Preparation method of low-temperature composite flora, cold-resistant microbial agent for low-temperature sewage treatment and application of cold-resistant microbial agent

Abstract

The invention belongs to the technical field of sewage treatment, and particularly relates to a preparation method of a low-temperature composite flora, a cold-resistant microbial agent for low-temperature sewage treatment and application of the cold-resistant microbial agent. The invention provides a low-temperature complex microbial community, which is prepared from flavobacterium (Flavobacterium), aeromonas (Aeromonas), sphingomonas (Sphingobacterium), pseudomonas (Pseudomonas), Comamonas (Comamonas), Massiella (Massiella) and Delftia (Delftia), and is characterized in that the low-temperature complex microbial community is prepared from the following raw materials: the flavobacterium (Flavobacterium), the aeromonas (Aeromonas), the sphingomonas (Sphingobacterium), the pseudomonas (Pseudomonas (Pseudomonas (Pseudomonas (Pseudomonas (Pseudomonas (Pseudomonas (Pseudomonas (Pseudomonas (Pseudomonas (Pseudomonas (Pseudomonas (Pseudomonas The low-temperature composite flora can treat domestic wastewater under a low-temperature condition, and is high in nitrogen removal rate, short in treatment time, wide in application range and better in impact resistance.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment technology, specifically relating to a method for preparing a low-temperature composite bacterial community, a cold-resistant bacterial agent for low-temperature wastewater treatment, and its application. Background Technology

[0002] In domestic wastewater treatment, biotechnology has become a primary method due to its advantages such as low cost, high efficiency, and no secondary pollution. Currently, most urban and rural wastewater treatment plants in my country still rely on A... 2 Traditional processes such as nitrogen removal and biofilm removal are the primary methods used in wastewater treatment. Under ideal operating conditions, these processes offer good nitrogen and phosphorus removal capabilities. However, in some parts of southern my country, winter temperatures can drop to -5°C, causing influent temperatures to fall to 0-15°C. Since biological treatment technologies are highly sensitive to temperature, when water temperatures are between 4-15°C, the metabolism and reproduction of some microorganisms slow down or even die, severely impacting the sludge settling performance and degradation efficiency of the treatment system. This results in high nitrogen and phosphorus content in the treated wastewater, failing to meet discharge standards. In the treatment of low-temperature domestic wastewater, the types of carbon and nitrogen sources in the low-temperature influent directly affect the efficiency of the entire low-temperature denitrification system. Simultaneously, the activity of nitrifying and denitrifying microorganisms is inhibited under low-temperature conditions, leading to substandard effluent quality. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a method for preparing a low-temperature composite microbial community, a cold-resistant bacterial agent for low-temperature wastewater treatment, and its application. The provided low-temperature composite microbial community can efficiently treat domestic wastewater under low-temperature conditions.

[0004] To address the aforementioned technical problems, the following technical solutions are proposed: This invention provides a method for preparing a low-temperature complex microbial community, comprising: Low-temperature samples taken from low-temperature water bodies were enriched and cultured to obtain a low-temperature complex bacterial community; the low-temperature complex bacterial community included: Flavobacterium ( Flavobacterium Aeromonas ( Aeromonas ), Sphingosine mononucleosis ( Sphingobacterium ), Pseudomonas ( Pseudomonadales ), from Trichomonas vaginalis ( Comamonas ), Masseilles ( Massilia ) and Delfollicle bacteria ( Delftia Each 1L of enrichment medium consists of: 0.5g ammonium sulfate, 5.62g sodium succinate, and 50mL Vickers salt solution.

[0005] Preferably, the enrichment culture is performed once, and each enrichment culture includes three enrichment processes.

[0006] Preferably, the volume ratio of the bacterial solution to the enrichment medium in each enrichment process is 1:9.

[0007] Preferably, the enrichment culture temperature is 12°C, and the enrichment culture is carried out until the liquid in the culture medium becomes obviously turbid; the enrichment culture rotation speed is 125 rpm.

[0008] Preferably, the abundance percentages of Flavobacterium, Aeromonas, Sphingosomalidone, Pseudomonas, Trichomonas, Masséria, and Delftobacterium in the low-temperature complex microbiota are 12.41%, 1.54%, 0.28%, 0.09%, 3.62%, 6.32%, and 0.22%, respectively.

[0009] This invention provides the application of the low-temperature composite microbial community obtained by the preparation method described above in wastewater treatment.

[0010] Preferably, the type of wastewater includes domestic sewage.

[0011] Preferably, the wastewater treatment includes reducing the nitrogen content in the wastewater; the nitrogen includes one or more of total nitrogen, ammonium nitrogen, nitrate nitrogen and nitrite nitrogen; the application temperature is 12°C, the application time is ≥5h, and the inoculation amount of the low-temperature composite microbial community is 2%~4% of the wastewater volume.

[0012] This invention provides a method for applying a low-temperature composite microbial community, comprising: mixing the low-temperature composite microbial community with wastewater and then degrading it; wherein the low-temperature composite microbial community is the low-temperature composite microbial community obtained by the preparation method described in the above technical solution.

[0013] This invention provides a cold-resistant bacterial agent for low-temperature wastewater treatment, comprising a low-temperature composite bacterial group obtained by the preparation method described above.

[0014] The beneficial effects of this invention are: This invention provides a method for preparing a low-temperature composite bacterial community, which can treat domestic wastewater under low-temperature conditions with a high nitrogen removal rate. In this invention, *Flavobacterium* in the low-temperature composite bacterial community can secrete extracellular polysaccharides (EPS). These high-molecular polymers form a protective layer on the cell surface, which helps cells aggregate into flocs or biofilms, thereby enhancing biofilm stability. The EPS produced by *Aeromonas* in the low-temperature composite bacterial community has antibacterial activity, inhibiting the growth of pathogens and promoting biofilm formation. The fatty acids in the cellular lipids of *Sphingosine Monomer* in the low-temperature composite bacterial community are mainly oleic acid and tetradecanoic acid, which can withstand low-temperature shock. *Flavobacterium*, *Aeromonas*, and *Sphingosine Monomer* can improve the low-temperature tolerance of the composite bacterial community and protect the biofilm from damage. Furthermore, the Pseudomonas bacteria in the low-temperature complex can produce cytochromes to withstand low-temperature shock and have the function of removing nitrogen; Trichomonas bacteria participate in the denitrification of nitrates into nitrogen gas; Massébium participates in the carbon-nitrogen cycle and converts organic matter into inorganic matter through metabolic activities; and Delford bacteria, with its low-temperature resistance and strong degradation ability, can effectively remove these organic substances.

[0015] The existing single-species Pseudomonas bacteria have nitrate reductase activity of 0.048 U / mg protein and nitrite reductase activity of 0.021 U / mg protein, resulting in low nitrification and denitrification efficiencies. The low-temperature composite microbial community of this invention is composed of multiple bacterial species with high nitrate and nitrite reductase activities, possessing both nitrification and denitrification functions. Among them, Flavobacterium, Aeromonas, and Sphingosine monocytogenes can improve the low-temperature tolerance of the composite microbial community and protect the biofilm from damage. Pseudomonas, Trichomonas, Masséria, and Delftobacter can degrade organic matter and remove nitrogen. There is a synergistic degradation effect among different bacteria, which can efficiently remove nitrogen from domestic sewage and reduce the COD value of domestic sewage. Moreover, the low-temperature composite microbial community has a short treatment time, a wide range of adaptability, and better shock resistance. Detailed Implementation

[0016] This invention provides a method for preparing a low-temperature complex microbial community, comprising: Enrichment culture was performed on sediment mixture samples taken from low-temperature water bodies to obtain a low-temperature complex bacterial community; the low-temperature complex bacterial community includes: Flavobacterium ( Flavobacterium Aeromonas ( Aeromonas ), Sphingosine mononucleosis ( Sphingobacterium ), Pseudomonas ( Pseudomonadales ), from Trichomonas vaginalis ( Comamonas ), Masseilles ( Massilia ) and Delfollicle bacteria ( Delftia Each 1L of enrichment medium consists of: 0.5g ammonium sulfate, 5.62g sodium succinate, and 50mL Vickers salt solution.

[0017] This invention utilizes a low-temperature composite bacterial flora enriched from low-temperature water bodies. This low-temperature composite bacterial flora is a psychrogenic agent capable of normal growth at low temperatures, thus addressing the problem of substandard wastewater treatment in winter. The technical solution of this invention successfully enriches Flavobacterium, Aeromonas, Sphingosoma, Pseudomonas, Trichomonas, Masséria, and Delftobacterium by collecting a mixture of sediment and water at a depth of approximately 10 cm in low-temperature water bodies and using a specific enrichment medium. The mixture is then cultured at 125 rpm at 12°C for 48 hours. Sodium succinate in the specific enrichment medium serves as a carbon source, allowing for the screening of bacterial flora that utilize sodium succinate as a carbon source for nitrification and denitrification under low-temperature conditions.

[0018] This invention involves enriching and culturing a mixture of sediment and mud taken from a low-temperature water body to obtain a low-temperature complex bacterial community. The enrichment culture is preferably performed once. Preferably, this single enrichment culture includes three enrichment processes. In each enrichment process, the volume ratio of the inoculated bacterial solution to the enrichment medium is 1:9. In a specific embodiment of this invention, the sediment mixture is inoculated into the enrichment medium for a first enrichment process to obtain a first enriched bacterial solution; the volume ratio of the sediment mixture to the enrichment medium is 1:9. The first enriched bacterial solution is then inoculated into the enrichment medium for a second enrichment process to obtain a second enriched bacterial solution; the volume ratio of the first enriched bacterial solution to the enrichment medium is 1:9. The second enriched bacterial solution is then inoculated into the enrichment medium for a third enrichment process to obtain a third enriched bacterial solution; the volume ratio of the second enriched bacterial solution to the enrichment medium is 1:9. The third enriched bacterial solution is the low-temperature complex bacterial community, which is stored in a -80°C freezer. This invention utilizes multiple enrichment cultures to maximize the screening of microorganisms with highly efficient nitrification and denitrification capabilities. Each enrichment culture is conducted at a temperature of 12°C, a rotation speed of 125 rpm, and a carbon source concentration of 115-120 mg / L. Each enrichment culture continues until the medium becomes noticeably turbid. The OD value of the noticeably turbid medium in this invention is... 600 The concentration is 0.5~0.6. The rotation speed for each enrichment culture in this invention is 125 rpm. Each 1L of enrichment medium in this invention comprises: 0.5g ammonium sulfate, 5.62g sodium succinate, and 50mL Vickers salt solution.

[0019] As an optional implementation, the abundance percentages of Flavobacterium, Aeromonas, Sphingosomalidone, Pseudomonas, Trichomonas, Masséria, and Delfordia in the low-temperature composite microbial community of the present invention are 12.41%, 1.54%, 0.28%, 0.09%, 3.62%, 6.32%, and 0.22%, respectively. The Flavobacterium, Aeromonas, Sphingosomalidone, and Pseudomonas described in this invention can improve the low-temperature tolerance of the composite microbial community and protect the biofilm from damage. The Pseudomonas, Trichomonas, Masséria, and Delfordia can degrade organic matter and remove nitrogen. There is a synergistic degradation effect among the different bacteria, enabling efficient removal of nitrogen and reduction of COD in domestic sewage. The treatment time is short, the adaptability is wide, and the shock resistance is better.

[0020] This invention provides the application of the low-temperature composite microbial community obtained by the preparation method described above in wastewater treatment.

[0021] As an optional implementation, the type of wastewater described in this invention includes domestic sewage.

[0022] As an optional implementation, the wastewater treatment method of the present invention includes reducing the nitrogen content in the wastewater; the nitrogen includes one or more of total nitrogen, ammonium nitrogen, nitrate nitrogen and nitrite nitrogen.

[0023] As an optional implementation, the method of application of the present invention includes: mixing the low-temperature composite microbial community with wastewater and then degrading it. The application temperature of the low-temperature composite microbial community of the present invention is 12°C, the application time is ≥5h, or ≥10h, more preferably ≥10h and <40h, and the inoculation amount is 2%~4% of the wastewater volume, or 2%~3%, more preferably 2%.

[0024] This invention provides a method for applying a low-temperature composite microbial community, which involves mixing the low-temperature composite microbial community with wastewater and then subjecting it to degradation. The low-temperature composite microbial community is the one prepared by the method described in the above technical solution. The degradation temperature is 12°C, and the degradation time is ≥5 hours, ≥10 hours, more preferably ≥10 hours and <40 hours. The inoculum amount is 2%~4% of the wastewater volume, 2%~3%, more preferably 2%.

[0025] This invention provides a refrigerated bacterial agent for low-temperature wastewater treatment, comprising a low-temperature composite bacterial community obtained by the preparation method described above. The refrigerated bacterial agent of this invention is used for low-temperature wastewater treatment.

[0026] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Detection methods 1. The total nitrogen content was determined using the alkaline potassium persulfate digestion ultraviolet spectrophotometric method. The specific steps are as follows: (1) Required medicines: Total nitrogen standard stock solution: After drying potassium nitrate (KNO3) at 105~110℃ for 4h, take 0.7218g, dissolve it in water, transfer it to a 1000mL volumetric flask, and add 2mL of chloroform as a preservative (can be stored for 6 months).

[0028] Standard working solution: Dilute the total nitrogen standard stock solution 10 times.

[0029] Alkaline potassium persulfate solution: Prepare a solution by drying and purifying 40g of potassium persulfate and 15g of sodium hydroxide. After cooling the NaOH solution to room temperature, mix the potassium persulfate solution and NaOH solution, and then make up to 1000mL. It can be stored for a maximum of 5 days.

[0030] 1+9 Hydrochloric Acid (Prepare fresh for immediate use): Take 10 mL of hydrochloric acid into a 100 mL volumetric flask and dilute to 100 mL.

[0031] (2) Plotting the standard curve: Take 0 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, 5 mL, 7 mL, and 8 mL of standard working solution respectively into 25 mL test tubes, and dilute to 10 mL. Add 5 mL of alkaline potassium persulfate solution for sterilization for 30 min. After cooling, add 1 mL of (1+9) hydrochloric acid and dilute to 25 mL. Use water as a reference and measure the absorbance at dual wavelengths of 220 nm and 275 nm. The standard curve is as follows: y =9.5748 x -0.541, of which x The absorbance of the sample to be tested is... y The concentration of the sample to be tested is denoted as .

[0032] (3) Experimental steps: 1) Take 10 mL of the sample to be tested into a 25 mL test tube; 2) Add 5 mL of alkaline potassium persulfate solution; 3) Sterilize in a high-pressure steam sterilizer for 30 minutes; 4) After cooling, add 1 mL of (1+9) hydrochloric acid; 5) Make up to 25 mL, and measure the absorbance at dual wavelengths of 220 nm and 275 nm, using water as a reference.

[0033] The formula for calculating the total nitrogen removal rate is: Total nitrogen removal rate = (Initial total nitrogen concentration of bacterial culture - Total nitrogen concentration of bacterial culture after culture) / Initial total nitrogen concentration of bacterial culture.

[0034] 2. The ammonia nitrogen content was determined using Nessler's reagent spectrophotometric method. The specific steps are as follows: (1) Required medicines: Ammonia nitrogen standard stock solution: Dissolve 3.819 g of superior grade ammonium chloride (NH4Cl) dried at 100℃ and bring the volume to 1000 mL.

[0035] Ammonia nitrogen standard working solution: Take 5 mL of standard stock solution and dilute to 500 mL.

[0036] Potassium sodium tartrate: Add 50g of potassium sodium tartrate to 100mL of water, boil to remove ammonia, and then bring the volume to 100mL.

[0037] Nessler's reagent: Dissolve 7g of potassium iodide in 10mL of water, and dissolve 10g of mercuric iodide in 15mL of water (mercuric iodide is insoluble in water). Mix the two solutions thoroughly to obtain a mixed solution. Dissolve 16g of sodium hydroxide in 35mL of water. After the sodium hydroxide solution cools, slowly pour the mixed solution into the sodium hydroxide solution while stirring. Finally, make up to 100mL and store in a polyethylene bottle.

[0038] (2) Plotting the standard curve: Take 0 mL, 0.5 mL, 1 mL, 3 mL, 5 mL, 7 mL, and 10 mL of ammonia nitrogen standard working solution respectively into 50 mL test tubes, dilute to 50 mL, add 1 mL of potassium sodium tartrate, add 1.5 mL of Nessler's reagent, shake well, and after 10 min, measure the absorbance at a single wavelength of 420 nm using water as a reference. The standard curve is as follows. y =6.5394 x -0.6855, of which x The absorbance of the sample to be tested is... y The concentration of the sample to be tested is denoted as .

[0039] (3) Experimental steps: 1) Take 20 mL of the sample to be tested into a 50 mL test tube and bring the volume up to 50 mL; 2) Add 1 mL of potassium sodium tartrate; 3) Add 1.5 mL of Nessler's reagent and shake well; 4) After 10 minutes, the absorbance was measured at a single wavelength of 420 nm using water as a reference.

[0040] The formula for calculating the ammonium nitrogen removal rate is: Ammonium nitrogen removal rate = (Initial ammonium nitrogen concentration in bacterial culture - Ammonium nitrogen concentration in bacterial culture after cultivation) / Initial ammonium nitrogen concentration in bacterial culture.

[0041] 3. The content of nitrate nitrogen was determined by ultraviolet spectrophotometry. The specific steps are as follows: (1) Required medicines: Standard stock solution: Use total nitrogen standard stock solution.

[0042] 1 mol / L hydrochloric acid (prepared fresh): Add 8.4 mL of hydrochloric acid to 100 mL of water.

[0043] 0.8% Sulfamic acid: Dissolve 0.8g of sulfamic acid in water and bring the volume up to 100mL.

[0044] (2) Standard curve: Take 0 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, and 4 mL of total nitrogen standard stock solution respectively into 200 mL volumetric flasks, and dilute to 200 mL. Add 1 mL of 1 mol / L hydrochloric acid and 0.8% aminosulfonic acid (not added for blank samples) sequentially. Using the blank sample as a reference, measure the absorbance at wavelengths of 220 nm and 275 nm. y =4.1531 x +0.0015, of which x The absorbance of the sample to be tested is... y The concentration of the sample to be tested is denoted as .

[0045] (3) Experimental steps: 1) Take 20 mL of the filtered sample and bring the volume up to 50 mL; 2) Add 1 mL of 1 mol / L hydrochloric acid; 3) Add 0.1 mL of 0.8% aminosulfonic acid (do not add to blank sample); 4) Using water as a reference, absorbance was measured at dual wavelengths of 220 nm and 275 nm.

[0046] Nitrate nitrogen removal rate = (initial nitrate nitrogen concentration in bacterial culture - nitrate nitrogen concentration in bacterial culture after culture) / initial nitrate nitrogen concentration in bacterial culture 4. The nitrite nitrogen content was determined using the N-(1-phenyl)-ethylenediamine spectrophotometric method. The specific steps are as follows: (1) Required medicines: Nitrite nitrogen standard stock solution: Dissolve 1.232g sodium nitrite (NaNO2) in 150mL of water, bring the volume up to 1000mL, and add 1mL of chloroform as a preservative (it can be stably stored at 2℃~5℃ for at least one month).

[0047] Intermediate solution: 50 mL of nitrite nitrogen standard stock solution was brought to a final volume of 250 mL (diluted 5 times).

[0048] Standard working solution: Dilute 10 mL of intermediate solution to 500 mL (50-fold dilution).

[0049] Colorimetric reagent: Take 250 mL of water and 50 mL of phosphoric acid in a 500 mL beaker, add 20 g of p-aminobenzenesulfonamide and stir well, then dissolve 1 g of N-(1-phenyl)-ethylenediamine dihydrochloric acid in the mixed solution, make up to 500 mL and store in a brown bottle.

[0050] (2) Plotting the standard curve: Take 0 mL, 1 mL, 3 mL, 5 mL, 7 mL, and 10 mL of nitrite nitrogen standard stock solution into 25 mL test tubes, respectively, dilute to 25 mL, add 0.5 mL of colorimetric reagent, let stand for 20 min, and measure the absorbance at a single wavelength of 540 nm with water as a reference. y =0.5244 x -0.0616, of which x The absorbance of the sample to be tested is... y The concentration of the sample to be tested is denoted as .

[0051] (3) Experimental steps: 1) Take 25 mL of the filtered sample to be tested; 2) Add 0.5 mL of colorimetric reagent and let stand for 20 min; 3) Using water as a reference, the absorbance was measured at a single wavelength of 540 nm.

[0052] Nitrite nitrogen removal rate = (initial nitrite nitrogen concentration in bacterial culture - nitrite nitrogen concentration in bacterial culture after cultivation) / initial nitrite nitrogen concentration in bacterial culture.

[0053] Example 1 1. Preparation method of low-temperature complex microbial community: A sediment mixture was sampled from a low-temperature water body and allowed to stand for 2 hours. 10 mL of the supernatant was then transferred to 90 mL of enrichment medium and incubated at 12℃ with shaking at 125 rpm for a specified time. Once the medium became noticeably turbid, 10 mL of the bacterial suspension from the turbid medium was inoculated into 90 mL of fresh enrichment medium. The same incubation process was repeated at 12℃ with shaking at 125 rpm until the medium became noticeably turbid. This yielded a cold-resistant bacterial agent for low-temperature domestic wastewater treatment, namely a low-temperature composite bacterial group. The composition of each 1000 mL enrichment medium was: 0.5 g ammonium sulfate, 5.62 g sodium succinate, 50 mL Vickers salt solution, and the remainder water, with a pH of 7.0–7.2. The Vickers salt solution consists of: 2.5 g / L sodium chloride, 0.05 g / L manganese sulfate tetrahydrate, 6.5 g / L dipotassium hydrogen phosphate trihydrate, 2.5 g / L magnesium sulfate heptahydrate, and 0.05 g / L ferrous sulfate heptahydrate.

[0054] 2. The growth curve of the low-temperature complex bacterial culture was detected by inoculating it into the enrichment medium. The results are shown in Table 1. The culture entered the logarithmic growth phase approximately 5 hours after inoculation. Between 10 and 12 hours, the bacterial culture developed a certain degree of resistance, leading to a decrease in OD... 600It rose slightly, then entered a stable period around the 28th hour.

[0055] Table 1 Growth curves of the low-temperature complex microbial community

[0056] Example 2 The low-temperature complex bacterial group prepared in Example 1 was inoculated at a volume ratio of 2% into ammonium nitrogen (NH4). + The bacteria were cultured in an enrichment medium with ammonium nitrogen (NH3) as the sole nitrogen source for 40 hours. The concentrations of total nitrogen, ammonia nitrogen, and nitrate nitrogen in the medium were simultaneously measured to assess nitrogen removal. The results are shown in Table 2. It can be seen that under low-temperature conditions (12℃), the bacterial community almost completely removed ammonium nitrogen within 40 hours, with a removal rate of 97.34%. The maximum removal rate of ammonium nitrogen within 40 hours was 4.51 mg / h, and the removal rate of total nitrogen was 94.43%.

[0057] Ammonium nitrogen (NH4) + The enrichment medium with ammonium sulfate (N) as the sole nitrogen source consists of 0.5 g of ammonium sulfate, 5.62 g of sodium succinate, 50 mL of Vickers salt solution, and the remainder water per 1000 mL of enrichment medium, with a pH of 7.0-7.2.

[0058] Table 2. Nitrogen concentration in the culture medium at different culture times.

[0059] Example 3 The low-temperature complex bacterial culture prepared in Example 1 was inoculated at a 2% inoculum with nitrate nitrogen (NO3). - The bacteria were cultured for 40 h in an enrichment medium (denoted as denitrification medium 1) with N2 as the sole nitrogen source. The total nitrogen, ammonia nitrogen, and nitrate nitrogen concentrations in the medium were measured simultaneously to assess nitrogen removal. The results are shown in Table 3. It was found that under low temperature conditions (12℃), the bacterial community achieved a nitrate removal rate of 97.13%; the maximum nitrate removal rate within 40 h was 3.97 mg / h; and the total nitrogen removal rate was 92.43%. Denitrification medium 1 consisted of: 0.36 g / L potassium nitrate, 5.62 g / L sodium succinate, 50 mL / L Vickers salt solution, and the remainder water, with a pH of 7.0–7.2.

[0060] Table 3. Nitrogen concentration in the culture medium at different culture times.

[0061] Example 4 The low-temperature complex bacterial group prepared in Example 1 was inoculated at a volume ratio of 2% into a solution containing nitrite nitrogen (NO2). -The bacteria were cultured in an enrichment medium (denoted as denitrification medium 2) with sodium nitrite (N-N) as the sole nitrogen source. Nitrogen removal in the medium was measured at different culture times, and the results are shown in Table 4. It can be seen that under low temperature conditions (12℃), the bacterial community removed 59.56% of nitrite, and ammonium salt accumulation was detected during the removal process; the bacterial community removed 97.86% of total nitrogen, with a maximum removal rate of 8.79 mg / h. The composition of denitrification medium 2 was: 0.172 g / L sodium nitrite, 5.62 g / L sodium succinate, 50 mL / L Vickers salt solution, and the balance being water, with a pH of 7.0–7.2.

[0062] Table 4 Nitrogen content at different culture times

[0063] Example 5 The nitrate reductase and nitrite reductase activities of the low-temperature complex bacterial group prepared in Example 1 were determined using the following method: Treatment 1 (M2 group): The low-temperature complex bacterial group M2 prepared in Example 1 was cultured in enrichment medium (the composition of the enrichment medium is the same as in Example 1) for 28 h. The resulting bacterial solution was inoculated with NH4 at a volume ratio of 2%. + In nitrified medium with -N as the sole nitrogen source, the cells were cultured at 14°C and 200 rpm until the end of the logarithmic growth phase. The cells were collected by centrifugation and resuspended in 25 mM phosphate buffer (PBS, pH 7.5) containing 1 mg / mL lysozyme and 2 mM EDTA, and incubated at 30°C for 2 h. Cell disruption was then performed using an ultrasonic cell disruptor, and the crude enzyme solution was obtained by centrifugation at 4°C and 8000 × g for 30 min.

[0064] Ammonium nitrogen (NH4) + The nitrification medium, with ammonium sulfate (NH3) as the sole nitrogen source, consists of 0.5 g ammonium sulfate, 5.62 g sodium succinate, 50 mL Vickers salt solution, and the remainder water per 1000 mL of enrichment medium, with a pH of 7.0–7.2. The Vickers salt solution comprises: 2.5 g / L sodium chloride, 0.05 g / L manganese sulfate tetrahydrate, 6.5 g / L dipotassium hydrogen phosphate trihydrate, 2.5 g / L magnesium sulfate heptahydrate, and 0.05 g / L ferrous sulfate heptahydrate.

[0065] For the determination of nitrate reductase (NR) and nitrite reductase (NIR) activities in the crude enzyme solution, the specific reaction system (1.2 mL) was as follows: 0.35 mL of 40 mM PBS buffer (pH 7.5), 0.4 mL of crude enzyme solution, and 0.8 mM NO3. - / 0.04 mMNO2 -0.3 mL of solution, 0.1 mL of 5 mM methyl viologen (MV+), and 0.05 mL of sodium sulfite solution (50 mg dissolved in 2 mL of 0.3 M sodium bicarbonate solution) were added. The reaction system was subjected to a reaction at 30 °C for 2 h, and the reaction was terminated by vigorous shaking. The NO3 levels before and after the reaction were calculated. - and NO2 - The reduction in the amount of enzyme activity was used to characterize the enzyme activities of NR and NIR.

[0066] Control 1: Similar to Treatment 1, the only difference being that the low-temperature complex microbial community M2 was replaced with... Pseudomonadales .

[0067] Control 2: Similar to Treatment 1, the only difference being that the low-temperature complex microbial community was replaced with M2. Bacillus simplex.

[0068] Control 3: Similar to Treatment 1, the only difference being that the low-temperature complex bacterial group M2 was replaced with... Comamonas .

[0069] Group M1: Similar to treatment 1, the only difference is that the low-temperature complex microbial community M2 is replaced with M1. Shewanella and Pseudomonas aeruginosa are included. Pseudomonas aeruginosa After being mixed with live bacteria at a ratio of 1:1, the mixture was inoculated into enrichment medium and cultured for 28 hours to obtain M1.

[0070] The results are shown in Tables 5 and 6. It can be seen that the enzyme activity of the low-temperature complex M2 is higher than that of other single bacteria.

[0071] Table 5. Nitrate reductase and nitrite reductase activities of low-temperature complex bacterial groups

[0072] Table 6. Nitrate reductase and nitrite reductase activities of single bacteria in the control group.

[0073] In summary, the various bacteria in the low-temperature composite microbial community of this invention exhibit synergistic degradation effects, thereby enhancing the degradation efficiency. The mixed microbial community of this invention is superior to single-strain microorganisms, and also offers shorter treatment times, wider applicability, and better shock resistance.

[0074] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a low-temperature complex microbial community, characterized in that, include: Enrichment culture was performed on sediment mixture samples taken from low-temperature water bodies to obtain low-temperature complex bacterial communities. The low-temperature complex microbial community includes: Flavobacterium ( Flavobacterium Aeromonas ( Aeromonas ), Sphingosine mononucleosis ( Sphingobacterium ), Pseudomonas ( Pseudomonadales ), from Trichomonas vaginalis ( Comamonas ), Masseilles ( Massilia ) and Delfollicle bacteria ( Delftia Each 1L of enrichment medium consists of: 0.5g ammonium sulfate, 5.62g sodium succinate, and 50mL Vickers salt solution.

2. The preparation method according to claim 1, characterized in that, The enrichment culture is performed once, and each enrichment culture includes three enrichment processes.

3. The preparation method according to claim 1, characterized in that, The volume ratio of the bacterial solution to the enrichment medium in each enrichment process was 1:

9.

4. The preparation method according to claim 1 or 2, characterized in that, The enrichment culture was conducted at a temperature of 12°C until the liquid in the culture medium became noticeably turbid; the enrichment culture was conducted at a rotation speed of 125 rpm.

5. The preparation method according to claim 1, characterized in that, The abundance percentages of Flavobacterium, Aeromonas, Sphingosomal, Pseudomonas, Trichomonas, Masséria and Delftobacterium in the low-temperature complex were 12.41%, 1.54%, 0.28%, 0.09%, 3.62%, 6.32% and 0.22%, respectively.

6. The application of the low-temperature composite microbial community obtained by the preparation method according to any one of claims 1 to 5 in wastewater treatment.

7. The application according to claim 6, characterized in that, The types of wastewater include domestic sewage.

8. The application according to claim 6 or 7, characterized in that, The wastewater treatment includes reducing the nitrogen content in the wastewater; the nitrogen includes one or more of total nitrogen, ammonium nitrogen, nitrate nitrogen and nitrite nitrogen; the application temperature is 12℃, the application time is ≥5h, and the inoculation amount of the low-temperature compound microbial community is 2%~4% of the wastewater volume.

9. A method for applying a low-temperature complex microbial community, characterized in that, include: The low-temperature composite microbial community was mixed with wastewater and then degraded. The low-temperature composite microbial community is the low-temperature composite microbial community obtained by the preparation method according to any one of claims 1 to 5.

10. A cold-resistant bacterial agent for low-temperature wastewater treatment, characterized in that, Includes the low-temperature composite microbial community obtained by the preparation method according to any one of claims 1 to 5.

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