 25 mg / L.

[0016] 3. Decolorization and denitrification medium: the basic components are the same as the enrichment domestication medium, when the concentrations of nitrate nitrogen and nitrite nitrogen are explored in the examples, the concentrations are increased accordingly and the concentration of trisodium citrate is controlled (100 mg / L of nitrate nitrogen or nitrite nitrogen in the system matches 1.5 g / L of trisodium citrate). 4. Single dye decolorization and denitrification medium: 1.5 g / L trisodium citrate, 1.5 g / L yeast extract, 0.72 g / L KNO3, 10 mg / L K2Cr2O7, 1 g / L NaH2PO4, 1 g / L Na2HPO4, 0.05 g / L MgSO4, solvent is water.

[0017] The above medium components are filtered to remove bacteria or sterilized by high-temperature steam.

[0018] The experimental index detection method involved in the present application is as follows: 1. Detection of nitrate nitrogen (NO3-N): ultraviolet spectrophotometry; 2. Detection of nitrite nitrogen (NO2-N): N-(1-naphthyl)-ethylenediamine spectrophotometry; Nitrate nitrogen removal rate (%) = (C0-Ct) / C0 C0 is the initial concentration of nitrate nitrogen, and Ct is the concentration of nitrate nitrogen after t hours of treatment.

[0019] 3. Determination of dye decolorization rate: maximum absorption wavelength spectrophotometry; Decolorization rate (%) = (A0-At) / A0 A0 is the absorbance of the dye before treatment, and At is the absorbance of the dye after t hours of treatment. The maximum absorption wavelengths of the dyes acid orange, carmine B, direct blue 1, orange yellow G and congo red are 480 nm, 500 nm, 590 nm, 480 nm and 490 nm, respectively.

[0020] Example 1: Chromium-resistant, decolorization and full denitrification strain enrichment screening The activated sludge was collected from the biochemical tank of the Xindu printing and dyeing wastewater station in Keqiao, Shaoxing, Zhejiang, inoculated into the enrichment and domestication medium at an inoculation amount of 2%, and then cultured. After the culture system was obviously decolorized and the removal rate of nitrate nitrogen was higher than 90%, the culture was transferred into a new enrichment and domestication medium. After three rounds of enrichment and domestication operation, the enrichment liquid was coated and streaked to obtain a purified strain. After single-bacterium function verification, the strain S8 with the abilities of chromium tolerance, decolorization and full denitrification was obtained. The strain S8 was amplified by 16s bacterial universal primers 27F and 1492R, and the full-length sequence was 1449 bp. The sequence was submitted to the NCBI database for comparison and analysis. The results showed that the strain S8 had the highest homology with Pseudomonas stutzeri. Therefore, the strain S8 was determined to be Pseudomonas stutzeri. The Pseudomonas stutzeri is Pseudomonas stutzeri S8 (Pseudomonas stutzeri), which is preserved in the China Center for Type Culture Collection (CCTCC) located at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with a postal code of 430072. The preservation date is June 23, 2025, and the preservation number is CCTCC NO: M 20251451.

[0021] Example 2: Synchronization decolorization and denitrification characteristics of Pseudomonas stutzeri S8 under different concentrations of nitrate nitrogen The Pseudomonas stutzeri S8 was inoculated into the LB liquid medium and cultured at 30°C with 150 rpm shaking for 24 h. After the culture was completed, the culture was centrifuged at 8000xg for 5 min, and then diluted with sterile water to OD600=1.5 as a seed liquid for standby. Then, the seed liquid was inoculated into the decolorization and denitrification medium with different concentrations of nitrate nitrogen (100-500 mg / L) at an inoculation amount of 5% and cultured (30°C, static) for 48 h. Then, the supernatant was detected for decolorization rate, nitrate nitrogen removal rate and nitrite nitrogen content.

[0022] The research results show that Figure 2 As the concentration of nitrate nitrogen (100-500 mg / L) increases, the removal rate of nitrate nitrogen of the Pseudomonas stutzeri S8 can remain above 97%, and the decolorization rate is higher than 80%. When the concentration of nitrate nitrogen is 100-300 mg / L, the strain S8 basically does not accumulate nitrite nitrogen during denitrification, and the residual nitrite nitrogen concentration in the system is lower than 6 mg / L after 48 h. When the initial concentration of nitrate nitrogen increases to 400 mg / L and 500 mg / L, the residual nitrite nitrogen in the system is only 23 mg / L and 41 mg / L after 48 h. The results show that the strain S8 can still maintain high-efficiency full denitrification and decolorization ability under different concentrations of nitrate nitrogen.

[0023] Example 3: S8 (Pseudomonas stutzeri) decolorization and denitrification characteristics under different concentrations of nitrite nitrogen To verify the decolorization and denitrification effect of S8 (Pseudomonas stutzeri) under different concentrations of nitrite nitrogen, the strain seed liquid was inoculated into the decolorization and denitrification medium modified by 100-500 mg / L nitrite nitrogen at an inoculation amount of 5% according to Example 2, and the supernatant was sampled and centrifuged after static culture at 30°C for 48 h to detect the decolorization rate, nitrite nitrogen removal rate and nitrate nitrogen content.

[0024] As shown in Figure 3 When the initial nitrite nitrogen is 100-300 mg / L, the nitrite nitrogen removal rate of S8 (Pseudomonas stutzeri) is higher than 96%, and the decolorization rate is higher than 82%, and further increasing the concentration of nitrite nitrogen will inhibit the decolorization and denitrification effect of the strain, but the nitrite nitrogen removal rate and decolorization rate can still remain 83.7% and 73.1% at 400 mg / L, and 76.1% and 66.8% at 500 mg / L, and under the stress of 100-500 mg / L nitrite nitrogen, the initial 60 mg / L or so of nitrate nitrogen in the mixed system does not exist residual (less than 1 mg / L). Therefore, the strain has the ability of full denitrification and decolorization under high concentration of nitrite nitrogen.

[0025] Example 4: S8 (Pseudomonas stutzeri) decolorization and denitrification characteristics under different concentrations of dye wastewater Dyes and their metabolites have certain toxic effects on microbial decolorization and denitrification, so the full denitrification and decolorization efficiency under different initial concentrations of dye wastewater is explored. The S8 (Pseudomonas stutzeri) seed liquid with an inoculation amount of 5% was inoculated into the medium with different concentrations of dye wastewater, and the concentration of nitrate nitrogen was controlled at 100 mg / L, the concentration of trisodium citrate and yeast extract was 1.5 g / L, and the rest of the inorganic salt components were the same as the decolorization and denitrification medium. After inoculation, the culture was statically cultured at 30°C for 48 h, and then the supernatant was sampled and centrifuged to detect the dye decolorization rate, nitrate nitrogen removal rate and nitrite nitrogen content.

[0026] As shown in Figure 4, with the increase of initial concentration of dye wastewater (20%-100%), Pseudomonas stutzeri S8 can still maintain high efficiency of full denitrification, and the system nitrate nitrogen can be completely removed (the removal rate is higher than 97%), and basically will not accumulate nitrite nitrogen, and the final concentration is lower than 4 mg / L. In addition, with the increase of dye wastewater concentration, the strain can still maintain high efficiency of decolorization, and the response value of dye wastewater treated by the strain at the maximum absorption wavelength can reach about 0.2, which may be due to the accumulation of metabolites caused by single static condition. Therefore, the strain has certain tolerance to actual dye wastewater, and has the ability of decolorization and denitrification of different concentrations of dye wastewater.

[0027] Example 5: Pseudomonas stutzeri S8 (Pseudomonas stutzeri) under the stress of different concentrations of hexavalent chromium ions for simultaneous decolorization and denitrification characteristics Hexavalent chromium ions are an important pollutant in textile printing and dyeing wastewater, and their biological toxicity is much higher than that of trivalent chromium ions, and they have strong inhibition to microorganisms. In order to explore the effect of different concentrations of hexavalent chromium ions on Pseudomonas stutzeri S8 decolorization and denitrification, the strain was inoculated into different concentrations (10-50 mg / L) of hexavalent chromium ion modified decolorization and denitrification medium at an inoculation amount of 5%, and the supernatant was detected after centrifugation after 48 h of static culture at 30°C. The decolorization rate, nitrate nitrogen removal rate and nitrite nitrogen content.

[0028] The results show that Figure 5 ) the strain can still maintain high efficiency of decolorization and full denitrification in the presence of hexavalent chromium ions. After 48 h of culture, 10-50 mg / L Cr 6+ The system nitrate nitrogen removal rate can reach 98%, the nitrite nitrogen accumulation is lower than 5 mg / L, and the wastewater decolorization rate is as high as 80%, so the strain has the ability of chromium-resistant decolorization and full denitrification.

[0029] Example 6: Pseudomonas stutzeri S8 (Pseudomonas stutzeri) under the stress of different single dyes for simultaneous decolorization and denitrification characteristics In order to verify the full denitrification and decolorization ability of Pseudomonas stutzeri S8 under the condition of different single dyes, the S8 strain seed liquid was inoculated into the decolorization and denitrification medium containing 50 mg / L different single dyes at an inoculation amount of 5%, and the supernatant was detected after centrifugation after 48 h of static culture at 30°C. The decolorization rate, nitrate nitrogen removal rate and nitrite nitrogen content.

[0030] As Figure 6As shown, Pseudomonas stutzeri S8 can still perform efficient full denitrification and decolorization under the stress of different single dyes and hexavalent chromium ions. Within 48 h, the decolorization rates of acid orange, orange yellow G, direct blue 1, congo red and scarlet B were 80.9%, 95.3%, 72.5%, 92.6% and 96.1%, respectively. The removal rates of nitrate nitrogen were all higher than 98%, and the denitrification process did not accumulate nitrite nitrogen at the end, with the final content being lower than 3 mg / L. Therefore, the strain has the functions of chromium-tolerant decolorization and full denitrification under different single dye conditions.

[0031] Example 7: Simultaneous decolorization and denitrification characteristics of Pseudomonas stutzeri S8 in an anoxic bioreactor As shown in Figure 7 Pseudomonas stutzeri S8 seed liquid 120 mL was inoculated into an anoxic bioreactor containing 2.4 L of decolorization and denitrification medium, and the reactor was operated at room temperature for 48 h. The supernatant was centrifuged every 24 h to detect the contents of nitrate nitrogen, nitrite nitrogen and color.

[0032] After 24 h of treatment by Pseudomonas stutzeri S8, the removal rate of nitrate nitrogen in the raw water was higher than 95%, the accumulation of nitrite nitrogen was about 30 mg / L, and the decolorization rate was higher than 55%. After 24 h of continuous reaction, the contents of nitrate nitrogen and nitrite nitrogen in the reactor were reduced to below 5 mg / L, and the decolorization rate was higher than 80%.

[0033] The above only describes the preferred embodiments of the present application, and the protection scope of the present application is not limited to the above examples. Any technical solutions falling within the concept of the present application shall fall within the protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, some improvements and refinements without departing from the principles of the present application shall also be considered as the protection scope of the present application.

Claims

1. A fully denitrifying Pseudomonas, which is Pseudomonas stutzeri S8 (Pseudomonas stutzeri), deposited in the China Center for Type Culture Collection (CCTCC), with a deposit date of June 23, 2025, and a deposit number of CCTCC NO: M20251451.

2. The method of claim 1, wherein the denitrifying Pseudomonas aeruginosa and its application in textile printing and dyeing wastewater are characterized in that: The 16SrDNA gene sequence of the Pseudomonas stutzeri S8 is shown in SEQ ID NO.

1.

3. Use of a full-process denitrifying Pseudomonas according to claim 1 in textile printing and dyeing wastewater.

4. The use of a full-process denitrifying Pseudomonas in textile printing and dyeing wastewater according to claim 3, characterized in that: The Pseudomonas stutzeri S8 can grow and metabolize in textile printing and dyeing wastewater that actually contains hexavalent chromium, dyes, and nitrate nitrogen or nitrite nitrogen. While tolerating the toxicity of hexavalent chromium, it uses organic carbon sources and yeast extracts as co-metabolites to carry out full-process denitrification and decolorization, thereby achieving simultaneous decolorization and denitrification of textile printing and dyeing wastewater.

5. The use of a full-process denitrifying Pseudomonas according to claim 4, characterized in that: By adding the seed liquid of Pseudomonas stutzeri S8 to the actual textile printing and dyeing wastewater containing hexavalent chromium, dyes and nitrate nitrogen, and adding an appropriate amount of organic carbon source and yeast extract for static cultivation, the simultaneous removal of dyes and nitrates in the textile printing and dyeing wastewater can be achieved.

6. The use of a full-process denitrifying Pseudomonas according to claim 5, characterized in that: The application is carried out according to the following steps: inoculating Pseudomonas stutzeri S8 into LB liquid culture medium, culturing on a shaker, centrifuging after the culture is completed, and diluting with sterile water to an OD600 of 1.3-1.7 to obtain a seed solution for standby use; adding the seed solution at a ratio of 4-6% to a decolorization and denitrification culture medium containing different concentrations of nitrate nitrogen, nitrite nitrogen, and hexavalent chromium ions, a wastewater culture medium containing different dye concentrations, a decolorization and denitrification culture medium containing different single dyes, and an anoxic bioreactor, controlling the nitrate nitrogen concentration and nitrite nitrogen concentration to be between 100-500 mg / L, the hexavalent chromium ion concentration to be between 10-50 mg / L, and the dye wastewater concentration to be between 20-100%, and culturing at 30-40°C.

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