A Paracoccus pantotrophus strain and its applications

Abstract

The present invention belongs to the field of biotechnology, and provides a Paracoccus pantotrophus and its application. The Paracoccus pantotrophus ( Paracoccus pantotrophus ) YJY23-09 has a preservation number of CGMCC No. 26983. This strain can degrade trace organic nitrogen in seawater aquaculture wastewater and medical wastewater, filling the blank that existing biological treatment systems are difficult to degrade trace organic nitrogen, which is of great significance for the further harmless treatment of wastewater and will not cause secondary pollution to the environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and provides a Paracoccus pantotrophus and its application. Background Art

[0002] Total nitrogen is an index that is difficult to meet the standard during the wastewater treatment process. Total nitrogen mainly includes organic nitrogen and inorganic nitrogen. Inorganic nitrogen is easier to degrade than organic nitrogen. The organic nitrogen remaining in the wastewater after conventional biochemical treatment is at a trace level, generally not higher than 100 ppm, and its biodegradability is poor. Moreover, organic nitrogen belongs to organic matter and also has a certain impact on the COD index. The physical and chemical methods have poor treatment effects on this kind of low-concentration wastewater and high costs. In addition, due to the complex composition of the wastewater, it is difficult to distinguish the components of the remaining undegradable organic nitrogen by liquid chromatography-mass spectrometry. Therefore, it is also impossible to use a single substance for the directional screening of effective strains.

[0003] In recent years, with the development of the marine aquaculture industry, organic nitrogen pollution is a difficult point in the marine aquaculture industry. The high salt content and low C / N in seawater pose certain obstacles to biochemical treatment. Moreover, due to the large volume of seawater, the organic nitrogen is at a trace level like the above-mentioned wastewater, which also makes this part of organic nitrogen difficult to treat. Therefore, how to effectively treat the trace organic nitrogen in seawater and various existing wastewaters has become one of the problems that need to be solved urgently by those skilled in the art. Summary of the Invention

[0004] In view of the above problems existing in the technology, the present invention provides a Paracoccus pantotrophus and its application. The Paracoccus pantotrophus ( Paracoccus pantotrophus ) YJY23-09, with the preservation number of CGMCC No. 26983. This strain can degrade the trace organic nitrogen in seawater and medical wastewater, filling the blank that the existing biological treatment system is difficult to degrade trace organic nitrogen, which has important significance for the further harmless treatment of wastewater and will not cause secondary pollution to the environment.

[0005] The specific technical solution provided by the present invention is as follows:

[0006] First, 6 strains with the effect of degrading trace organic nitrogen were isolated from the aerobic tank and anoxic tank of a wastewater treatment company in Weifang, Shandong, and were respectively isolated and purified. Finally, a strain with high trace degradation efficiency, easy to culture and stable subculture characteristics was screened out, and it was named YJY23-09. Its morphological characteristics are as follows: there is a thick capsule around the bacterial cells, the spore wall is thick, oval, central or subterminal, and the sporangium does not expand or slightly expands; the colony is round, colorless, raised, gelatinous and viscous, transparent or semi-transparent, with a neat edge; it grows aerobically or facultatively anaerobically. When growing in LB liquid medium, white insoluble particles will appear at the bottom of the bottle.

[0007] The 16S rDNA sequence of this strain was subjected to BLAST alignment. The results showed that the nucleotide sequence of the 16S rDNA of this strain had a homology of more than 99% with the nucleotide sequences of different strains of the genus Paracoccus ( Paracoccus Davis ), and a homology of 100% with the strain clearly labeled as Paracoccus pantotrophus. Therefore, it was identified as Paracoccus pantotrophus( Paracoccus pantotrophus ). The inventor deposited it in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms in March 2023, and the deposit number was CGMCC No. 26983.

[0008] After experiments, the inventor found that this strain had good degradation characteristics for trace organic nitrogen, and would not cause secondary pollution to the environment, improving the treatment efficiency of trace organic nitrogen in wastewater, and the content of organic nitrogen in the wastewater was ≤ 100 ppm; the wastewater was seawater aquaculture wastewater or medical wastewater.

[0009] After obtaining the above strain, the inventor also provided a corresponding method for producing microbial agents, including the following steps:

[0010] (1) Strain activation: Take out the preserved strain and activate it at room temperature for 3h - 4h. Since the strain is preserved on a test tube slant nutrient agar medium, no additional nutrients need to be added during the activation process;

[0011] (2) Seed liquid preparation: In a sterilized aseptic workbench, add 10 ml of sterilized distilled water to the test tube containing the preserved bacteria, and repeatedly pipette to make a bacterial suspension. Then, transfer the bacterial suspension into 100 ml of sterilized LB liquid medium and shake culture for 16 - 20 h to prepare the seed liquid;

[0012] (3) Fermentation: Add the fermentation medium to the fermenter. After sterilization, inoculate the seed liquid at a volume ratio of 2‰. During the fermentation process, control the temperature at 30 - 35 °C, the tank pressure at 0.02 - 0.05 MPa, the initial rotation speed at 200 rpm, the dissolved oxygen ≥ 20%, and the gas - liquid ratio at 1:1; when the dissolved oxygen drops to 10 - 20% and the pH rises to 7.0 - 8.0, the fermentation is completed, and the effective viable bacteria count in the fermentation broth is 2 - 3 billion / ml.

[0013] The above sterilization method is preferably sterilization at 121 °C for 0.5 hours.

[0014] The fermentation medium formula is composed by weight percentage as follows: corn flour 1.5 - 2.5%, maltose 0.8 - 1.5%, dry corn steep liquor 2 - 3%, soybean meal 1.5 - 2.5%, urea 0.1 - 0.2%, magnesium sulfate 0.02 - 0.04%, sodium chloride 0.1 - 0.3%, potassium dihydrogen phosphate 0.2 - 0.4%, manganese sulfate 0.002 - 0.004%, pH 7.2 - 7.4, polyether defoamer 0.1%, and the balance is water.

[0015] During application, the bacterial agent obtained by the above fermentation can be directly added to the aerobic tank of sewage treatment. After adding the above bacterial agent, the initial effective viable bacterial count in the aerobic tank is 10 5 -10 6 cfu / ml. The treatment conditions of the aerobic tank are as follows: temperature 31 - 37°C, preferably 33°C; pH 6 - 8, preferably pH 7; dissolved oxygen content 2 - 4 mg / L, preferably 4 mg / L.

[0016] Among them, in the aerobic tank of sewage treatment, it is necessary to meet the conditions that organic nitrogen ≤ 300 ppm and B / C ≤ 0.3, so as to enable the bacterial agent to play a better role. After meeting the above conditions, 3 - 5 days after adding the bacterial agent, the organic nitrogen in the aerobic tank of sewage treatment can be removed by 100%.

[0017] Compared with the prior art, the beneficial effects of this application are as follows:

[0018] The bacterial agent prepared by using the strain provided by the present invention is applied to the biological treatment of pharmaceutical wastewater containing trace organic nitrogen. The strain of the present invention has very good degradation characteristics for trace organic nitrogen in pharmaceutical wastewater, and the degradation amount of trace organic nitrogen below 100 mg / L in pharmaceutical wastewater reaches 100%. By using the strain and method of the present invention, the treatment efficiency of trace organic nitrogen in pharmaceutical wastewater is significantly improved, the impact of trace organic nitrogen on the pharmaceutical wastewater treatment system can be significantly alleviated, and the wastewater meets the discharge standards.

[0019] The inventor has preserved the above strain, and the preservation information is as follows:

[0020] Preservation time: March 31, 2023

[0021] Name of the preservation unit: General Microbiology Center of China Microbial Culture Collection Management Committee

[0022] Preservation number: CGMCC No.26983

[0023] Address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0024] Taxonomic name: Paracoccus pantotrophus ( Paracoccus pantotrophus ). Description of the Drawings

[0025] Figure 1 It is a schematic diagram of the pH screening experiment result in Example 3;

[0026] Figure 2 It is a schematic diagram of the temperature screening experiment result in Example 3;

[0027] Figure 3 It is a degradation curve graph of the bacterial agent for trace organic nitrogen in pharmaceutical wastewater in Example 5;

[0028] Figure 4 It is the degradation curve of the microorganic nitrogen in the seawater aquaculture wastewater by the microbial agent in Example 7. Specific Embodiments

[0029] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Unless otherwise specified, all conventional prior arts are used in the following examples, and the bacterial strains used in the following examples are all bacterial strains with the preservation number CGMCC No. 26983. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0030] Example 1 Obtaining of Strains:

[0031] Six strains of bacteria with the effect of degrading microorganic nitrogen with a content of less than 100 mg / L were isolated from the aerobic tank and anoxic tank of a wastewater treatment company in Weifang, Shandong, and were separately isolated and purified.

[0032] Taking the aerobic effluent (organic nitrogen content less than 100 mg / L) of a wastewater treatment company in Weifang, Shandong as the experimental object, subsequent studies on the degradation of microorganic nitrogen by the six candidate strains were carried out respectively. Finally, a strain with high degradation efficiency, easy to culture and stable subculture characteristics was screened out, and it was named YJY23-09.

[0033] Morphological characteristics of this strain: There is a thick capsule around the bacterial cells, the spore wall is thick, oval in shape, central or subterminal, and the sporangium does not expand or slightly expands; the colony is round, colorless, raised, gelatinous and viscous, transparent or semi-transparent, with a neat edge; it grows aerobically or facultatively anaerobically. When growing in LB liquid medium, white insoluble particles will appear at the bottom of the flask.

[0034] The inventor carried out 16S rDNA sequencing on it, and the obtained 16S rDNA sequence was subjected to BLAST alignment. The alignment results showed that the nucleotide sequence of the 16S rDNA of this strain had more than 99% homology with the nucleotide sequences of different strains of the genus Paracoccus ( Paracoccus Davis ), and had 100% homology with the strain clearly labeled as Paracoccus pantotrophus ( Paracoccus pantotrophus ).

[0035] The inventor carried out biological preservation on it, and its biological preservation number is CGMCC No. 26983.

[0036] Example 2 Preparation of Paracoccus pantotrophus Microbial Agent:

[0037] (1)Activation of strains: Take out the preserved strains and activate them at room temperature for 3h - 4h. Since the strains are preserved on the slant nutrient agar medium in test tubes, no additional nutrients need to be added during the activation process.

[0038] (2)Preparation of seed liquid: In a sterilized laminar flow hood, add 10 ml of sterilized distilled water to the test tube containing the preserved bacteria, and repeatedly pipette to make a bacterial suspension. Then, transfer the bacterial suspension into 100 ml of sterilized conventional LB liquid medium and shake culture for 16 - 20 h to prepare the seed liquid;

[0039] (3)Fermentation: Add the fermentation medium into the fermenter. After sterilization, inoculate the seed liquid at a volume ratio of 2‰. During the fermentation process, control the temperature at 35°C, the tank pressure at 0.05 MPa, the initial rotation speed at 200 rpm, the dissolved oxygen ≥ 20%, and the gas - liquid ratio at 1:1; When the dissolved oxygen drops to 20% and the pH rises to 8.0, the fermentation is completed; The number of effective viable bacteria in the obtained fermentation broth is about 2 billion / ml.

[0040] The sterilization method is preferably sterilization at 121°C for 0.5 hours.

[0041] The formula of the fermentation medium in step (3) is as follows: by weight (w / w): corn flour 2.5%, maltose 1.5%, corn steep liquor dry powder 3%, soybean meal 2.5%, urea 0.2%, magnesium sulfate 0.04%, sodium chloride 0.3%, potassium dihydrogen phosphate 0.4%, manganese sulfate 0.004%, polyether defoamer 0.1%, the balance is water, and the pH is 7.2.

[0042] Example 3 Screening of conditions for the degradation of trace organic nitrogen in water by the bacterial agent:

[0043] Use the fermentation broth obtained in Example 2 to conduct the following experiments:

[0044] ① pH screening experiment

[0045] Take 50 ml of aerobic effluent from a wastewater treatment company in Shandong, and detect its organic nitrogen content as 83.25 mg / L. Measure 4% of the bacterial liquid by volume percentage. After centrifuging the bacterial liquid, add the centrifuged bacterial cells to the above - mentioned aerobic effluent. Set the system pH to 4.0, 5.0, 6.0, 7.0, and 8.0 respectively, and shake - culture at 30°C and 180 rpm for 24 h to determine the remaining organic nitrogen, and set a blank control at the same time.

[0046] The results are as Figure 1 shown. When the pH is 7.0, the degradation effect of the strain on trace organic nitrogen is the best, and the degradation rate reaches 100% in 24 h.

[0047] ② Temperature screening experiment

[0048] The experiment was carried out according to the above pH screening experiment, except that the temperatures were set at 24 °C, 27 °C, 30 °C, 33 °C, and 36 °C respectively, pH 7.0, and shaken at 180 rpm for 24 h for the determination of the remaining organic nitrogen, and a blank control was set at the same time.

[0049] The results are as Figure 2 shown. The degradation effect of the strain on trace organic nitrogen is the best at 33 °C, and the degradation rate reaches 100% in 24 h.

[0050] Example 4 Verification of the strain effect:

[0051] Pick a single colony of the preserved strain and inoculate it into 100 ml of sterile LB liquid medium in a laminar flow hood, shake culture at 35 °C and 180 rpm for 16 - 20 h. Measure 4% of the cultured bacterial liquid by volume percentage. After centrifugation of the bacterial liquid, take the centrifuged cells and inoculate them into 50 ml of the aerobic effluent of a wastewater treatment company in Shandong. Adjust the pH to 7.0, place the flask in a constant temperature shaker at 33 °C and 180 r / min for 24 h, set a blank control at the same time, sample and detect the mass concentration of the remaining organic nitrogen in the flask, and calculate the degradation rate of the strain on organic nitrogen. The results are shown in Table 1. The degradation rate of strain YJY23 - 09 on the solution containing 91.55 mg / L reaches 100% in 24 h.

[0052] Table 1 Verification of the degradation effect of the strain on trace organic nitrogen

[0053] 。

[0054] Example 5 Application of the strain in the treatment of a pharmaceutical wastewater in Shandong:

[0055] Due to the influence of the remaining trace organic nitrogen in the wastewater on the system in a pharmaceutical wastewater treatment company in Shandong, the wastewater could not meet the discharge standards. The bacterial liquid after fermentation of the strain (the same as in Example 2) was added to the aerobic tank at a ratio of one ten - thousandth by volume for verification of the organic nitrogen degradation effect. After detection, the initial bacterial count of the trace organic nitrogen - degrading bacteria in the aerobic tank was about 10 6 cfu / ml, the temperature of the aerobic tank was 31 - 33 °C, the dissolved oxygen was 3.5 - 4 mg / L, the pH was 6.8 - 7.0, the hydraulic retention time was 12 h, and the organic nitrogen content in the system was tracked and detected every day; at the same time, the aerobic tank without adding the bactericide was used as a control.

[0056] The initial concentrations of organic nitrogen in both tanks were adjusted to 125.35 mg / L, and the treatment results are as Figure 3As shown in the figure, the content of organic nitrogen in the aerobic tank of the experimental group with the addition of bacterial agents showed a linear downward trend. The content of organic nitrogen was 0 mg / L on the 6th day, and the degradation rate reached 100%. While the content of organic nitrogen in the control tank remained basically flat, showing no degradation effect on the organic nitrogen in the wastewater. It can be seen that the strain provided by the present invention has an obvious degradation effect on trace organic nitrogen in pharmaceutical wastewater.

[0057] Example 6: Application of the strain in the treatment of a pharmaceutical wastewater in Jiangsu:

[0058] A pharmaceutical wastewater treatment company in Jiangsu still had 58.31 mg / L of organic nitrogen in the treated wastewater, which affected the normal discharge of the wastewater. The bacterial liquid after fermentation of the strain (the same as in Example 2) was added to the aerobic tank at a ratio of one ten-thousandth by volume. The initial effective bacteria amount was detected to be about 10 5 cfu / ml, the temperature was 31 - 33 °C, pH was 7.0 - 7.2, dissolved oxygen was 3.8 - 4 mg / L, and the hydraulic retention time was 15 h. The remaining organic nitrogen content in the aerobic effluent was tracked and detected every day. The results showed that the organic nitrogen content in the aerobic tank decreased from the initial 58.31 mg / L to 0 mg / L after 72 h, and the effluent index returned to stability. The strain greatly alleviated the impact of trace organic nitrogen on the wastewater treatment system.

[0059] Example 7: Application of the strain in the treatment of a seawater aquaculture wastewater in Lianyungang, Jiangsu:

[0060] After pretreatment, a seawater aquaculture wastewater in Lianyungang, Jiangsu still had 220 mg / L of residual organic nitrogen, which affected the recycling of the aquaculture water and would be toxic to the aquaculture organisms. In the experimental group, the bacterial liquid after fermentation of the strain (the same as in Example 2) was added to the wastewater treatment system at a ratio of two ten-thousandths by volume. The initial effective bacteria amount was detected to be about 10 6 cfu / ml, the temperature was 32 - 33 °C, pH was 7.0 - 7.5, dissolved oxygen was 3.5 - 4 mg / L, and the hydraulic retention time was 10 h. The remaining organic nitrogen content in the system effluent was tracked and detected every day; at the same time, a wastewater treatment system without adding bacterial agents was used as the control group.

[0061] Figure 4 The results showed that the organic nitrogen content in the effluent of the experimental group system decreased from the initial 220 mg / L to 0 after 5 days, and the effluent index was stable, ensuring the recycling of the aquaculture wastewater, while the organic nitrogen content in the control group did not change significantly.

[0062] The above embodiments facilitate those skilled in the art to better understand the present invention. The implementation manners of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A strain of Paracoccus pantrophus ( Paracoccus pantotrophus )YJY23-09, characterized by: The deposit number is CGMCC No. 26983, and it was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on March 31, 2023.

2. The use of the Paracoccus pantotrophicus according to claim 1 in degrading organic nitrogen in wastewater, characterized in that: The organic nitrogen content in the wastewater is ≤100ppm, and the wastewater is marine aquaculture wastewater or medical wastewater.

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