Pseudomonas strain as well as culture method and application thereof

Abstract

The invention discloses a Pseudomonas sp. And a culture method and application thereof, the Pseudomonas sp. Is named as 320-7 and preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number of the Pseudomonas sp. Is CGMCC No.33204, the preservation date is December 25, 2024, and the 16S rDNA sequence of the Pseudomonas sp. Is as shown in SEQ ID NO.1. The Pseudomonas sp. Is named as 320-7 and preserved in the China General Microbiological Culture Collection Center (CGMCC). The pseudomonas strain (Pseudomonas sp.) 320-7 provided by the invention has a remarkable removal effect on COD (Chemical Oxygen Demand), ammonia nitrogen and total nitrogen in the sulfur-containing petrochemical wastewater, and also has good sulfur tolerance, broad spectrum of substrate and heavy metal resistance at the same time. The pseudomonas can remarkably reduce main pollution indexes of water and improve the treatment efficiency, shows huge application potential in the aspect of refining and chemical sewage treatment, and has a relatively good industrial application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of environmental microorganisms, and in particular to a Pseudomonas strain and a culture method and application thereof. Background Art

[0002] The petroleum and petrochemical industry is a key pillar of modern industry in the global economic landscape, starting from crude oil extraction, going through multiple links such as refining to the synthesis of chemical products. But at the same time, this process also produces a large amount of petroleum and petrochemical wastewater. With the continuous expansion of the scale of the petroleum and petrochemical industry and the increasingly complex production processes, the amount of wastewater discharged has shown an increasing trend year by year. This type of wastewater has complex components and contains a variety of difficult-to-degrade organic compounds. These pollutants are highly toxic and chemically stable, posing extremely severe challenges and threats to the ecological environment and human health, and have become a major problem that needs to be solved urgently.

[0003] Among the many pollutants in petroleum and petrochemical wastewater, the rising sulfide content has become a prominent problem. The crude oil extracted in some areas has a high sulfur content. The thermal cracking, catalytic reforming and other processes will cause the organic sulfides in the crude oil to decompose and generate inorganic sulfides such as hydrogen sulfide. At the same time, the desulfurization efficiency of some old production equipment and processes gradually decreases when dealing with high-sulfur crude oil, causing more sulfides to enter the subsequent production process and eventually enter the wastewater. The wastewater treatment facilities of some petroleum and petrochemical enterprises are relatively backward, and the treatment process is difficult to cope with the ever-changing wastewater quality, especially the limited removal capacity of sulfides, resulting in the inability to fully degrade and remove sulfides during the treatment process, causing the sulfide concentration in the discharged wastewater to remain high.

[0004] At present, a variety of technical means have been developed for the treatment of petroleum and petrochemical wastewater. The biological method uses the metabolism of microorganisms to degrade organic pollutants in wastewater. It has the advantages of low cost and environmental friendliness. It is one of the mainstream technologies for the treatment of petroleum and petrochemical wastewater. However, when facing high-concentration and difficult-to-degrade organic pollutants in petroleum and petrochemical wastewater, traditional biological treatment processes still have problems such as low treatment efficiency, long treatment cycle, and microorganisms being easily inactivated by shock. This is mainly because the organic pollutants in petroleum and petrochemical wastewater have complex structures, and it is difficult for ordinary microorganisms to effectively use them as carbon sources and energy, thus limiting the effectiveness of biological treatment processes.

[0005] Microorganisms have unique advantages and great potential in the degradation and transformation of environmental pollutants. Different types of microorganisms have diverse metabolic pathways and enzyme systems, enabling them to adapt to and degrade specific types of pollutants. Among various microbial groups, Pseudomonas sp. has attracted much attention due to its wide substrate adaptability, strong environmental tolerance, and rich metabolic diversity. Pseudomonas sp. can secrete various extracellular enzymes, such as lipase, protease, laccase, etc., which play a key role in the decomposition and transformation of organic pollutants. At the same time, Pseudomonas sp. also has good growth and reproduction ability and genetic plasticity, and can survive under complex and changeable environmental conditions and adapt to new pollutants through gene regulation. Previous studies have confirmed that Pseudomonas sp. has a certain ability to degrade petroleum hydrocarbon pollutants, but for the complex organic pollutant system in petrochemical wastewater, specific Pseudomonas sp. strains with stronger adaptability and high degradation ability still need to be screened and cultivated to meet the increasingly demanding requirements for petrochemical wastewater treatment, fill the deficiencies in treatment efficiency and effect of existing technologies, and provide strong technical support for the sustainable development of the petrochemical industry. Summary of the Invention

[0006] The present invention provides a Pseudomonas sp. strain, its cultivation method, and its application.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] On the one hand, the present invention provides a Pseudomonas sp. strain (Pseudomonas sp.) 320-7, which has been deposited in the General Microbiological Center of the National Committee for the Management of Microbiological Culture Collections. The deposit number is CGMCC No. 33204. Address of the deposit unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Deposit date: December 25, 2024.

[0009] The colony of the Pseudomonas sp. strain (Pseudomonas sp.) 320-7 is semi-transparent light yellow and irregularly round, with a moist and smooth surface.

[0010] The Pseudomonas sp. strain (Pseudomonas sp.) 320-7 was isolated from an activated sludge sample, and the activated sludge sample was taken from the biochemical tank of a petrochemical sewage treatment plant.

[0011] On the other hand, the present invention provides a cultivation method for the Pseudomonas sp. strain (Pseudomonas sp.) 320-7. After inoculating the activated sludge sample into a screening medium for screening, a Pseudomonas sp. strain is obtained through multiple purifications on a solid medium. The purified strain is then expanded and cultured in a liquid medium to obtain a bacterial suspension. The culture conditions are an appropriate pH of 6.5-7.5 and an appropriate temperature of 28-32°C.

[0012] Furthermore, the components of the screening medium are: NH 4 NO 3 1.0 g / L, KH 2 PO 4 0.5 g / L, MgSO 4 ·7H 2 O 0.5 g / L, NaCl 0.5 g / L, FeSO4·7H 2 O 0.01 g / L, petrochemical wastewater 1000 mL, adjust the pH to 6.5 - 7.5 with 1 mol / L sodium hydroxide, and sterilize at 121 °C for 30 min before use.

[0013] Furthermore, the components of the solid medium are: NH 4 NO 3 1.0 g / L, KH 2 PO 4 0.5 g / L, MgSO 4 ·7H 2 O 0.5 g / L, NaCl 0.5 g / L, FeSO4·7H 2 O 0.01 g / L, petrochemical wastewater 1000 mL, agar 15 g / L, adjust the pH to 6.5 - 7.5 with 1 mol / L sodium hydroxide, and sterilize at 121 °C for 30 min before use.

[0014] Furthermore, for the liquid medium for the enlarged culture of Pseudomonas, its components are: tryptone 5.0 g / L, yeast extract 2.5 g / L, NaCl 5.0 g / L, KH 2 PO 4 0.5 g / L, MgSO 4 ·7H 2 O 0.1 g / L, petrochemical wastewater 1000 mL, adjust the pH to 6.5 - 7.5 with 1 mol / L sodium hydroxide, and sterilize at 121 °C for 30 min before use.

[0015] The treatment of sulfide by the above-mentioned strain, the effects of different substrates and heavy metal resistance are determined by the strain sulfide treatment experiment, the strain different substrate broad-spectrum experiment, and the strain different heavy metal resistance experiment. Set the sulfide (S 2- ) concentrations at 40 mg / L, 80 mg / L, 120 mg / L, 160 mg / L, 200 mg / L, select ethylene, cyclohexane, toluene, phenol for the substrate broad-spectrum experiment for different substrates, and the strain heavy metal resistance experiment includes the effects of lead nitrate, copper sulfate, cadmium chloride, and zinc sulfate on the growth of the strain.

[0016] The present invention further provides an application of a Pseudomonas sp. strain 320-7 in treating petrochemical wastewater containing high concentrations of sulfide.

[0017] Further, the sulfur concentration in the petrochemical wastewater containing high concentrations of sulfide is 40-200 mg / L.

[0018] The present invention further provides an application of a Pseudomonas sp. strain in treating petrochemical wastewater containing heavy metals.

[0019] Further, the heavy metals in the petrochemical wastewater containing heavy metals include lead nitrate, zinc sulfate, and cadmium chloride.

[0020] The beneficial effects of the present invention compared with the prior art are as follows:

[0021] A Pseudomonas sp. strain 320-7 provided by the present invention is a strain with high sulfide tolerance obtained by directional screening, isolation, and purification from an activated sludge sample. This strain can grow using a variety of substrates, has diverse metabolic modes, can survive under different nutritional conditions, and can better adapt to environmental changes. The strain has strong resistance to a variety of heavy metals and can treat petrochemical wastewater containing heavy metals. During the treatment of petrochemical wastewater, the Pseudomonas sp. strain has a significant removal effect on COD, ammonia nitrogen, and total nitrogen in the water body, can overall improve the treatment efficiency of petrochemical wastewater, and has good prospects for industrial application. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 It is a colony photo of the Pseudomonas sp. strain 320-7;

[0024] Figure 2 An optical microscope photo of the Pseudomonas sp. strain 320-7;

[0025] Figure 3 It is a growth curve graph of the Pseudomonas sp. strain 320-7;

[0026] Figure 4Results of the tolerance experiment of Pseudomonas sp. 320-7 to different concentrations of sulfide;

[0027] Figure 5 Results of the utilization experiment of Pseudomonas sp. 320-7 on different substrates;

[0028] Figure 6 Results of the resistance experiment of Pseudomonas sp. 320-7 to different heavy metals;

[0029] Figure 7 Removal effects of COD by the control group and the experimental group in Example 6;

[0030] Figure 8 Removal effects of NH 3 -N by the control group and the experimental group in Example 6;

[0031] Figure 9 Removal effects of TN by the control group and the experimental group in Example 6;

[0032] Figure 10 Removal effects of COD by the control group and the experimental group in Example 7;

[0033] Figure 11 Removal effects of NH 3 -N by the control group and the experimental group in Example 7;

[0034] Figure 12 Removal effects of TN by the control group and the experimental group in Example 7. Detailed implementation manners

[0035] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] Example 1: Isolation and screening of strains

[0037] The Pseudomonas strain 320-7 was isolated and screened from the activated sludge sample in the biochemical pool of a certain petrochemical sewage treatment plant, specifically as follows:

[0038] Take 1 ml of the sludge sample and add it to sterile physiological saline, shake well to make a mud-water mixture. Using the gradient dilution method, the mixture was successively diluted to 10 -1 -10-8 Different gradients such as... were taken. 0.1 mL of each gradient dilution was taken and evenly spread on the solid medium plate containing petrochemical wastewater, and then cultured in a constant temperature incubator at 30 °C for 2 - 3 days. After the colonies grew out, preliminary screening was carried out.

[0039] Single colonies were picked and inoculated into the screening medium. After culturing for 48 h, the growth of the strains was measured. For the strains with good growth, screening and separation were carried out again. An appropriate amount of bacterial liquid was used for streak plating on the solid medium, and then sealed and inverted for constant temperature culture.

[0040] The components of the above screening medium are as follows: NH 4 NO 3 1.0 g / L, KH 2 PO 4 0.5 g / L, MgSO 4 ·7H 2 O 0.5 g / L, NaCl 0.5 g / L, FeSO4·7H 2 O 0.01 g / L, 1000 mL of petrochemical wastewater; The components of the solid medium for purifying the strain: NH 4 NO 3 1.0 g / L, KH 2 PO 4 0.5 g / L, MgSO 4 ·7H 2 O 0.5 g / L, NaCl 0.5 g / L, FeSO4·7H 2 O 0.01 g / L, 15 g / L of agar, 1000 mL of petrochemical wastewater.

[0041] The above steps were repeated multiple times, and the content of petrochemical wastewater in the medium could be increased during the screening process until strains with good growth were screened and isolated. Strain 320 - 7 was obtained. As Figure 1 shown, its colony characteristics are semi - transparent light yellow, irregular round, with a moist and smooth surface.

[0042] Example 2: Identification of the strain

[0043] The strain 320 - 7 obtained in Example 1 was subjected to Gram staining. According to the results of Gram staining, strain 320 - 7 is a Gram - negative bacterium, and its cell morphology is rod - shaped, arranged singly or in pairs, as Figure 2 shown;

[0044] This strain was enlarged and cultured in a liquid medium, and its growth curve is as Figure 3 shown. The components of the liquid medium: 5.0 g / L of tryptone, 2.5 g / L of yeast powder, 5.0 g / L of NaCl, KH 2 PO4 0.5 g / L, MgSO 4 ·7H 2 O 0.1 g / L, 1000 mL of petrochemical wastewater, adjust the pH to 6.5 - 7.5 with 1 mol / L sodium hydroxide, and sterilize the medium at 121 °C for 30 min before use.

[0045] After the cultivation, observe the growth of the bacterial solution. Transfer the bacterial solution to a centrifuge tube, centrifuge at 4000 rpm for 20 min to precipitate the bacteria. Discard the supernatant and collect the bacteria. Add an appropriate amount of PBS buffer to the centrifuge tube to resuspend the bacteria, then centrifuge again and repeat the washing 2 - 3 times to remove the medium components and impurities on the surface of the bacteria. Resuspend the washed bacteria with an appropriate amount of sterile glycerol, aliquot the bacterial solution into sterile cryotubes, 0.5 - 1 ml per tube, make good marks, and store in a -80 °C refrigerator for use in strain identification.

[0046] Through 16S rDNA sequencing and gene sequence alignment, this strain has the highest similarity with Pseudomonas veronii. Name this strain Pseudomonas sp. 320 - 7. Its 16S rDNA gene sequence is shown in the sequence as SEQ ID NO.1. Pseudomonas sp. 320 - 7 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on December 25, 2024, with the deposit number CGMCC No. 33204 and the address at No. 3, Yard 1, Beichen West Road, Beijing.

[0047] Example 3: Tolerance experiment of Pseudomonas sp. 320 - 7 to different concentrations of sulfide

[0048] Aliquot the liquid medium into multiple sterile conical flasks in equal volumes (the components are the same as those of the liquid medium in Example 1). Add different amounts of sulfide standard solution to each conical flask to make the sulfide concentration in the medium 40 mg / L, 80 mg / L, 120 mg / L, 160 mg / L, and 200 mg / L in sequence. Inoculate the Pseudomonas sp. 320 - 7 bacterial solution into the above media with different sulfide concentrations in the same volume (2%). The inoculation process strictly follows the aseptic operation specifications to avoid contamination.

[0049] Place the inoculated conical flasks in a constant temperature shaker, set the temperature to 30 °C and the rotation speed to 180 rpm for cultivation. During the cultivation, take an appropriate amount of bacterial solution (about 1 mL) from each conical flask every 6 h, transfer it to a cuvette, and measure the absorbance (OD 600 ) of the bacterial solution at a wavelength of 600 nm using an ultraviolet - visible spectrophotometer to characterize the growth of Pseudomonas sp. 320 - 7.

[0050] It can be seen from Figure 4 that as the culture time prolongs, the growth of Pseudomonas 320-7 under different concentrations of sulfide all shows an upward trend. Under the conditions of low-concentration sulfide (40 mg / L, 80 mg / L), the growth of Pseudomonas is good, and the OD 600 value of the strain at 48 hours is relatively high and still has an upward trend. As the sulfide concentration increases to 120 mg / L, the growth of Pseudomonas is inhibited to a certain extent, but it still has a good growth state. When the sulfide concentration reaches 160 mg / L and 200 mg / L, the growth of Pseudomonas is significantly inhibited, and the OD 600 value increases slowly and remains at a relatively low level within 48 hours. Generally speaking, Pseudomonas 320-7 has a certain tolerance to sulfide, and as the sulfide concentration increases, the inhibitory effect on its growth gradually strengthens.

[0051] Example 4: Utilization experiment of Pseudomonas sp. 320-7 on different substrates

[0052] Prepare a screening medium (the components are the same as those of the liquid medium in Example 1), weigh a certain amount of ethylene, cyclohexane, toluene, and phenol respectively, and add them to the medium through appropriate dissolution methods to prepare media with substrate concentrations of 5 mg / L, 10 mg / L, 20 mg / L, 40 mg / L, and 80 mg / L respectively. Each substrate-concentration combination is sub-packed into 150 mL conical flasks, 50 mL per flask. Inoculate the Pseudomonas 320-7 bacterial solution into the above media containing different substrates and concentrations in the same volume (2%), ensuring the asepticity of the inoculation operation. Place the inoculated conical flasks in a constant temperature shaker at 30 °C and 180 rpm for oscillating culture for 48 h. After culturing for 48 h, take 1 mL of the bacterial solution from each conical flask and measure the absorbance value (OD 600 ) of the bacterial solution using an ultraviolet-visible spectrophotometer.

[0053] As shown by the growth of Pseudomonas 320-7 under different substrates and concentrations Figure 5 , when ethylene is used as the substrate, the growth amount is good and the difference is small at low concentrations (5 mg / L and 10 mg / L). Although the growth amount decreases at high concentrations, the overall utilization ability still ranks first among several substrates, indicating that the bacterium has strong adaptability and utilization ability to ethylene, but high-concentration ethylene has a certain inhibitory effect on its growth. When cyclohexane is used as the substrate, the OD 600 value is overall lower than that of the ethylene group, and as the substrate concentration increases, the OD 600 value shows a downward trend, indicating that the utilization ability of Pseudomonas 320-7 for cyclohexane weakens with the increase in concentration. When toluene and phenol are used as substrates, at higher substrate concentrations (40 mg / L and 80 mg / L), the OD600 The value decreased significantly, and Pseudomonas 320-7 had poor utilization ability for toluene and phenol because these two substrates were highly toxic to this bacterium.

[0054] Generally speaking, the utilization ability of Pseudomonas 320-7 for different substrates was ethylene, cyclohexane, toluene, and phenol in sequence, and the influence of substrate concentration on its utilization ability varied with substrate types.

[0055] Example 5: Resistance experiment of Pseudomonas sp. strain 320-7 to different heavy metals

[0056] Weigh a certain amount of lead nitrate, cadmium chloride, zinc sulfate, and copper sulfate respectively, dissolve them and add them to the basic liquid medium to prepare media with metal concentrations of 10 mg / L, 20 mg / L, 40 mg / L, 80 mg / L, and 120 mg / L respectively. Each metal-concentration combination was aliquoted into 150 mL sterile conical flasks, 50 mL per flask. The bacterial solution of Pseudomonas 320-7 was inoculated into the above media containing different metals and concentrations in the same volume (2%), and the inoculated conical flasks were placed in a constant temperature shaker at 30 °C and 180 rpm for shaking culture for 48 h. During the culture process, conditions such as temperature and rotation speed were strictly controlled to ensure the consistency of the experiment. After 48 h of culture, take the bacterial solution to measure the absorbance value (OD 600 ).

[0057] From Figure 6 it can be seen that when lead nitrate was used as the metal stress factor, the OD 600 value of Pseudomonas 320-7 was relatively high at each concentration. Even when the metal concentration was as high as 120 mg / L, it still maintained a certain growth amount, indicating that this bacterium had strong resistance to lead nitrate. As the metal concentration of zinc sulfate increased, the decrease in the OD 600 value was relatively gentle, indicating that Pseudomonas 320-7 also had good resistance to zinc sulfate. When cadmium chloride was used as the metal stress factor, the OD 600 value was relatively low, and as the metal concentration increased from 10 mg / L to 120 mg / L, the decrease in the OD 600 value was relatively obvious, indicating that this bacterium had weak resistance to cadmium chloride. As the metal concentration of copper sulfate increased, the OD 600 value decreased sharply, especially at high concentrations (80 mg / L and 120 mg / L), and the OD 600 value approached 0, indicating that Pseudomonas 320-7 had the worst resistance to copper sulfate.

[0058] Example 6: Experiment 1 on treating the pretreated wastewater of a petrochemical wastewater treatment plant by Pseudomonas sp. strain 320-7

[0059] Take the pretreated wastewater from an oil and petrochemical sewage treatment plant and measure its initial water quality parameters (COD 466 mg / L, NH 3 -N 16.7 mg / L, TN 24.9 mg / L, sulfide 80 mg / L). Set up an experimental group and a control group. The experimental group is added with activated sludge and Pseudomonas sp. 320-7, and the control group is only added with activated sludge without adding strains. Place the wastewater of the experimental group and the control group in 250 mL conical flasks respectively, and adjust the pH to about 6.6-7.5. For the experimental group, add the strain according to the inoculation amount of 2%, and culture it by shaking in a constant temperature shaker at 30 °C and 160 rpm; the control group is placed under the same conditions but without adding the strain. During the treatment process, samples are taken from the experimental group and the control group every 12 hours to measure the water quality parameters of the samples (chemical oxygen demand COD, ammonia nitrogen NH 3 -N, total nitrogen TN).

[0060] The experimental results are as Figure 7 shown. From 12 hours to 48 hours, the COD removal rate of the control group slowly increased from 47% to 55%, only increasing by 8 percentage points within 48 hours, indicating that under natural conditions (control group), the ability of the strain to remove COD from oil and petrochemical wastewater improved relatively slowly. The COD removal rate of the experimental group rapidly increased from 57% at 12 hours to 76% at 48 hours, increasing by 19 percentage points within 48 hours, indicating that after adding the strain Pseudomonas sp. 320-7, the removal effect of COD was significantly improved, and the effect became more obvious over time.

[0061] As Figure 8 shown, the NH 3 -N removal rate of the control group gradually increased from 62% at 12 hours to 72% at 48 hours, increasing by 10 percentage points within 48 hours. Its increase range was slightly larger than that of the COD removal rate, but the overall upward trend was still relatively gentle. The NH 3 -N removal rate of the experimental group increased from 71% at 12 hours to 85% at 48 hours, increasing by 14 percentage points within 48 hours, showing that the strain had a strong and increasing ability to remove NH 3 -N.

[0062] As Figure 9 shown, the TN removal rate of the control group increased from 69% at 12 hours to 77% at 48 hours, increasing by 8 percentage points within 48 hours. Its change trend was similar to that of the COD removal rate. The TN removal rate of the experimental group increased from 81% at 12 hours to 87% at 48 hours, increasing by 6 percentage points within 48 hours. Although the increase range was relatively small, the initial removal rate was high and it always maintained a high removal rate.

[0063] It can be seen that the COD removal rate, NH3 The removal rates of -N and TN are both higher than those of the control group. When treating petrochemical wastewater with a sulfide concentration of 80 mg / L, the treatment effect of the experimental group is significantly better than that of the control group, indicating that the strain Pseudomonas 320-7 has a significant effect on the treatment of sulfur-containing petrochemical wastewater. This Pseudomonas strain has the potential for treating sulfur-containing petrochemical wastewater and can be applied to the treatment of wastewater containing high COD and nitrogen pollutants.

[0064] Example 7: Experiment II on treating the pretreated wastewater of a certain petrochemical wastewater treatment plant with Pseudomonas sp. 320-7

[0065] Take the pretreated wastewater from a certain petrochemical plant and measure its initial water quality parameters (COD 629 mg / L, NH 3 -N 32.7 mg / L, TN 58.4 mg / L, sulfide 120 mg / L). Set up an experimental group and a control group. The experimental group is added with activated sludge and Pseudomonas 320-7, and the control group is only added with activated sludge without adding the strain. Place the wastewater of the experimental group and the control group in 250 mL conical flasks respectively, and adjust the pH to about 6.5 - 7.5. For the experimental group, add the strain according to the inoculation amount of 3%, and oscillate and culture in a constant temperature shaker at 30 °C and 160 rpm; the control group is placed under the same conditions but without adding the strain. During the treatment process, samples are taken from the experimental group and the control group every 12 hours to measure the water quality parameters (COD, NH 3 -N, TN) of the samples.

[0066] The experimental results are as Figure 10 shown. From 12 hours to 48 hours, the COD removal rate of the control group slowly rises from 46% to 59%, only increasing by 13 percentage points within 48 hours, indicating that under natural conditions (the control group), the removal effect of the strain on COD in petrochemical wastewater is relatively slow. The COD removal rate of the experimental group rapidly rises from 63% at 12 hours to 82% at 48 hours, increasing by 19 percentage points within 48 hours, indicating that after adding the strain Pseudomonas 320-7, the removal effect on COD is significantly improved, and the effect becomes more obvious over time.

[0067] As Figure 11 shown, the NH 3 -N removal rate of the control group gradually rises from 61% at 12 hours to 71% at 48 hours, increasing by 10 percentage points within 48 hours, and the overall treatment effect trend is still relatively gentle. The NH 3 -N removal rate of the experimental group rises from 77% at 12 hours to 91% at 48 hours, increasing by 14 percentage points within 48 hours, showing that the strain has a strong and increasing ability to remove NH 3 -N.

[0068] As Figure 12 shown, the TN removal rate of the control group increased from 70% at 12 hours to 79% at 48 hours, an increase of 9 percentage points within 48 hours. The TN removal rate of the experimental group increased from 80% at 12 hours to 89% at 48 hours. Although the increase was relatively small, the starting point was high, and a relatively high TN removal rate was maintained throughout.

[0069] The data indicate that the traditional activated sludge has an unsatisfactory treatment effect under the influence of sulfide, while the strain Pseudomonas 320 - 7 has good application prospects in the treatment of sulfur - containing wastewater from the petroleum and petrochemical industries. In actual wastewater treatment projects, this strain can be considered to improve the treatment efficiency of sulfur - containing wastewater from the petroleum and petrochemical industries and reduce the environmental pollution caused by such wastewater. Compared with traditional treatment methods, using the strain Pseudomonas 320 - 7 has the advantages of low cost and good treatment effect.

[0070] In summary, the present invention provides a strain of Pseudomonas, which has the ability to efficiently treat petrochemical wastewater, and has good sulfur tolerance, substrate broad - spectrum characteristics, and heavy - metal resistance. At the same time, the present invention also relates to the cultivation method and application technology of this strain, aiming to reduce the treatment cost of petrochemical wastewater and environmental pollution through this efficient microbial technology, and provide a new solution for the sustainable development of the petrochemical industry.

[0071] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Pseudomonas sp. 320-7 strain, characterized in that: The bacteria is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No. 33204 and a deposit date of December 25, 2024. The 16SrDNA sequence of the Pseudomonas strain 320-7 is shown in SEQ ID NO.

1.

2. The method for culturing the Pseudomonas strain according to claim 1, characterized in that: The Pseudomonas strain is derived from an activated sludge sample from a petroleum and petrochemical wastewater treatment plant. The activated sludge sample is inoculated in a screening culture medium for screening, and then purified multiple times through a solid culture medium to obtain a Pseudomonas strain. The purified strain is expanded and cultured using a liquid culture medium to obtain a bacterial suspension. The culture conditions are a suitable pH of 6.5-7.5 and a suitable temperature of 28-32°C.

3. The culture method according to claim 2, characterized in that The screening medium comprises the following components: 1.0 g / L NH4NO3, 0.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L NaCl, 0.01 g / L FeSO4·7H2O, 1000 mL petrochemical wastewater, pH value adjusted to 6.5-7.5 with 1 mol / L sodium hydroxide, and sterilized at 121° C. for 30 min before use.

4. The culture method according to claim 2, characterized in that The solid culture medium comprises the following components: 1.0 g / L NH4NO3, 0.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L NaCl, 0.01 g / L FeSO4·7H2O, 1000 mL petrochemical wastewater, 15 g / L agar, pH value is adjusted to 6.5-7.5 with 1 mol / L sodium hydroxide, and the solid culture medium is sterilized at 121° C. for 30 min before use.

5. The culture method according to claim 2, characterized in that: The liquid culture medium for expanding the culture of Pseudomonas has the following culture medium components: 5.0 g / L tryptone, 2.5 g / L yeast powder, 5.0 g / L NaCl, 0.5 g / L KH2PO4, 0.1 g / L MgSO4·7H2O, 1000 mL petrochemical wastewater, pH value adjusted to 6.5-7.5 with 1 mol / L sodium hydroxide, and sterilized at 121°C for 30 min before use.

6. Use of the Pseudomonas strain according to claim 1 in treating petrochemical wastewater containing high concentration of sulfide.

7. The use of the Pseudomonas strain according to claim 6 in treating petrochemical wastewater containing high concentrations of sulfide, characterized in that: The sulfur concentration in the petrochemical wastewater containing high-concentration sulfides is 40-200 mg / L.

8. Use of the Pseudomonas strain according to claim 1 in treating petrochemical wastewater containing heavy metals.

9. The use of the Pseudomonas strain in treating high-salt refinery wastewater according to claim 8, characterized in that: The heavy metals in the heavy metal-containing petrochemical wastewater include lead nitrate, zinc sulfate and cadmium chloride.

Images