Pseudogulbenkiania soli and application thereof in degrading sulfide

Abstract

The present application relates to a strain of Pseudogulbenkiania non-saccharolytica and its application, the strain is preserved in China General Microbiological Culture Collection Center, the address is: No. 1, Beichen West Road, Hua-yuan District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the preservation number is: CGMCC No. 28059, and the preservation date is: July 28, 2023, the beneficial effect of the Pseudogulbenkiania non-saccharolytica provided by the present application is: strong and fast effect of sulfide degradation, can efficiently and quickly degrade ferrous sulfide existing in wastewater, and oxidize it into white sulfur element, effectively solve the black and odorous problem of water body and river sediment, and then can be used as a microbial inoculant for river sediment improvement.

Description

Technical Field

[0001] This invention relates to a strain of Pseudomonas agalactiae, a microbial agent containing the strain, and its application in the field of environmental remediation, belonging to the field of environmental microbiology technology. Background Technology

[0002] With the acceleration of urbanization and industrialization, the amount of sewage discharge is also increasing day by day, and the large amount of sewage discharge has seriously affected the self-cleaning ability of urban water bodies.

[0003] Increasing amounts of sulfides generated during production processes are dissolved in wastewater and discharged, posing a significant new challenge to these industries by treating high-concentration, difficult-to-degrade sulfur-containing organic wastewater. This type of wastewater contains high concentrations of sulfur compounds, is highly toxic, and causes severe environmental pollution, potentially leading to natural disasters and even posing health risks, such as acid rain.

[0004] Pollutant discharge has resulted in high levels of organic matter in river water, with ammonia nitrogen and total nitrogen levels significantly exceeding standards, leading to rapid deterioration of river water quality and even causing it to become foul-smelling and black. River dredging is currently a primary measure to ensure water resource security and a healthy aquatic environment; however, the disposal of dredged river sediment remains a significant social problem. Because the sediment contains large amounts of organic matter and nutrients, improper dumping or treatment can easily cause secondary pollution of the water body. Furthermore, sediment with excessive pollutants cannot be reused as agricultural fertilizer. Therefore, the importance of sediment remediation is gradually attracting the attention of environmentalists.

[0005] The main cause of the black and smelly bottom sediment is that the corpses and feces of aquatic organisms settle to the bottom and are utilized by harmful bacteria in the anaerobic environment of the sediment to produce hydrogen sulfide gas. The gas releases and produces a foul odor, and at the same time, black ferrous sulfide precipitates are formed in the water.

[0006] There are many common methods for sediment improvement, such as physical, chemical, and microbial methods. Common physical improvement methods include screening and compression. These physical methods remove impurities and pollutants from the sediment, thus purifying the water. Chemical methods involve adding oxidants and reducing agents to adjust the pH balance, causing organic matter and pollutants in the sediment to react and be removed. However, both of these methods typically face challenges such as high costs, requiring significant manpower and resources, and the use and residue of chemical reagents may cause secondary pollution to the environment. Therefore, microbial treatment methods are becoming increasingly widely used.

[0007] Microbial agents, combined with the beneficial bacteria already present in the river, can quickly establish a dominant microbial community in the original aquatic environment. This community transforms, decomposes, and discharges various organic matter and pollutants, thereby purifying the water, repairing damaged ecosystems, and restoring the original ecological functions of the sediment. The treated sediment can also be used as fertilizer for irrigation, achieving secondary utilization.

[0008] In summary, by isolating sulfur-oxidizing bacteria with strong sulfur ion oxidation function, free sulfur ions can be converted into water-insoluble and sedimentable elemental sulfur under anaerobic, hypoxic, and aerobic conditions using the microbial metabolic mechanism itself. This has significant practical and applied significance for improving bottom sediment and alleviating black and odorous water bodies. Summary of the Invention

[0009] This invention addresses the shortcomings of existing methods for treating sulfides in wastewater and improving sediment. It provides a strain of Pseudomonas aeruginosa, a microbial agent containing it, and its application. This strain can utilize its own metabolic mechanism to degrade sulfides in wastewater and ferrous sulfide precipitates that cause sediment to turn black.

[0010] A strain of *Pseudochrobactrum asaccharolyticum* BC-1 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 28059 and deposit date of July 28, 2023. Its 16S rDNA sequence is shown in SEQ ID No: 1. In this invention, *Pseudochrobactrum asaccharolyticum* refers to strain BC-1.

[0011] The beneficial effects of the non-glycolytic Pseudomonas aeruginosa provided by this invention are:

[0012] (1) It has strong sulfide degradation ability and fast effect. Under the condition of 30℃, for an initial sulfide concentration of about 100mg / L, the degradation rate can reach 96% in as little as 24 hours.

[0013] (2) It can efficiently and quickly degrade ferrous sulfide in wastewater and oxidize it into white elemental sulfur, effectively solving the black and odorous problems of water bodies and riverbed sediments, and can then be used as a microbial agent for improving riverbed sediments.

[0014] (3) The strain has a simple culture method, strong environmental adaptability, rapid reproduction and high safety.

[0015] The present invention also claims protection for microbial agents containing the non-glycolytic pseudoanthobacterium.

[0016] This invention also claims protection for a fermentation method of the non-glycolytic Pseudomonas aeruginosa, comprising the following steps:

[0017] (1) Primary seed culture: Under aseptic conditions, take unsaccharitrophs Pseudomonas aeruginosa and inoculate them into the nutrient culture medium. Culture them at 25-35℃ and 150-300rpm for 12-24h to obtain the primary seed culture solution.

[0018] (2) Secondary seed culture: Under aseptic conditions, the primary seed culture solution is inoculated into the nutrient medium at an inoculation rate of 1-10 vol%, and cultured at 25-35℃ and 150-300 rpm for 12-24 h to obtain the secondary seed culture solution.

[0019] (3) Fermentation: After the fermentation medium is sterilized, the secondary seed culture solution is inoculated into the fermentation medium at an inoculation rate of 1-10 vol%. Fermentation is carried out under the conditions of temperature of 25-35℃, rotation speed of 150-300 rpm, and aeration ratio of 1:(1-2). Fermentation is stopped when dissolved oxygen begins to rise, and fermentation liquid is obtained.

[0020] The aeration ratio mentioned in the fermentation method of this invention refers to the ratio of the volume of air introduced into the fermenter per minute to the total volume of the fermentation liquid.

[0021] The nutrient culture medium consists of: 5-15 g / L peptone, 3-8 g / L yeast extract, 8-12 g / L sodium chloride, water as solvent, and pH = 6.5-7.5.

[0022] The fermentation medium consists of: 5-15 g / L carbon source, 3-8 g / L nitrogen source, and PO4. 3- 1-3g / L, K + 1-2g / L, Mg 2+ 0.05-0.10 g / L, Ca 2+ 0.5-1.5g / L, Mn 2+ 0.05-0.1 g / L, solvent is water, pH = 6-8.

[0023] Preferably, the PO4 3- and K + The source is dipotassium hydrogen phosphate or potassium dihydrogen phosphate, wherein the Mg 2+ The source is a mixture of one or more of magnesium sulfate, magnesium nitrate, or magnesium chloride, wherein the Ca... 2+ The source is one or more of calcium chloride and calcium nitrate, wherein the Mn 2+ The source is one or more of manganese sulfate monohydrate, manganese sulfate tetrahydrate, manganese nitrate, or manganese chloride.

[0024] Furthermore, the carbon source is selected from one or more of glucose, sucrose, starch, sodium acetate, or sodium succinate.

[0025] Furthermore, the nitrogen source is selected from one or more of yeast extract, peptone, urea, ammonium sulfate, or potassium nitrate.

[0026] In practical applications, the final form of the *Pseudomonas aeruginosa* product can be determined according to actual usage and storage needs. When liquid products are required, the fermentation broth can be diluted to the required concentration for direct use. When solid products are required, the fermentation broth can be centrifuged to obtain bacterial sludge, and then freeze-dried to obtain solid bacterial powder.

[0027] The present invention also claims protection for a method of purifying wastewater using an activated solution of *Pseudomonas saccharolyticus* or a microbial agent containing *Pseudomonas saccharolyticus*, comprising the step of inoculating the wastewater with an activated solution of *Pseudomonas saccharolyticus* or a microbial agent containing *Pseudomonas saccharolyticus*.

[0028] Furthermore, the concentration of sulfides in the wastewater is below 400 mg / L, preferably below 300 mg / L, and most preferably below 200 mg / L.

[0029] Furthermore, the inoculation amount of the activated liquid of *Pseudomonas sucrastinata* or the microbial agent containing *Pseudomonas sucrastinata* is 100 ppm or more, preferably 100-1000 ppm.

[0030] Furthermore, the temperature during the purification process is 25-35℃.

[0031] This invention also claims protection for the application of *Pseudomonas agallochae* and microbial agents containing *Pseudomonas agallochae* in the field of environmental remediation.

[0032] Preferably, the *Pseudomonas aeruginosa* and the microbial agent containing *Pseudomonas aeruginosa* are used to degrade sulfides; more preferably, the sulfides are ferrous sulfide, H2S, and HS-. - and S 2- . Attached Figure Description

[0033] Figure 1 The growth curve of *Pseudomonas aeruginosa* as determined in Example 2;

[0034] Figure 2 It is factory wastewater containing a large amount of black ferrous sulfide precipitate;

[0035] Figure 3 Factory wastewater after inoculation with Pseudomonas sacchariformis BC-1 and culture for 72 hours. Detailed Implementation

[0036] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0037] Example 1. Isolation, purification and identification of *Pseudomonas aeruginosa*

[0038] (1) Screening and isolation of strains:

[0039] Sludge and sludge suspension were collected from a chemical plant. 10 mL of the sludge-water mixture was inoculated into a 250 mL headspace bottle containing 100 mL of enrichment medium (5 g yeast powder, 10 g peptone, 10 g sodium chloride, 1 L distilled water, pH = 7.0, sterilized at 121℃ for 20 min). The mixture was then cultured on a constant temperature shaker at 30℃ and 200 r / min for one week to obtain the enriched solution.

[0040] The enrichment solution was serially diluted to 10. -3 10 -4 10 -5 and 10 -6 The diluted solutions were then spread onto SOB solid selective medium (KH2PO4 1g, NH4Cl 0.8g, MgCl2·6H2O 0.8g, CaCl2·2H2O 0.01g, FeCl3·6H2O 0.01g, MnCl2·4H2O 0.04g, Na2S·9H2O 1.2g, beef extract 2g, peptone 10g, agar 20g, distilled water 1L, pH=7.0, sterilized at 121℃ for 20min). The spread plates were placed in a 30℃ incubator and incubated until single colonies grew. Single colonies with different morphologies were picked and transferred to test tube slant medium and incubated at 30℃ for about 48h. Then, they were transferred to a 4℃ refrigerator for storage.

[0041] A total of 6 strains were isolated using the above method and named BC-1, BC-2, BC-3, BC-4, BC-5 and BC-6, respectively.

[0042] (2) Evaluation of strains

[0043] The six obtained strains were inoculated into activation medium (5g yeast extract, 10g peptone, 10g sodium chloride, 1L distilled water, pH=7.0) and cultured for 48 hours at 30℃ with a shaker at 200 rpm to obtain activation solutions. Then, 100mL of liquid selective medium (1g KH₂PO₄, 0.8g NH₄Cl, 0.8g MgCl₂·6H₂O, 0.01g CaCl₂·2H₂O, 0.01g FeCl₃·6H₂O, 0.04g MnCl₂·4H₂O, Na₂S·9H₂O) was prepared. 0.4g of beef extract, 2g of beef extract, 10g of peptone, 1L of distilled water (pH=7.0, sterilized at 121℃ for 20min) were used to inoculate the activation solutions of each strain into the headspace vials (250mL). The inoculation volume of the activation solution was 100ppm. The vials were cultured at 200r / min and 30℃ on a shaker. The changes in sulfide content after 0, 24, 48, 72, 96 and 120 hours were measured using an LH-S3H sulfide analyzer manufactured by Beijing Lianhua Yongxing Technology Development Co., Ltd., along with special measuring reagents. The results are shown in Table 1.

[0044] Table 1 shows the degradation of sulfides by the selected strains.

[0045] BC-1 BC-2 BC-3 BC-4 BC-5 BC-6 24h 97.92 269.01 269.55 272.00 217.60 269.28 48h 8.16 268.74 272.00 269.28 225.22 269.01 72h 0.00 268.74 272.00 272.00 214.61 272.00 96h 0.00 267.65 271.18 263.02 209.44 268.46 120h 0.00 268.46 272.00 265.74 207.81 270.10

[0046] As can be seen from the data in Table 1, compared with other strains, strain BC-1 has a faster onset and better effect in removing sulfides. The sulfide concentration of about 270 ppm in the evaluation medium can be completely removed in 72 hours. The strain was re-inoculated into LB medium to prepare an activation solution, and then stored in glycerol tubes at -80℃.

[0047] (3) Testing and identification

[0048] The bacterial slant was identified by 16S rDNA gene sequence detection as *Pseudochrobactrum asaccharolyticum*. The 16S rDNA gene sequence determination results of this strain are shown in SEQ ID No:1.

[0049] Example 2. Investigation of growth conditions of *Pseudomonas saccharolyticus*

[0050] (1) Determination of the optimal carbon source for strain growth

[0051] Experimental methods:

[0052] Fermentation media were prepared using yeast extract as the nitrogen source and various carbon sources including glucose, molasses, brown sugar, maltodextrin, sodium acetate, starch, and corn flour, with a carbon-to-nitrogen ratio of 2:1 and a concentration of 1 g / L. Two inorganic salt solutions were prepared, and the experiment was divided into two groups. The fermentation medium using inorganic salt 1 contained the following micronutrient concentrations: dipotassium hydrogen phosphate 0.3%, magnesium sulfate 0.2%, manganese sulfate 0.2%, and calcium carbonate 0.2%. The fermentation medium using inorganic salt 2 contained the following micronutrient concentrations: magnesium sulfate 0.5%, manganese sulfate 0.3%, calcium chloride 1.5%, and dipotassium hydrogen phosphate 1.5%. NA and LB media were also added to the experimental groups to identify the optimal culture medium for *Pseudomonas aeruginosa* seed culture.

[0053] After inoculation with BC-1 strain, all samples were cultured in a shaker at 30°C and 180 rpm, and their OD values ​​were measured periodically. 600 (OD 600 This value represents the absorbance of the solution at a wavelength of 600 nm. It is used to measure the concentration of bacterial culture media and is commonly used to indicate bacterial cell density. Bacterial growth can be monitored using OD (Obstruction Discharge). 600 The growth extent of the strain was determined by monitoring the values ​​of the carbon source and the viable cell count. The results are shown in Table 2. In Table 2, the numbers 1 and 2 after the carbon source indicate the ratio of inorganic salt 1 or inorganic salt 2 used in the inorganic salt formulation of the culture medium.

[0054] Table 2. OD of strain BC-1 cultured in media with different carbon sources 600 Value and viable count

[0055]

[0056] The data in Table 2 show that the BC-1 strain grows best when glucose is the carbon source in the culture medium. Therefore, glucose is determined to be the optimal carbon source for *Pseudomonas saccharigenes*.

[0057] (2) Determination of the optimal nitrogen source for strain growth

[0058] Experimental methods:

[0059] Fermentation medium was prepared using glucose as the carbon source and soy protein isolate, soy protein, yeast extract, peptone, ammonium sulfate, and soybean meal as the nitrogen sources. The carbon-to-nitrogen ratio was 2:1, and inorganic salts were prepared according to formula 2.

[0060] After inoculation with BC-1 strain, all samples were cultured in a shaker at 30°C and 180 rpm, and their OD values ​​were measured periodically. 600 (OD 600 This value represents the absorbance of the solution at a wavelength of 600 nm. It is used to measure the concentration of bacterial culture media and is commonly used to indicate bacterial cell density. Bacterial growth can be monitored using OD (Obstruction Discharge).600 The growth extent of the bacterial strain was determined by monitoring the values ​​of the bacterial count and viable count. The results are shown in Table 3.

[0061] Table 3. OD of strain BC-1 cultured in different nitrogen source media 600 Value and viable count

[0062]

[0063] The data in Table 3 show that the BC-1 strain exhibits the best growth when yeast extract is used as the nitrogen source in the culture medium. Therefore, yeast extract is determined to be the optimal nitrogen source for *Pseudomonas aeruginosa*.

[0064] (3) Determination of bacterial growth curve

[0065] Experimental methods:

[0066] Pick up slant culture and inoculate it into LB medium. After culturing on a shaker for 48 hours, the activated solution is obtained. Inoculate the activated solution into fresh LB medium to start the experiment. The inoculation amount is 2%.

[0067] The experiment was divided into two experimental groups, with three replicates prepared for each group. Experimental group one was inoculated at 9:00 AM, resulting in three replicates. After inoculation, the primary activation solution was refrigerated. OD 600 Initial values ​​were measured. OD of Experimental Group 1. 600 Measurements were taken at 9:00 AM, 11:00 AM, and 1:00 PM, 3:00 PM, at a dilution of 10-fold. Group 2 was inoculated at 5:00 PM, with a total of three replicates. OD 600 Initial measurement, OD 600 Measurements were taken at 5 PM and 7 PM, and at 9 AM, 11 PM, 1 AM, 3 AM, and 5 AM the following day, with a dilution factor of tenfold. After each sampling, microscopic examination was performed to check for bacterial contamination. The results are shown in Table 4. Figure 1 As shown.

[0068] Table 4. Growth curve data of strain BC-1

[0069] Incubation time (h) <![CDATA[OD 600 ]]> Incubation time (h) <![CDATA[OD 600 ]]> 0 0.027 30 0.393 2 0.044 32 0.410 4 0.124 40 0.537 6 0.195 42 0.534 8 0.222 48 0.648 22 0.244 50 0.650 24 0.244 52 0.653 26 0.332 54 0.657 28 0.361

[0070] From Table 4 and Figure 1 It can be seen that the logarithmic growth phase of *Pseudomonas saccharolyticus* is approximately 30-40 hours. Note: The measurement of the growth curve may not be entirely accurate; the experimental logarithmic growth phase is longer than the actual growth phase.

[0071] Example 3. Production and post-processing technology of *Pseudomonas aeruginosa* (a type of bacteria) that does not solubilize sugars.

[0072] (1) Fermentation by strain

[0073] 1) Primary shake flask activation

[0074] In a sterile environment, one loopful of *Pseudomonas sucrastinata* BC-1 strain was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of enrichment medium (5 g yeast extract, 10 g peptone, 10 g sodium chloride, pH = 7.0, sterilized at 121 °C for 20 min). The flask was then incubated at 30 °C and 180 r / min for 24 h to obtain the primary activation solution.

[0075] 2) Secondary shake flask culture

[0076] In a sterile environment, 10 mL of the primary activation solution was transferred to four 1 L Erlenmeyer flasks containing 500 mL of enrichment medium (5 g yeast powder, 10 g peptone, 10 g sodium chloride, 1 L distilled water, pH = 7.0, sterilized at 121 °C for 20 min). The flasks were then incubated at 30 °C and 180 r / min for 24 h to obtain the secondary activation solution.

[0077] 3) 20L tank fermentation culture

[0078] The fermentation medium was sterilized at 121℃ for 20 min. The fermentation medium formula was: glucose 10 g / L, yeast powder 5 g / L, MgSO4 0.4 g / L, MnSO4 0.2 g / L, CaCl2 3 g / L, K2HPO4 3 g / L. The secondary seed culture was inoculated into the fermentation medium at an inoculation rate of 5-10 vol%. The fermentation tank was filled to 70% capacity. The initial pH was adjusted to 7.0 using sodium hydroxide, and the aeration ratio was 1:1.25 (m³). 3 ·min / m 3 Fermentation was carried out at 180 rpm and 30℃. During the fermentation process, ammonia was added to control the pH to 6.5. The fermentation cycle was about 40 hours. Fermentation was stopped immediately when dissolved oxygen began to rise. At this time, the fermentation was in the late logarithmic phase, with a viable cell count as high as 30 billion CFU / mL. The cell activity was the strongest, the residual fermentation nutrients were the least, and the decline in viable cell count during storage was minimal.

[0079] 2. Post-treatment process of fermentation broth

[0080] To obtain bacterial powder, the fermentation broth can be treated with freeze-drying. The viable bacterial count of the freeze-dried bacterial powder can reach 2 trillion CFU / g.

[0081] Example 4. Wastewater evaluation of *Pseudomonas saccharolyticus*

[0082] A single colony of *Pseudomonas sucralis* BC-1 was inoculated into an activation medium (5g yeast extract, 10g peptone, 10g sodium chloride, 1L distilled water, pH=7.0) and cultured for 48 hours at 30°C and 200r / min to obtain an activation solution with a viable count of 6 billion CFU / mL.

[0083] The sulfide oxidation effect of strain BC-1 was repeatedly evaluated using sulfur-containing wastewater from two factories with initial sulfide concentrations of 90 mg / L and 119 mg / L. Headspace bottles (250 mL each) containing 200 mL of factory wastewater were prepared, and the strain's activation solution was inoculated into each bottle at a concentration of 100 ppm. The cultures were incubated on a shaker at 200 rpm and 30°C. The changes in sulfide content after 0, 24, 48, and 72 hours were measured using an LH-S3H sulfide analyzer manufactured by Beijing Lianhua Yongxing Technology Development Co., Ltd., along with specialized reagents. The results are shown in Table 5.

[0084] Table 5. Degradation of sulfides in industrial wastewater by strain BC-1

[0085]

[0086] Example 5. Evaluation of ferrous sulfide degradation by *Pseudomonas sucrastinata*

[0087] A single colony of *Pseudomonas sacchariformis* BC-1 was inoculated into an activated liquid culture medium (5g yeast extract, 10g peptone, 10g sodium chloride, 1L distilled water, pH=7.0) and cultured for 48 hours at 30°C and 200r / min to obtain an activated solution with a viable count of 6 billion CFU / mL.

[0088] Next, prepare a headspace bottle (250mL) containing 250mL of factory wastewater, such as... Figure 2 As shown, the activated solution of the strain was inoculated into headspace bottles at a concentration of 100 ppm. The bottles were incubated at 200 rpm and 30°C on a shaker, and the changes in the liquid were observed. The sulfide content in the clarified water was measured using an LH-S3H sulfide analyzer manufactured by Beijing Lianhua Yongxing Technology Development Co., Ltd., along with specialized reagents. The results after 72 hours of incubation are shown below. Figure 3 As shown.

[0089] Experiments have shown that after inoculating the factory wastewater with the activated solution of strain BC-1, the ferrous sulfide in the wastewater was oxidized into white elemental sulfur by the functional bacteria, and the concentration of sulfide in the wastewater was measured to be zero.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of *Pseudomonas pallida* that cannot hydrolyze sugars ( Pseudochrobactrum asaccharolyticum )BC-1, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 28059.

2. A microbial inoculant, characterized in that, The active ingredient comprises *Pseudomonas sucrastinata* as described in claim 1.

3. The fermentation method of *Pseudomonas aeruginosa* according to claim 1, characterized in that, Includes the following steps: (1) Primary seed culture: Under aseptic conditions, take unsaccharitrophs Pseudomonas aeruginosa and inoculate them into the nutrient culture medium. Culture them at 25-35℃ and 150-300rpm for 12-24h to obtain the primary seed culture solution. (2) Secondary seed culture: Under aseptic conditions, the primary seed culture solution is inoculated into the nutrient medium at an inoculation rate of 1-10 vol%, and cultured at 25-35℃ and 150-300 rpm for 12-24 h to obtain the secondary seed culture solution. (3) Fermentation: After the fermentation medium is sterilized, the secondary seed culture solution is inoculated into the fermentation medium at an inoculation rate of 1-10 vol%. Fermentation is carried out under the conditions of temperature of 25-35℃, rotation speed of 150-300 rpm, and aeration ratio of 1:(1-2). Fermentation is stopped when dissolved oxygen begins to rise, and fermentation liquid is obtained.

4. The fermentation method according to claim 3, characterized in that, The nutrient culture medium consists of: 5-15 g / L peptone, 3-8 g / L yeast extract, 8-12 g / L sodium chloride, water as solvent, and pH = 6.5-7.

5. The fermentation medium consists of: 5-15 g / L carbon source, 3-8 g / L nitrogen source, and PO4. 3- 1-3g / L, K + 1-2g / L, Mg 2+ 0.05-0.10 g / L, Ca 2+ 0.5-1.5g / L, Mn 2+ 0.05-0.1 g / L, solvent is water, pH=6~8.

5. The fermentation method according to claim 4, characterized in that, The carbon source is selected from one or more of glucose, sucrose, starch, sodium acetate, or sodium succinate; The nitrogen source is selected from one or more of yeast extract, peptone, urea, ammonium sulfate, or potassium nitrate.

6. A method for purifying wastewater, characterized in that, The method includes the step of inoculating wastewater with the activated solution of *Pseudomonas aeruginosa* as described in claim 1 or the microbial agent as described in claim 2.

7. The method according to claim 6, characterized in that, The concentration of sulfides in the wastewater is below 400 mg / L.

8. The method according to claim 7, characterized in that, The concentration of sulfides in the wastewater is below 300 mg / L.

9. The method according to claim 8, characterized in that, The concentration of sulfides in the wastewater is below 200 mg / L.

10. The method according to any one of claims 6-9, characterized in that, The inoculation amount of the activated liquid of *Pseudomonas aeruginosa* or the microbial agent containing *Pseudomonas aeruginosa* is 100 ppm or more.

11. The method according to claim 10, characterized in that, The inoculation amount of the activated solution of *Pseudomonas agallochae* or the microbial agent containing *Pseudomonas agallochae* is 100-1000 ppm.

12. The application of the non-glycolytic Pseudomonas aeruginosa according to claim 1 and the microbial agent according to claim 2 for the degradation of sulfides.

13. The application according to claim 12, characterized in that, The sulfide is ferrous sulfide, H2S, or HS. - and S 2- .

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