Method for repairing naphthalene contaminated wastewater based on microbial co-culture induced biological calcification

Abstract

The application discloses a method for repairing naphthalene contaminated wastewater based on microbial co-culture induced biological calcification, which comprises the following steps: step one, inoculating naphthalene contaminated wastewater with polycyclic aromatic hydrocarbon degrading bacteria and urea bacteria to obtain a repaired wastewater system; step two, oscillating the repaired wastewater system at 25-35 DEG C for 10-14 hours to obtain a co-cultured system; step three, adding urea and calcium chloride to the co-cultured system to obtain an induced system; and step four, oscillating the induced system at 25-35 DEG C for more than or equal to 24 hours to complete the repair of the naphthalene contaminated wastewater. The method has the characteristics of high naphthalene pollutant repair efficiency and strong stability of the repaired system.

Description

Technical Field

[0001] This invention belongs to the field of environmental engineering technology, specifically relating to a method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biological calcification. Background Technology

[0002] Polycyclic aromatic hydrocarbons (PAHs) in the atmosphere and water bodies mainly originate from incomplete combustion of petrochemical products, leaks during storage, transportation, and production of crude oil, as well as sewage discharge. Through rainfall, surface runoff, and seepage, PAHs in the atmosphere enter the soil and groundwater, and eventually enter organisms through bioaccumulation.

[0003] PAHs are highly toxic due to their recalcitrant nature, strong bioaccumulation, semi-volatile nature, and carcinogenic, teratogenic, and mutagenic effects. Naphthalene is the most typical polycyclic aromatic hydrocarbon pollutant and was listed by the U.S. Environmental Protection Agency (USEPA) in 1979 as one of the 16 priority polycyclic aromatic hydrocarbon pollutants to be controlled.

[0004] Currently, many methods have been developed for remediating PAHs, such as physical remediation (incineration, adsorption, and gas-phase extraction), chemical remediation (photocatalysis and advanced oxidation), phytoremediation, and microbial remediation. Compared with other remediation technologies, microbial remediation technology has the advantages of simple operation, low cost, and minimal environmental impact, and can simultaneously remediate contaminated soil and groundwater.

[0005] Biotechnology can be used to remediate polycyclic aromatic hydrocarbons (PAHs), but its remediation effectiveness decreases significantly with increasing PAH concentration and the number of benzene rings formed. Naphthalene's thermal decomposition temperature is around 1077–1377 °C. Due to its long-term degradation characteristics, PAHs can migrate and transform through abiotic pathways (such as volatilization), significantly increasing the difficulty of PAH removal. Furthermore, PAH metabolism produces complex toxic intermediates, posing even greater challenges to removal methods. Therefore, a collaborative and innovative strategy is urgently needed to enhance the bioremediation of PAHs. Summary of the Invention

[0006] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification. This method involves inoculating polycyclic aromatic hydrocarbon (PAH) degrading bacteria and urea-releasing bacteria sequences into the naphthalene-contaminated wastewater for co-culture, followed by the addition of urea and calcium chloride to induce remediation and complete the treatment of the naphthalene-contaminated wastewater. This method features high naphthalene remediation efficiency and strong post-remediation system stability.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification, characterized in that it includes:

[0008] Step 1: Sequentially inoculate naphthalene-contaminated wastewater with bacterial suspensions of polycyclic aromatic hydrocarbon degrading bacteria and urea-degrading bacteria to obtain a wastewater remediation system;

[0009] Step 2: Shake the remediated wastewater system at 25-35℃ for 10-14 hours to obtain the co-cultured system;

[0010] Step 3: Add urea and calcium chloride to the co-culture system to obtain the induction system;

[0011] Step 4: Shake the induction system at 25-35℃ for ≥24 hours to complete the remediation of naphthalene-contaminated wastewater.

[0012] The above-mentioned method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification is characterized in that, in step one, the concentration of naphthalene in the naphthalene-contaminated wastewater is 50–200 mg / L.

[0013] The above-mentioned method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification is characterized in that, in step one, the sequence inoculation involves first adding a bacterial solution of polycyclic aromatic hydrocarbon degrading bacteria to the naphthalene-contaminated wastewater, shaking and culturing for 20-30 minutes, and then adding a bacterial solution of urea-degrading bacteria.

[0014] The above-mentioned method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification is characterized in that, in step one, the volume percentage of polycyclic aromatic hydrocarbon degrading bacteria and urea-degrading bacteria in the wastewater remediation system is 5%.

[0015] The above-mentioned method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification is characterized in that, in step one, the polycyclic aromatic hydrocarbon degrading bacteria are *Microbacterium paraoxidans*, and the urea-degrading bacteria are *Bacillus pasteurellii*.

[0016] The above-mentioned method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification is characterized in that, in step one, the preparation method of the bacterial solution of the polycyclic aromatic hydrocarbon (PAH) degrading bacteria includes: culturing the PAH degrading bacteria in a lysate broth medium with a pH of 6-7 for 22-26 hours; the culturing temperature is 25-35°C, and the shaking speed is 170-190 rpm.

[0017] The above-mentioned method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification is characterized in that the lysozyme culture medium contains 8-12 g / L tryptone, 4-6 g / L yeast extract, 9-11 g / L sodium chloride, and the remainder is water.

[0018] The above-mentioned method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification is characterized in that, in step one, the preparation method of the urea-releasing bacteria solution includes: culturing the urea-releasing bacteria in an ammonium chloride medium with a pH of 8-9 for 22-26 hours; the culture temperature is 25-35℃, and the shaking speed is 170-190 rpm.

[0019] The above-mentioned method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification is characterized in that the ammonium chloride culture medium contains 18-22 g / L yeast extract, 8-12 g / L ammonium chloride, 8-12 mg / L manganese sulfate monohydrate, 22-26 mg / L nickel chloride hexahydrate, and the remainder is water.

[0020] The above-mentioned method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification is characterized in that, in step three, the concentrations of urea and calcium chloride in the induction system are both 90–110 mM.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] 1. The present invention relates to a method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification. This method involves co-culturing polycyclic aromatic hydrocarbon (PAH) degrading bacteria and urea-degrading bacteria in naphthalene-contaminated wastewater, followed by the addition of urea and calcium chloride for induced remediation. This method effectively achieves rapid sealing and adsorption of naphthalene, and effectively reduces the impact of abiotic factors (such as naphthalene volatilization) or intermediate products on the remediation effect. It features high naphthalene remediation efficiency and strong post-remediation system stability.

[0023] 2. The method of the present invention for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification includes inoculating the wastewater with a bacterial suspension of polycyclic aromatic hydrocarbon degrading bacteria and a bacterial suspension of urea-degrading bacteria for co-culture. This co-culture system fully combines the adsorption characteristics of urea-degrading bacteria and the degradation characteristics of degrading bacteria. The urea-degrading bacteria reduce the toxicity of the environment, ensuring that the degrading bacteria complete the initial degradation of naphthalene. At the same time, the naphthalene degradation products are used as a nutrient source to support the growth and metabolism of the urea-degrading bacteria.

[0024] 3. The method of the present invention for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification includes adding urea and calcium chloride to the co-culture system for induction culture, using bacteria that adsorb calcium ions in the contaminated system as nucleation sites to induce biocalcification and generate precipitates, thereby rapidly sealing and solidifying the adsorbed naphthalene and degradation products to ensure efficient naphthalene remediation.

[0025] 4. The method of the present invention for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification has the characteristics of high remediation stability. The precipitate in the remediation system has a large specific surface area and a layered porous structure, which can further adsorb bacteria and stabilize pollutants, and prevent naphthalene volatilization.

[0026] 5. The method of this invention is reasonably conceived and provides new ideas for enriching the solutions for naphthalene wastewater treatment.

[0027] 6. The method of the present invention is simple to operate, the device is easy to build, the process scale-up is minimal, and it is easy to promote and apply.

[0028] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0029] Instruction manual illustrations

[0030] Figure 1 This is a graph showing the relationship between the concentration of naphthalene in naphthalene-contaminated wastewater with a naphthalene concentration of 50 mg / L and the measurement time, as shown in Example 1.

[0031] Figure 2 This is a graph showing the relationship between the concentration of naphthalene in naphthalene-contaminated wastewater with a naphthalene concentration of 100 mg / L and the measurement time, as shown in Example 2.

[0032] Figure 3 This is a graph showing the relationship between the concentration of naphthalene in naphthalene-contaminated wastewater with a naphthalene concentration of 200 mg / L and the measurement time, as shown in Example 3.

[0033] Figure 4 The image shows the X-ray diffraction pattern of the mineral precipitate when the naphthalene concentration was 100 mg / L in Example 2.

[0034] Figure 5 The Fourier transform infrared spectrum of the mineral precipitate when the naphthalene concentration was 100 mg / L in Example 2 is shown.

[0035] Figure 6 This is a scanning electron microscope image of the mineral precipitate when the naphthalene concentration is 100 mg / L, as shown in Example 2. Detailed Implementation

[0036] Example 1

[0037] This embodiment provides a method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification, including:

[0038] Step 1: Sequentially inoculate polycyclic aromatic hydrocarbon (PAH)-degrading bacteria and urea-lying bacteria into naphthalene-contaminated wastewater with a naphthalene concentration of 50 mg / L to obtain a remediation wastewater system. The sequential inoculation involves first adding PAH-degrading bacteria to the naphthalene-contaminated wastewater, shaking and culturing for 30 minutes, followed by adding urea-lying bacteria. In the remediation wastewater system, the volume percentage of PAH-degrading bacteria is 5%, and the volume percentage of urea-lying bacteria is 5%. The OD value of PAH-degrading bacteria in the PAH-degrading bacteria solution is... 600 The value is around 2.1; the OD of urealytic bacteria in the urealytic bacteria culture is... 600 The value is approximately 1.9; the polycyclic aromatic hydrocarbon (PAH) degrading bacteria is *Microbacterium paraoxidans*, sourced from the China Marine Microbial Culture Collection Center, with accession number MCCC 1A01491; the preparation method of the PAH degrading bacteria culture includes: culturing *Microbacterium paraoxidans* in a lysozyme broth medium at pH 7 for 24 hours, and obtaining a mixed system of *Microbacterium paraoxidans* and the lysozyme broth medium from which *Microbacterium paraoxidans* grows and metabolizes; the lysozyme broth medium contains 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the remainder is water; the culture temperature is 30℃, and the shaking speed is 180 rpm; the urea-relieving bacteria is *Bacillus pasteurellii*, and the preparation method of the urea-relieving bacteria culture includes: culturing *Bacillus pasteurellii* in an ammonium chloride medium at pH 9 for 24 hours, obtaining the *Bacillus pasteurellii* culture... A mixed system of ammonium chloride culture medium for the growth and metabolism of cocci and Bacillus pasteurellis; wherein the ammonium chloride culture medium contains 20 g / L yeast extract, 10 g / L ammonium chloride, 10 mg / L manganese sulfate monohydrate, 24 mg / L nickel chloride hexahydrate, and the remainder is water; the culture temperature is 30℃, and the shaking speed is 180 rpm; the urea-releasing bacteria are obtained from the China General Microbiological Culture Collection Center, with the accession number CGMCC1.3687; the preparation method of the naphthalene-contaminated wastewater includes: dispersing 0.5 g of naphthalene in 100 mL of acetone to obtain a mother liquor, storing the mother liquor at 4℃, and mixing it with water to prepare naphthalene-contaminated wastewater with different naphthalene concentrations before use;

[0039] Step 2: Shake the wastewater remediation system at 30℃ for 12 hours to obtain the co-cultured system; the shaking rate is 180 rpm.

[0040] Step 3: Add urea and calcium chloride to the co-culture system to obtain an induction system; to induce the precipitation of aragonite and spherulite minerals; the concentration of urea and calcium chloride in the induction system is 100 mM.

[0041] Step 4: Shake the induction system at 30°C for ≥24 hours to continue the remediation process and complete the remediation of the naphthalene-contaminated wastewater.

[0042] Comparative Example 1

[0043] This comparative example is the same as Example 1, except that only the bacterial solution of polycyclic aromatic hydrocarbon degrading bacteria is added in step one.

[0044] Example 2

[0045] This embodiment provides a method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification, including:

[0046] Step 1: Sequentially inoculate polycyclic aromatic hydrocarbon (PAH)-degrading bacteria and urea-free bacteria solutions into naphthalene-contaminated wastewater with a naphthalene concentration of 100 mg / L to obtain a remediation wastewater system. The sequential inoculation involves first adding PAH-degrading bacteria solution to the naphthalene-contaminated wastewater, shaking and culturing for 30 min, followed by adding urea-free bacteria solution. In the remediation wastewater system, the volume percentage of PAH-degrading bacteria solution is 5%, and the volume percentage of urea-free bacteria solution is 5%. The OD600 of PAH-degrading bacteria solution is approximately 2.1, and the OD600 of urea-free bacteria solution is approximately 1.9. The PAH-degrading bacteria are *Microbacterium paraoxidans*, sourced from the China Marine Microbiological Culture Collection Center (MCCC). 1A01491; The method for preparing the bacterial suspension of the polycyclic aromatic hydrocarbon degrading bacteria includes: culturing *Microbacterium paraoxidans* in a lysozyme broth medium at pH 7 for 24 hours, and obtaining a mixed system of *Microbacterium paraoxidans* and the lysozyme broth medium from which *Microbacterium paraoxidans* grows and metabolizes; the lysozyme broth medium contains 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the remainder is water; the culture temperature is 30°C, and the shaking speed is 180 rpm; the urea-relieving bacteria is *Bacillus pasteurellii*, and the method for preparing the bacterial suspension of the urea-relieving bacteria includes: culturing *Bacillus pasteurellii* in an ammonium chloride medium at pH 9 for 24 hours, obtaining the *Bacillus pasteurellii*... A mixed system of ammonium chloride culture medium for the growth and metabolism of cocci and Bacillus pasteurellis; wherein the ammonium chloride culture medium contains 20 g / L yeast extract, 10 g / L ammonium chloride, 10 mg / L manganese sulfate monohydrate, 24 mg / L nickel chloride hexahydrate, and the remainder is water; the culture temperature is 30℃, and the shaking speed is 180 rpm; the urea-releasing bacteria are obtained from the China General Microbiological Culture Collection Center, with the accession number CGMCC1.3687; the preparation method of the naphthalene-contaminated wastewater includes: dispersing 0.5 g of naphthalene in 100 mL of acetone to obtain a mother liquor, storing the mother liquor at 4℃, and mixing it with water to prepare naphthalene-contaminated wastewater with different naphthalene concentrations before use;

[0047] Step 2: Shake the wastewater remediation system at 30℃ for 12 hours to obtain the co-cultured system; the shaking rate is 180 rpm.

[0048] Step 3: Add urea and calcium chloride to the co-culture system to obtain an induction system; to induce the precipitation of aragonite and spherulite minerals; the concentration of urea and calcium chloride in the induction system is 100 mM.

[0049] Step 4: Shake the induction system at 30°C for ≥24 hours to continue the remediation process and complete the remediation of the naphthalene-contaminated wastewater.

[0050] Comparative Example 2

[0051] This comparative example is the same as Example 2, except that only the bacterial solution of polycyclic aromatic hydrocarbon degrading bacteria is added in step one.

[0052] Example 3

[0053] This embodiment provides a method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification, including:

[0054] Step 1: Sequentially inoculate polycyclic aromatic hydrocarbon (PAH)-degrading bacteria and urea-free bacteria solutions into naphthalene-contaminated wastewater with a naphthalene concentration of 200 mg / L to obtain a remediation wastewater system. The sequential inoculation involves first adding PAH-degrading bacteria solution to the naphthalene-contaminated wastewater, shaking and culturing for 30 minutes, followed by adding urea-free bacteria solution. In the remediation wastewater system, the volume percentage of PAH-degrading bacteria solution is 5%, and the volume percentage of urea-free bacteria solution is 5%. The OD600 of PAH-degrading bacteria solution is approximately 2.1, and the OD600 of urea-free bacteria solution is approximately 1.9. The PAH-degrading bacteria are *Microbacterium paraoxidans*, sourced from the China Marine Microbiological Culture Collection Center (MCCC). 1A01491; The method for preparing the bacterial suspension of the polycyclic aromatic hydrocarbon degrading bacteria includes: culturing *Microbacterium paraoxidans* in a lysozyme broth medium at pH 7 for 24 hours, and obtaining a mixed system of *Microbacterium paraoxidans* and the lysozyme broth medium from which *Microbacterium paraoxidans* grows and metabolizes; the lysozyme broth medium contains 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the remainder is water; the culture temperature is 30°C, and the shaking speed is 180 rpm; the urea-relieving bacteria is *Bacillus pasteurellii*, and the method for preparing the bacterial suspension of the urea-relieving bacteria includes: culturing *Bacillus pasteurellii* in an ammonium chloride medium at pH 9 for 24 hours, obtaining the *Bacillus pasteurellii*... A mixed system of ammonium chloride culture medium for the growth and metabolism of cocci and Bacillus pasteurellis; wherein the ammonium chloride culture medium contains 20 g / L yeast extract, 10 g / L ammonium chloride, 10 mg / L manganese sulfate monohydrate, 24 mg / L nickel chloride hexahydrate, and the remainder is water; the culture temperature is 30℃, and the shaking speed is 180 rpm; the urea-releasing bacteria are obtained from the China General Microbiological Culture Collection Center, with the accession number CGMCC1.3687; the preparation method of the naphthalene-contaminated wastewater includes: dispersing 0.5 g of naphthalene in 100 mL of acetone to obtain a mother liquor, storing the mother liquor at 4℃, and mixing it with water to prepare naphthalene-contaminated wastewater with different naphthalene concentrations before use;

[0055] Step 2: Shake the wastewater remediation system at 30℃ for 12 hours to obtain the co-cultured system; the shaking rate is 180 rpm.

[0056] Step 3: Add urea and calcium chloride to the co-culture system to obtain an induction system; to induce the precipitation of aragonite and spherulite minerals; the concentration of urea and calcium chloride in the induction system is 100 mM.

[0057] Step 4: Shake the induction system at 30°C for ≥24 hours to continue the remediation process and complete the remediation of the naphthalene-contaminated wastewater.

[0058] Comparative Example 3

[0059] This comparative example is the same as Example 3, except that only the bacterial solution of polycyclic aromatic hydrocarbon degrading bacteria is added in step one.

[0060] Example 4

[0061] This embodiment provides a method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification, including:

[0062] Step 1: Sequentially inoculate polycyclic aromatic hydrocarbon (PAH)-degrading bacteria and urea-free bacteria suspensions into naphthalene-contaminated wastewater with a naphthalene concentration of 50 mg / L to obtain a remediation wastewater system. The sequential inoculation involves first adding PAH-degrading bacteria suspension to the naphthalene-contaminated wastewater, shaking and culturing for 20 min, followed by adding urea-free bacteria suspension. In the remediation wastewater system, the volume percentage of PAH-degrading bacteria suspension is 5%, and the volume percentage of urea-free bacteria suspension is 5%. The OD600 of PAH-degrading bacteria in the PAH-degrading bacteria suspension is approximately 2.1, and the OD600 of urea-free bacteria in the urea-free bacteria suspension is approximately 1.9. The PAH-degrading bacteria are *Microbacterium paraoxidans*, sourced from the China Marine Microbial Culture Collection Center (MCCC). 1A01491; The method for preparing the bacterial suspension of the polycyclic aromatic hydrocarbon degrading bacteria includes: culturing *Microbacterium paraoxidans* in a lysozyme broth medium at pH 6 for 26 hours, and obtaining a mixed system of *Microbacterium paraoxidans* and the lysozyme broth medium from which *Microbacterium paraoxidans* grows and metabolizes; the lysozyme broth medium contains 8 g / L tryptone, 4 g / L yeast extract, 11 g / L sodium chloride, and the remainder is water; the culture temperature is 25℃, and the shaking speed is 190 rpm; the urea-relieving bacteria is *Bacillus pasteurellii*, and the method for preparing the bacterial suspension of the urea-relieving bacteria includes: culturing *Bacillus pasteurellii* in an ammonium chloride medium at pH 8 for 26 hours, obtaining the *Bacillus pasteurellii*... A mixed system of ammonium chloride culture medium for the growth and metabolism of cocci and Bacillus pasteurellis; wherein the ammonium chloride culture medium contains 22 g / L yeast extract, 8 g / L ammonium chloride, 12 mg / L manganese sulfate monohydrate, 26 mg / L nickel chloride hexahydrate, and the remainder is water; the culture temperature is 25℃, and the shaking speed is 170 rpm; the urea-releasing bacteria are obtained from the China General Microbiological Culture Collection Center, with the accession number CGMCC1.3687; the preparation method of the naphthalene-contaminated wastewater includes: dispersing 0.5 g of naphthalene in 100 mL of acetone to obtain a mother liquor, storing the mother liquor at 4℃, and mixing it with water to prepare naphthalene-contaminated wastewater with different naphthalene concentrations before use;

[0063] Step 2: Shake the reclaimed wastewater system at 25℃ for 14 hours to obtain the co-cultured system; the shaking rate is 180 rpm.

[0064] Step 3: Add urea and calcium chloride to the co-culture system to obtain an induction system; to induce the precipitation of aragonite and spherulite minerals; the concentration of urea and calcium chloride in the induction system is 90 mM.

[0065] Step 4: Shake the induction system at 25°C for ≥24 hours to continue the remediation process and complete the remediation of the naphthalene-contaminated wastewater.

[0066] The remediation effect of this embodiment on naphthalene-contaminated wastewater is basically the same as that in Example 1.

[0067] Example 5

[0068] This embodiment provides a method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification, including:

[0069] Step 1: Sequentially inoculate polycyclic aromatic hydrocarbon (PAH)-degrading bacteria and urea-free bacteria solutions into naphthalene-contaminated wastewater with a naphthalene concentration of 200 mg / L to obtain a remediation wastewater system. The sequential inoculation involves first adding PAH-degrading bacteria solution to the naphthalene-contaminated wastewater, shaking and culturing for 25 min, followed by adding urea-free bacteria solution. In the remediation wastewater system, the volume percentage of PAH-degrading bacteria solution is 5%, and the volume percentage of urea-free bacteria solution is 5%. The OD600 of PAH-degrading bacteria solution is approximately 2.1, and the OD600 of urea-free bacteria solution is approximately 1.9. The PAH-degrading bacteria are *Microbacterium paraoxidans*, sourced from the China Marine Microbiological Culture Collection Center (MCCC). 1A01491; The method for preparing the bacterial suspension of the polycyclic aromatic hydrocarbon degrading bacteria includes: culturing *Microbacterium paraoxidans* in a lysozyme broth medium at pH 6.5 for 22 hours, and obtaining a mixed system of *Microbacterium paraoxidans* and the lysozyme broth medium from which *Microbacterium paraoxidans* grows and metabolizes; the lysozyme broth medium contains 12 g / L tryptone, 6 g / L yeast extract, 9 g / L sodium chloride, and the remainder is water; the culture temperature is 35℃, and the shaking speed is 170 rpm; the urea-relieving bacteria is *Bacillus pasteurellii*, and the method for preparing the bacterial suspension of the urea-relieving bacteria includes: culturing *Bacillus pasteurellii* in an ammonium chloride medium at pH 8.5 for 22 hours, and obtaining the *Bacillus pasteurellii*... A mixed system of ammonium chloride culture medium for the growth and metabolism of *Dystrophus occulta* and *Bacillus pasteurellii*; wherein the ammonium chloride culture medium contains 18 g / L yeast extract, 12 g / L ammonium chloride, 8 mg / L manganese sulfate monohydrate, 22 mg / L nickel chloride hexahydrate, and the remainder is water; the culture temperature is 35℃, and the shaking speed is 190 rpm; the urea-releasing bacteria are obtained from the China General Microbiological Culture Collection Center, with accession number CGMCC1.3687; the preparation method of the naphthalene-contaminated wastewater includes: dispersing 0.5 g of naphthalene in 100 mL of acetone to obtain a mother liquor, storing the mother liquor at 4℃, and mixing it with water to prepare naphthalene-contaminated wastewater with different naphthalene concentrations before use;

[0070] Step 2: Shake the wastewater remediation system at 35℃ for 10 hours to obtain the co-cultured system; the shaking rate is 180 rpm.

[0071] Step 3: Add urea and calcium chloride to the co-culture system to obtain an induction system; to induce the precipitation of aragonite and spherulite minerals; the concentration of urea and calcium chloride in the induction system is 110 mM.

[0072] Step 4: Shake the induction system at 35°C for ≥24 hours to continue the remediation process and complete the remediation of the naphthalene-contaminated wastewater.

[0073] The remediation effect of this embodiment on naphthalene-contaminated wastewater is basically the same as that of Example 3.

[0074] Performance Evaluation

[0075] The effects of Example 1 and Comparative Example 1 on naphthalene-contaminated wastewater are as follows: Figure 1 As shown, the horizontal axis represents the total time for step two (oscillation repair) and step four (continued repair), while the blank control group represents naphthalene-contaminated wastewater without any bacterial inoculation. According to... Figure 1 It is evident that the remediation method using the sequence of the present invention to inoculate polycyclic aromatic hydrocarbon degrading bacteria and urea-degrading bacteria infusions has a significantly higher remediation efficiency than the blank control group and the comparative example 1 inoculated only with degrading bacteria. Moreover, the residual naphthalene concentration was 1.06 mg / L after two days of remediation, indicating that the remediation efficiency was close to 100%. Furthermore, subsequent observation of the naphthalene-contaminated wastewater after remediation revealed that the naphthalene concentration remained essentially unchanged, demonstrating that the method of the present invention has the characteristics of long-lasting and stable remediation of naphthalene-contaminated wastewater.

[0076] The effects of Example 2 and Comparative Example 2 on naphthalene-contaminated wastewater are as follows: Figure 2 As shown, the effects of Example 3 and Comparative Example 3 on naphthalene-contaminated wastewater are as follows: Figure 3 As shown, the blank control group and the meaning of the horizontal axis are... Figure 1 Same. According to Figure 2 and Figure 3 As can be seen, the remediation effect of the method of the present invention in high-concentration naphthalene-contaminated wastewater follows a pattern that is basically consistent with that of Example 1, and still exhibits a significantly higher remediation effect than the comparative example. Figure 2 In the study, the residual concentration two days after the initial repair was 4.55 mg / L, with a repair efficiency of 97.75%. Figure 3 In the study, the residual amount after two days of remediation was 8.00 mg / L, and the remediation efficiency was 93.83%. Compared with the treatment of low-concentration naphthalene-contaminated wastewater (Example 1), the time for complete remediation of high-concentration naphthalene-contaminated wastewater was slightly longer, ranging from 2 to 3 days. It can also be seen that the method of the present invention still exhibits the characteristics of long-lasting stability for high-concentration naphthalene-contaminated wastewater.

[0077] Figure 4 This is a schematic diagram of the phase morphology of the mineralized products from the naphthalene-contaminated wastewater after 36 hours of continued remediation in Example 2. The detection method was as follows: The mineralized products from the naphthalene-contaminated wastewater system after 36 hours of continued remediation were analyzed using XRD patterns. Figure 4 As can be seen, the presence of characteristic peaks belonging to aragonite and aragonite in the sample indicates that the precipitate generated in the wastewater using the treatment method of the present invention is aragonite and aragonite.

[0078] Figure 5This is the FTIR spectrum of the mineralized products from the naphthalene-contaminated wastewater remediation process of Example 2, after 36 hours. The detection method was as follows: mineralized products from the naphthalene-contaminated wastewater system after 36 hours of remediation were collected and their FTIR spectra were measured. According to... Figure 5 It can be seen that 711 cm in the sample -1 871cm -1 1040cm -1 and 1404cm -1 The absorption peak is attributed to biocalcified calcium carbonate precipitate, 1651 cm⁻¹. -1 1791cm -1 and 3000~3200cm -1 The absorption peaks are attributed to naphthalene and its degradation intermediates, indicating that the mineralized products obtained by the remediation method of the present invention contain naphthalene and its degradation intermediates bound by biocalcification precipitation, demonstrating that the method of the present invention can promote the adsorption and stabilization of naphthalene and its intermediates.

[0079] Figure 6 This is a scanning electron microscope (SEM) image of the mineralization products from the naphthalene-contaminated wastewater after 36 hours of continued remediation, as shown in Example 2. The testing method included: taking mineralization products from the naphthalene-contaminated wastewater system after 36 hours of continued remediation and performing SEM analysis. Figure 6 It is evident that aragonite and spheroidite are present in a certain area of ​​this mineralization product, which is consistent with... Figure 3 The consistent characteristic peaks shown by XRD in the samples indicate that the method of this invention can obtain mineralized aragonite and spheroidized minerals. In another region of this mineralized product, a layered porous structure and surface-formed chambers capable of supporting bacteria are observed. In other regions of this mineralized product, biomineralized products induced by bacteria adsorbing naphthalene as nucleation sites are also present, indicating that the method of this invention successfully generates biomineralized precipitates adsorbed with naphthalene and its intermediates. The surface of this mineralized product exhibits multiple morphological characteristics, namely, the simultaneous presence of bacterial-loaded chambers, the obtained final mineralized products of aragonite and spheroidized aragonite, and induced biocalcification regions during the nucleation process, indicating continuous remediation. The method of this invention possesses more stable naphthalene contaminant remediation characteristics.

[0080] Tables 1-3 show the GC / MS analysis results of the mineralized products from the naphthalene-contaminated wastewater after 36 hours of continued remediation in Example 2. The testing method included: taking the mineralized products from the naphthalene-contaminated wastewater system after 36 hours of continued remediation, performing thermogravimetric analysis (TGA) in a nitrogen atmosphere, and then qualitatively analyzing the products using GC / MS. The samples analyzed by GC / MS were the volatile substances produced by the thermal decomposition of the mineralized products at 50℃, 230℃, and 730℃. The results are shown in Tables 1-3. As can be seen from Tables 1-3, the products after thermal decomposition of the minerals are small-molecule organic acids, alcohols, aldehydes, alkanes, etc., indicating that naphthalene or its metabolites have undergone degradation in the minerals. Due to the inherent physical properties of naphthalene, under appropriate pyrolysis conditions, pure polycyclic aromatic hydrocarbon naphthalene itself cannot undergo thermal decomposition, but will generate naphthalene vapor through volatilization. Gas chromatography-mass spectrometry (GC-MS) results show that no naphthalene vapor is present in the products of mineralization, indicating the absence of volatile naphthalene in the minerals. This demonstrates that the method of this invention allows naphthalene in wastewater to be converted into biomineralized products through encapsulation or degradation, and the naphthalene present in the biomineralized products can be degraded into low-toxicity or non-toxic metabolites. This indicates that the method of this invention not only removes naphthalene from polluted wastewater but also encapsulates naphthalene in a degradable form. The method of this invention facilitates the complete degradation of naphthalene and effectively avoids secondary pollution.

[0081] Table 1. Gas chromatography-mass spectrometry (GC-MS) results of thermogravimetric decomposition products at 50℃

[0082] Degrading bacteria + urea-lysinic bacteria, 50℃

[0083]

[0084] Table 2. Gas chromatography-mass spectrometry (GC-MS) results of thermogravimetric decomposition products at 230℃

[0085] Degrading bacteria + urea-lysinic bacteria, 230℃

[0086]

[0087] Table 3. Gas chromatography-mass spectrometry (GC-MS) results of thermogravimetric decomposition products at 730℃

[0088] Degrading bacteria + urea-lysing bacteria, 730℃

[0089]

[0090] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the invention shall still fall within the protection scope of the present invention.

Claims

1. A method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification, characterized in that, include: Step 1: Sequentially inoculate naphthalene-contaminated wastewater with bacterial suspensions of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria and urea-lying bacteria to obtain a wastewater remediation system. The sequential inoculation involves first adding the PAH-degrading bacterial suspension to the naphthalene-contaminated wastewater, shaking and culturing for 20–30 minutes, and then adding the urea-lying bacterial suspension. The PAH-degrading bacteria are *Microbacterium paraoxidans*, and the urea-lying bacteria are *Bacillus pasteurellii*. Step 2: Shake the remediated wastewater system at 25-35℃ for 10-14 hours to obtain the co-cultured system; Step 3: Add urea and calcium chloride to the co-culture system to obtain the induction system; the concentration of urea and calcium chloride in the induction system are both 90-110 mM. Step 4: Shake the induction system at 25-35℃ for ≥24 hours to complete the remediation of naphthalene-contaminated wastewater.

2. The method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification according to claim 1, characterized in that, In step one, the concentration of naphthalene in the naphthalene-contaminated wastewater is 50–200 mg / L.

3. The method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification according to claim 1, characterized in that, In step one, the volume percentage of polycyclic aromatic hydrocarbon degrading bacteria and urea-degrading bacteria in the wastewater remediation system is 5%.

4. The method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification according to claim 1, characterized in that, In step one, the method for preparing the bacterial culture of the polycyclic aromatic hydrocarbon degrading bacteria includes: culturing the polycyclic aromatic hydrocarbon degrading bacteria in a lysate broth medium with a pH of 6-7 for 22-26 hours; the culture temperature is 25-35℃ and the shaking speed is 170-190 rpm.

5. The method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification according to claim 4, characterized in that, The lysozyme broth culture medium contains 8–12 g / L tryptone, 4–6 g / L yeast extract, 9–11 g / L sodium chloride, and the remainder is water.

6. The method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification according to claim 1, characterized in that, In step one, the method for preparing the urealytic bacteria culture includes: culturing the urealytic bacteria in an ammonium chloride medium with a pH of 8-9 for 22-26 hours; the culture temperature is 25-35℃ and the shaking speed is 170-190 rpm.

7. The method for remediating naphthalene-contaminated wastewater based on microbial co-culture-induced biocalcification according to claim 6, characterized in that, The ammonium chloride culture medium contains 18–22 g / L yeast extract, 8–12 g / L ammonium chloride, 8–12 mg / L manganese sulfate monohydrate, 22–26 mg / L nickel chloride hexahydrate, and the remainder is water.

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