Mineralizing bacterium, application thereof and preparation method of struvite

Abstract

The invention relates to the technical field of biology, in particular to an organophosphorus mineralizing bacterium and application thereof. According to the invention, an organophosphorus mineralizing bacterium is screened from activated sludge of a sewage treatment plant and is identified as Serratia sp. AS, and the preservation number of the Serratia sp. AS is CGMCC (China General Microbiological Culture Collection Center) NO.33590. The strain can efficiently recover nitrogen and phosphorus in wastewater to generate struvite, and alleviate and / or eliminate eutrophication of a water body. Meanwhile, the compound has relatively high tolerance to various heavy metal ions (Cu < 2 + >, Zn < 2 + >, Pb < 2 + >, Ni < 2 + > and Cd < 2 + >), and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a mineralizing bacterium, its application, and a preparation method of struvite. Background Art

[0002] The discharge of wastewater rich in phosphorus and nitrogen not only causes water eutrophication but also results in serious loss of nitrogen and phosphorus. At the same time, phosphorus, as a non-renewable resource, plays an irreplaceable role in global food production. Therefore, the lack of phosphorus will threaten the survival and development of humanity. Currently, the global phosphorus utilization efficiency can only reach 20%, and the remaining phosphorus enters wastewater or surface water, resulting in a large amount of loss. Relevant research shows that theoretically, recovering phosphorus from domestic sewage alone can meet 15 - 20% of the global phosphorus demand. To simultaneously solve the problems of phosphorus deficiency and water eutrophication, the struvite (MgNH4PO4·6H2O) crystallization method has emerged as the times require.

[0003] The struvite crystallization method can simultaneously recover phosphorus and nitrogen in wastewater, and the generated struvite can also be reused as a slow-release fertilizer. However, the struvite crystallization method requires adding an alkali source such as NaOH to the sewage to adjust the pH to an appropriate range to promote struvite crystallization, which greatly increases the recovery cost. In addition, the struvite crystallization method can only recover dissolved inorganic phosphorus in sewage and cannot recover dissolved organic phosphorus. In fact, dissolved organic phosphorus is more common in aquaculture and agricultural wastewater, accounting for 25 - 80% of the total phosphorus in wastewater. For this reason, researchers have proposed the method of microbial mineralization of struvite, that is, using microorganisms to metabolize phosphorus- and nitrogen-containing organic matter in sewage by themselves, releasing ammonium ions and phosphate ions, while increasing the pH of the system and promoting the precipitation of struvite. This method can overcome the problems of high cost and inability to treat organic nitrogen and phosphorus in the struvite crystallization method. Therefore, microbial mineralization of struvite is considered to be a potentially ideal way to recover nitrogen and phosphorus from sewage. In recent years, there have also been patent applications for recovering phosphorus using microbial mineralization of struvite. For example, Chinese Patent Application No. 201610476479.5, "A method for removing nitrogen and phosphorus from sewage and recovering struvite using Shewanella", Chinese Patent Application No. 201810296839.2, "A method for recovering struvite from wastewater", etc. However, there are a variety of heavy metal ions widely present in sewage, such as Pb 2+ 、Zn 2+ 、Cu 2+ 、Cd 2+ 、Ni 2+ etc. These heavy metal ions can bind to enzymes, destroy the structure and function of enzymes, induce the production of high-concentration reactive oxygen species to oxidize DNA, lipids, and proteins, and have an adverse impact on the growth and metabolism of microorganisms, thereby possibly reducing the yield of struvite and the phosphorus recovery efficiency. Therefore, isolating and screening heavy metal-tolerant organic phosphorus mineralizing bacteria and using them for struvite mineralization will be beneficial to expanding the application prospect of microbial mineralization of struvite for phosphorus recovery. Summary of the Invention

[0004] In view of this, the present invention provides a mineralizing bacterium, its application, and a method for preparing struvite. This strain is an organophosphorus mineralizing bacterium tolerant to heavy metals, which can mineralize nitrogen and phosphorus to produce struvite under the condition of the presence of various heavy metal ions.

[0005] A strain of bacterium AS was screened from activated sludge in the present invention, and it was identified as Serratia sp. by morphology and 16S rDNA, and its preservation number is CGMCC NO. 33590.

[0006] Experiments show that the metabolic activity of Serratia sp. AS provides the necessary environmental conditions for struvite precipitation, and nitrogen and phosphorus in wastewater are removed and recovered by precipitating struvite, realizing the recovery of struvite and effectively alleviating and eliminating water eutrophication.

[0007] The present invention also provides a bacterial agent including the above-mentioned Serratia sp. AS.

[0008] In some embodiments, the bacterial agent provided by the present invention further includes acceptable auxiliaries. The auxiliaries are commercially available products, and the present invention has no special restrictions on the specific types of auxiliaries, and those commonly used or common in the art can be used.

[0009] In some embodiments, the dosage forms of the bacterial agent include one or more of powder, liquid, or granule.

[0010] The present invention also provides the application of the above-mentioned Serratia sp. or the microbial bacterial agent in at least one of the following aspects:

[0011] Recycling nitrogen and phosphorus to prepare struvite;

[0012] Alleviating and / or eliminating water eutrophication;

[0013] Adsorbing and / or tolerating heavy metal ions.

[0014] In some embodiments, the present invention finds that strain AS has the function of adsorbing and / or tolerating heavy metal ions. The heavy metal ions include heavy metal ions in environments such as sewage, sludge, and / or soil.

[0015] In some embodiments, the heavy metal ions specifically include: Ni 2+ , Cd 2+ , Cu 2+ , Pb 2+ , Zn 2+ and at least one of them.

[0016] Research shows that the tolerance concentration of the strain AS of the present invention to heavy metal ions is: Pb 2+The concentration of Zn is 100 - 2000 mg / L; 2+ The concentration of Cu is 100 - 1200 mg / L, 2+ The concentration of Cd is 100 - 400 mg / L, 2+ The concentration of Ni is 100 - 400 mg / L, 2+ The concentration of which is 100 - 400 mg / L.

[0017] Research shows that the strain AS of the present invention has the function of recovering nitrogen and / or phosphorus to generate struvite. Among them, the nitrogen includes organic nitrogen; the phosphorus includes organic phosphorus.

[0018] The present invention also provides a method for preparing struvite, including: mixing the Serratia marcescens or the bacterial agent of the present invention with a sample containing nitrogen, phosphorus and magnesium, and reacting to obtain struvite.

[0019] The present invention also provides a sewage treatment method, including: adding the Serratia marcescens or the bacterial agent of the present invention to sewage.

[0020] Experiments show that the Serratia marcescens AS of the present invention has high tolerance to various heavy metal ions (Cu 2+ , Zn 2+ , Pb 2+ , Ni 2+ and Cd 2+ ), and the strain AS can efficiently mineralize struvite in a simulated wastewater containing only organic nitrogen and phosphorus such as peptone and yeast powder, and the conversion efficiency of magnesium ions is as high as 80%. It overcomes the limitation that the traditional struvite crystallization process can only treat inorganic nitrogen and phosphorus, and realizes more effective nitrogen and phosphorus removal and struvite recovery. It shows that the Serratia marcescens AS has strong heavy metal tolerance, can not only mineralize and recover struvite from sewage, but also be used to treat eutrophic water bodies.

[0021] Biological deposit description

[0022] Biological material: AS, classified and named: Serratia sp., deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 21, 2025, at the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC NO. 33590. Description of the drawings

[0023] Figure 1 It is the SEM photograph of the strain AS after culturing for 2 days in Example 1;

[0024] Figure 2 It is the 16S rDNA gene phylogenetic tree of the strain AS in Example 1;

[0025] Figure 3 XRD pattern (a) and SEM photograph (b) of the product obtained after culturing for 6 days in Example 3. Detailed implementation manners

[0026] The present invention provides a mineralizing bacterium, its application, and a method for preparing struvite. Those skilled in the art can draw on the content of this article and appropriately modify process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0027] In the present invention, among the technical features described in an open-ended manner (such as including, containing, having, etc.), it includes a closed technical solution composed of the listed features, and also includes an open technical solution containing the listed features.

[0028] The term "and / or" used in this article includes any and all combinations of one or more of the related listed items.

[0029] In the present invention, only some numerical ranges are specifically disclosed. However, any lower limit can be combined with any upper limit to form a range not explicitly recorded; and any lower limit can be combined with other lower limits to form a range not explicitly recorded, and similarly any upper limit can be combined with any other upper limit to form a range not explicitly recorded. In addition, each individually disclosed point or single numerical value itself can be used as a lower limit or an upper limit to be combined with any other point or single numerical value or with other lower limits or upper limits to form a range not explicitly recorded.

[0030] The Serratia marcescens AS of the present invention was obtained by separating, screening, and purifying from the activated sludge of Wangtang Wastewater Treatment Plant in Hefei, Anhui.

[0031] The experimental methods involved in the present invention are specifically as follows:

[0032] Isolation, screening, and purification of bacteria: Take a small amount of activated sludge sample and add it to a culture flask containing 100 mL of sterile water, culture it on a shaker at a temperature of 30 °C and a rotation speed of 200 rpm for 48 h, take 1 mL of the supernatant and add it to a test tube containing 9 mL of sterile water. This is the 10 -1 concentration, and then sequentially dilute it to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 concentration gradients. Select the concentrations of 10 -5 , 10 -6 , 10-7 The dilution solution was spread on an LB solid medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 g / L agar powder) containing 80 mg / L Cu 2+ and cultured in an incubator at 30 °C. After 48 h, 3 single colonies on the plate were selected and streaked on fresh LB solid medium containing 80 mg / L Cu 2+ and cultured in an incubator at 37 °C. The bacteria were separated and purified by streaking multiple times. The 3 strains of bacteria were respectively inoculated into LB solid media with different Cu 2+ concentrations (200 mg / L, 400, 600 mg / L) and cultured. After culturing at 30 °C for 48 h, the bacteria with the highest copper tolerance were found and named AS. Finally, the strain AS was stored in a cryogenic refrigerator using the glycerol freezing preservation method.

[0033] Activation of bacteria: The bacteria stored in the cryogenic refrigerator were thawed in a 4 °C refrigerator and inoculated into a culture flask containing 100 mL of LB medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl). The mixture was aerobically cultured on a shaker at 30 °C and 200 rpm for 24 - 48 hours to obtain the required activated bacterial liquid.

[0034] Determination of the minimum inhibitory concentration: That is, the lowest concentration at which bacteria do not grow at all in a medium containing heavy metals. Prepare heavy metal solutions containing Pb 2+ (lead nitrate), Zn 2+ (zinc sulfate), Cu 2+ (copper sulfate), Cd 2+ (cadmium chloride), Ni 2+ (nickel chloride) at 10 g / L. In a laminar flow hood, they were added to the LB solid medium by filtration sterilization to prepare heavy metal media with gradient concentrations of Pb 2+ (100 - 2000 mg / L), Zn 2+ (100 - 2000 mg / L), Cu 2+ (100 - 400 mg / L), Cd 2+ (100 - 400 mg / L), Ni 2+ (100 - 400 mg / L). Then the activated bacterial liquid was spread on the above media and cultured in an incubator at 37 °C for 48 - 72 h to observe the growth of the strains.

[0035] Bacterial synthesis of struvite: Add 100 mL of LB medium to a 250 mL culture flask, autoclave at 121 °C for 20 minutes, and then add 1 mL of 0.4 mol / L magnesium chloride solution by filtration sterilization. Then inoculate the activated bacterial solution into the above medium containing magnesium salt, and perform aerobic culture on a shaker at a rotation speed of 200 rpm and a temperature of 30 °C. Periodically check the culture flask to observe and record the precipitation. At the end of the culture, sediment and separate the product, then wash the product three times with absolute ethanol, and place it in a vacuum drying oven at room temperature for 48 hours. Scanning electron microscopy (SEM) observation of the product found a large number of crystals. The results of X-ray diffraction (XRD) showed that the product was a pure phase of struvite. Calculate the utilization rate of magnesium based on the yield of struvite and the dosage of magnesium added.

[0036] All the test materials used in the present invention are ordinary commercially available products and can be purchased in the market.

[0037] The present invention will be further described below in conjunction with embodiments:

[0038] Example 1

[0039] Take 1 g of activated sludge sample and add it to a culture flask containing 100 mL of sterile water, culture it on a shaker at a temperature of 30 °C and a rotation speed of 200 rpm for 48 h, take 1 mL of the supernatant and add it to a test tube containing 9 mL of sterile water, this is the 10 -1 concentration, and then sequentially dilute it to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 concentration gradients. Select the dilutions with concentrations of 10 -5 , 10 -6 , 10 -7 and coat them on LB solid medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 g / L agar powder) containing 80 mg / L Cu 2+ , and place it in an incubator at 30 °C for culture. After 48 h, select 3 single colonies on the plate and streak them on fresh LB solid medium containing 80 mg / L Cu 2+ , and culture them in an incubator at 37 °C. Streak and separate the bacteria multiple times. Inoculate these 3 strains of bacteria into different Cu 2+Cultured in LB solid medium with concentrations (200 mg / L, 400, 600 mg / L), after culturing at a constant temperature of 37 °C for 48 h, the bacteria with the highest tolerance to copper concentration were found and named AS. Finally, the strain AS was stored in an ultra-low temperature refrigerator using the glycerol cryopreservation method. The scanning electron microscopy results of the strain AS showed that the bacterial cells were rod-shaped, with a diameter of 0.5 - 0.8 μm and a length of 0.7 - 2.0 μm, as Figure 1 .

[0040] The 16S rDNA sequence of the strain AS was subjected to Blast alignment and homology analysis with the 16S rDNA sequences of other bacteria in the GenBank database. The results showed that the strain had the highest homology with the genus Serratia, with a similarity of over 99%. Thus, the strain was determined to be of the genus Serratia. Using the MEGA11 software, a phylogenetic tree of the strain AS was constructed by the neighbor-joining method, as Figure 2 shown.

[0041] Example 2

[0042] Prepared a heavy metal solution containing Pb 2+ (lead nitrate), Zn 2+ (zinc sulfate), Cu 2+ (copper sulfate), Cd 2+ (cadmium chloride), Ni 2+ (nickel chloride) at 10 g / L. In a laminar flow hood, it was added to the LB solid medium by filtration sterilization to prepare a heavy metal medium with gradient concentrations of Pb 2+ (100 - 2000 mg / L), Zn 2+ (100 - 1200 mg / L), Cu 2+ (100 - 400 mg / L), Cd 2+ (100 - 400 mg / L), Ni 2+ (100 - 400 mg / L). Then, the activated bacterial solution was spread on the above medium and placed in an incubator at a constant temperature of 37 °C for 48 - 72 h to observe the growth of the strain. The results showed that Serratia could tolerate multiple heavy metal ions at the above concentrations (Pb 2+ , Zn 2+ , Cu 2+ , Cd 2+ , Ni 2+ ). Beyond the above concentrations, the growth of the strain was inhibited. The order of inhibitory effects of these five heavy metal ions on Serratia was Ni 2+ >Cd 2+ >Cu 2+ >Pb 2+ >Zn 2+ , as shown in Table 1.

[0043] Table 1 Minimum inhibitory concentration of different heavy metal ions on strain AS

[0044]

[0045] Example 3

[0046] Add 100 mL of LB medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl) into a 250 mL conical flask, sterilize it at 121 °C under high pressure for 20 minutes, and then add 1 mL of 0.4 mol / L magnesium chloride solution by membrane filtration sterilization. Then inoculate 1 mL of bacterial solution into the above culture medium, and perform aerobic culture on a shaker at a rotation speed of 200 rpm and a temperature of 30 °C. After culturing for 6 days, sediment and separate the obtained product, then wash the product three times with absolute ethanol, and place it in a vacuum drying oven to dry at room temperature for 48 hours. The X-ray diffraction results show that the product is struvite, as Figure 3 shown in a. The scanning electron microscope results show that the product struvite presents micron-sized irregular block crystals, as Figure 3 shown in b. Characterize and analyze the results of three parallel experiments, and determine that the average yield of struvite obtained is 803 mg / L, and the conversion rate of magnesium is 81.9%. Since no additional inorganic nitrogen and inorganic phosphorus are added, it shows that Serratia can convert organic nitrogen and organic phosphorus into struvite.

[0047] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Serratia sp. with deposit number CGMCC NO. 33590.

2. Bacterial agent, characterized in that, Including the Serratia sp. described in claim 1.

3. The microbial agent according to the claim, characterized in that, Also including acceptable auxiliaries.

4. The bacterial agent according to claim 3, wherein The dosage form of the bacterial agent includes one or more of powder, liquid or granule.

5. Application of the Serratia sp. described in claim 1 or the bacterial agent described in any one of claims 2 to 4 in at least one of the following aspects: Recycling nitrogen and phosphorus to prepare struvite; Relieving and / or eliminating water eutrophication; Tolerating heavy metal ions.

6. The application according to claim 5, characterized in that, The heavy metal ions include: Ni 2+ , Cd 2+ , Cu 2+ , Pb 2+ , Zn 2+ and at least one of them.

7. The application according to claim 6, characterized in that, The heavy metals are heavy metals in sewage and / or sludge; in the sewage and / or sludge, the concentration of Pb 2+ is 100 - 2000 mg / L; the concentration of Zn 2+ is 100 - 1200 mg / L, the concentration of Cu 2+ is 100 - 400 mg / L, the concentration of Cd 2+ is 100 - 400 mg / L, and the concentration of Ni 2+ is 100 - 400 mg / L.

8. The application according to claim 5, wherein The nitrogen includes organic nitrogen; the phosphorus includes organic phosphorus.

9. Preparation method of struvite, characterized in that, Mixing the Serratia sp. described in claim 1 or the bacterial agent described in any one of claims 2 to 4 with a sample containing nitrogen, phosphorus and magnesium, and reacting to obtain struvite.

10. A sewage treatment method, characterized in that, Adding the Serratia sp. described in claim 1 or the bacterial agent described in any one of claims 2 to 4 to sewage.

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