A strain of Paracoccus mutans for degrading ammonia nitrogen in sewage and wastewater and its application

Abstract

The present invention relates to a strain of Paracoccus mutans and its application in the field of sewage and wastewater purification. The strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Committee, and the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is: CGMCC No. 23528, and the deposit date is: September 30, 2021. The beneficial effects of Paracoccus mutans provided by the present invention are: strong ammonia nitrogen degradation ability and fast onset, able to tolerate high concentrations of ammonia nitrogen and salt resistance, the strain can reproduce and grow normally in the water body when the salinity does not exceed 5%, and the growth condition is best under salinity conditions below 4%.

Description

Technical Field

[0001] The invention relates to a Paracoccus mutans, a microbial agent containing the Paracoccus mutans and application thereof in the field of sewage and wastewater purification, belonging to the technical field of environmental microorganisms. Background Art

[0002] In recent years, with the rapid development of my country's industry and the accelerated pace of life, urban domestic sewage and wastewater from agriculture and industry have made water pollution problems increasingly serious. These sewages have the characteristics of high COD value, high ammonia nitrogen content and strong odor, causing serious pollution to surface water and groundwater. Among them, ammonia nitrogen in water bodies is one of the main pollution factors. The large accumulation of ammonia nitrogen will lead to the aggravation of eutrophication of slow-flowing closed or semi-closed water bodies, causing environmental deterioration, and leading to poisoning or even death of aquatic organisms, seriously affecting the ecological balance.

[0003] Among the current sewage treatment methods, the removal methods of ammonia nitrogen mainly include physical method, chemical method and biological method. Compared with physical method and chemical method, biological method has the advantages of safety, economy and no secondary pollution, and has become the most widely used treatment method.

[0004] Among the biological treatment methods of ammonia nitrogen, using nitrifying bacteria to remove ammonia nitrogen pollution is the most convenient and efficient way to remove nitrogen. Nitrifying bacteria are divided into autotrophic nitrifying bacteria and heterotrophic nitrifying bacteria. Among them, autotrophic nitrifying bacteria are difficult to form dominant strains due to their slow reproduction rate, low biological density and harsh growth conditions, and it is difficult to expand production and promote their use. Since the heterotrophic nitrifying strains were isolated, more and more heterotrophic nitrifying bacteria have attracted widespread attention.

[0005] Based on the current research and development results, most heterotrophic nitrifying bacteria have problems such as too long time in treating high-ammonia nitrogen wastewater and unsatisfactory results in pilot-scale experiments. Therefore, screening microbial strains that can efficiently degrade ammonia nitrogen in water bodies is of great significance for environmental protection. Summary of the invention

[0006] Aiming at the deficiencies of existing heterotrophic nitrifying bacteria in treating high-ammonia nitrogen wastewater, the present invention provides a strain of Paracoccus variabilis, a microbial agent containing the same and application of the same in the field of sewage and wastewater purification.

[0007] A strain of Paracoccus versutus AOB133 is deposited in the General Microbiology Center of the China Culture Collection Administration, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 23528 and a deposit date of September 30, 2021. Its 16S rDNA sequence is shown in SEQID No: 1. The Paracoccus versutus referred to in the present invention refers to the Paracoccus versutus AOB133 strain.

[0008] The beneficial effects of the Paracoccus mutans provided by the present invention are:

[0009] 1) The ammonia nitrogen degradation ability is strong and the effect is fast. Under the condition of 30℃, for the initial ammonia nitrogen concentration of 100 mg / L, the ammonia nitrogen degradation rate in 72 hours reaches more than 94%; for the initial ammonia nitrogen concentration of 200 mg / L, the ammonia nitrogen degradation efficiency in 48 hours is about 75%, and the ammonia nitrogen degradation efficiency in 72 hours can reach more than 90%.

[0010] 2) Moderately salt-tolerant, it can reproduce and grow normally in water with salinity not exceeding 5%, and grows best in salinity below 4%;

[0011] 3) The strain has a simple culture method, strong environmental adaptability, rapid reproduction and high safety.

[0012] The present invention also claims a microbial agent comprising the Paracoccus variabilis.

[0013] The present invention also claims a fermentation method of the Paracoccus mutans, comprising the following steps:

[0014] (1) Primary seed culture: Under sterile conditions, Paracoccus variabilis is inoculated into a nutrient medium and cultured at 25-35° C. and 150-300 rpm for 12-24 h to obtain a primary seed culture solution;

[0015] (2) Secondary seed culture: Under sterile conditions, the primary seed culture solution is inoculated into a nutrient medium at an inoculum amount of 1-10 vol%, and cultured at 25-35° C. and 150-300 rpm for 12-24 h to obtain a secondary seed culture solution;

[0016] (3) Fermentation: After the fermentation medium is disinfected, the secondary seed culture solution is inoculated into the fermentation medium at an inoculation rate of 1-10vol%, and the fermentation is carried out under the conditions of controlling the temperature to 25-35°C, the rotation speed to 150-300rpm, and the ventilation ratio to 1:(1-2). When the dissolved oxygen begins to rise, the fermentation is stopped to obtain the fermentation liquid.

[0017] The ventilation ratio described in the fermentation method of the present invention refers to the ratio of the volume of air introduced into the fermentation tank per minute to the total volume of the fermentation liquid.

[0018] The nutrient medium is composed of 5-15 g / L peptone, 1-5 g / L beef powder, 3-8 g / L sodium chloride, water as solvent, and pH=7-7.5.

[0019] The fermentation medium consists of: 8-20 g / L carbon source, 2-5 g / L nitrogen source, PO4 3- 0.1-0.5g / L, K + 0.01-0.1g / L, Mg 2+ 0.01-0.05g / L, Fe 2+ (5-10)×10 -3 g / L、Mn 2+ (1-5)×10 -3 g / L, solvent is water, pH=6~8.

[0020] Preferably, the PO4 3- and K + The source is dipotassium hydrogen phosphate or potassium dihydrogen phosphate, the Mg 2+ The source is a combination of one or more of magnesium sulfate, magnesium nitrate or magnesium chloride, wherein the Fe 2+ The source is one or more of ferrous sulfate, ferrous chloride, and ammonium ferrous sulfate, wherein the Mn 2+ The source is one or more of manganese sulfate monohydrate, manganese sulfate tetrahydrate, manganese nitrate or manganese chloride.

[0021] Furthermore, the carbon source is selected from one or more of glucose, sucrose, starch, sodium acetate or sodium succinate.

[0022] Furthermore, the nitrogen source is selected from one or more of yeast extract, peptone, urea, ammonium sulfate or potassium nitrate.

[0023] In actual application, the form of the final Paracoccus labile product can be determined according to actual usage and storage needs. When a liquid product is needed, the fermentation broth can be diluted to the required concentration and used directly. When a solid product is needed, the fermentation broth can be centrifuged to obtain bacterial sludge, and then freeze-drying technology is used to prepare solid bacterial powder.

[0024] The present invention also claims a method for purifying sewage and wastewater using an activated solution of Paracoccus mutans or a microbial agent containing Paracoccus mutans, comprising the step of inoculating the activated solution of Paracoccus mutans or a microbial agent containing Paracoccus mutans into the sewage and wastewater.

[0025] Furthermore, the ammonia nitrogen concentration in the wastewater is below 400 mg / L, most preferably 100-200 mg / L.

[0026] Furthermore, the salinity of the wastewater is below 5%, preferably below 4%.

[0027] Furthermore, the inoculation amount of the activated solution of Paracoccus variabilis or the microbial agent containing Paracoccus variabilis is 100 ppm or more, preferably 100-2000 ppm, and most preferably 100-1000 ppm.

[0028] The present invention also claims to protect the use of Paracoccus variabilis and a microbial agent containing Paracoccus variabilis in the field of sewage and wastewater purification.

[0029] Preferably, the Paracoccus variabilis and the microbial agent comprising Paracoccus variabilis are used to degrade nitrogen-containing substances in sewage and wastewater, and more preferably, are used to degrade ammonia-nitrogen-containing substances in sewage and wastewater.

[0030] Furthermore, the ammonia nitrogen concentration in the wastewater is below 400 mg / L, most preferably 100-200 mg / L. DETAILED DESCRIPTION

[0031] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0032] Example 1. Isolation, purification and identification of Paracoccus mutans

[0033] (1) Screening and isolation of strains

[0034] The sewage from the aeration section of an industrial sewage treatment plant in Gaomi City, Weifang City, Shandong Province was collected and diluted to 10 -4 , absorb 10 -2 , 10 -3 , 10 -4 100 μL of each diluent was added to the separation medium (10 g peptone, 3 g beef powder, 5 g sodium chloride, 15 g agar powder, 1000 mL tap water), spread evenly and inverted to culture at 30°C. Single colonies grew in about 24 hours. Select single colonies with different morphologies and transfer them to the test tube slant separation medium, culture at 30°C for about 24 hours, and then transfer to a 4°C refrigerator for storage.

[0035] A total of 5 strains were obtained according to the above isolation method, and were numbered as: AOB131, AOB132, AOB133, AOB134 and AOB135.

[0036] (2) Evaluation of strains

[0037] The five strains obtained from the initial screening were inoculated into a basic culture medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L sodium chloride, water as solvent, pH = 7), and cultured at 30°C with shaking for 24 h. 10 mL of the culture solution was taken and centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and then inoculated into 100 mL of an evaluation culture medium (0.5 g ammonium sulfate, 5.62 g sodium succinate, 50 mL of Wei's salt solution, fixed to 1 L with sterile water, pH = 7, ammonia nitrogen content of 100 mg / L), and cultured at 30°C with shaking. Three parallel groups were set for each experimental group, and sterile water was used as a blank control. The changes in ammonia nitrogen concentration in the evaluation culture medium of each experimental group were regularly detected. The results are shown in Table 1.

[0038] The ammonia nitrogen detection method is carried out in accordance with "HJ535-2009 Determination of Ammonia Nitrogen-Nessler's Reagent Spectrophotometry".

[0039] Table 1 Degradation effect of various strains obtained from the initial screening on ammonia nitrogen

[0040]

[0041] The results in Table 1 show that compared with other strains, strain AOB133 degrades ammonia nitrogen quickly and has a significantly better removal effect than other strains, so it was further identified as a preferred strain.

[0042] (3) Detection and Identification

[0043] The AOB133 strain was identified by 16S rDNA gene sequence detection, and the identification result was Paracoccus versutus. The 16S rDNA gene sequence determination result of strain AOB133 is shown in SEQ ID No:1.

[0044] Example 2. Determination of ammonia nitrogen degradation efficiency of Paracoccus mutans

[0045] Under sterile conditions, Paracoccus mutans was inoculated into a 250mL conical flask containing 100mL of activation medium, the composition of the activation medium was: 10g / L tryptone, 5g / L yeast extract powder, 10g / L sodium chloride, the solvent was water and the pH was 6.5-7, and the strain was activated by shaking and culturing at 30°C and 200rpm in a shaker for 24h to obtain an activation solution of Paracoccus mutans. After testing, the number of viable bacteria in the activation solution was 5.5 billion cfu / mL.

[0046] 10 μl, 100 μl and 1000 μl of the activation solution of Paracoccus mutans were taken respectively and inoculated into 100 mL of sterilized evaluation culture medium (0.5 g of ammonium sulfate, 5.62 g of sodium succinate, 50 mL of Wei's salt solution, fixed to 1 L with sterile water, pH = 7, ammonia nitrogen content of 100 mg / L), and the inoculation amount of the activation solution of Paracoccus mutans was 100 ppm, 1000 ppm and 10000 ppm respectively. Then, each evaluation culture medium was placed in a shaking incubator at 30°C and 200 rpm for shaking culture. Three parallels were set for each group, and sterile water was used as a blank control. The ammonia nitrogen content in the evaluation culture medium was detected every 24 hours. The results are shown in Table 2.

[0047] The ammonia nitrogen detection method is carried out in accordance with "HJ535-2009 Determination of Ammonia Nitrogen-Nessler's Reagent Spectrophotometry".

[0048] Table 2 Ammonia nitrogen degradation effect of Paracoccus mutans

[0049]

[0050] From the results in Table 2, it can be seen that the degradation rate of ammonia nitrogen by Paracoccus mutans is fast and the degradation ability is strong. When the inoculation amount of Paracoccus mutans activation solution is above 100 ppm, the ammonia nitrogen degradation efficiency in 72 hours can reach above 94%, and when the inoculation amount reaches 100 ppm, the continued increase in the inoculation amount has a very weak effect on the ammonia nitrogen degradation effect. Therefore, based on economic considerations, the preferred inoculation amount of Paracoccus mutans activation solution is 100-1000 ppm.

[0051] Example 3: Salinity tolerance experiment of Paracoccus mutans

[0052] Under sterile conditions, Paracoccus mutans was inoculated into a 250mL Erlenmeyer flask containing 100mL of activation medium. The composition of the activation medium was: 10g / L tryptone, 5g / L yeast extract powder, 10g / L sodium chloride, the solvent was water and the pH was 6.5-7. The strain was activated by shaking and culturing at 30°C and 200rpm in a shaker for 24h to obtain an activation solution of Paracoccus mutans. After testing, the number of viable bacteria in the activation solution was 6 billion cfu / mL.

[0053] Using activated culture medium as basal culture medium (salinity of 1%), additional NaCl 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g were added to Erlenmeyer flasks containing 100 mL of activated culture medium, respectively, to set a salinity gradient of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%.

[0054] Take 5 mL of the activation solution of Paracoccus mutans and inoculate it into culture media with different salinity gradients. Then place it in a shaker at 30°C and culture it. The OD 600The values ​​and viable counts were used to determine the growth extent of the strain. The results are shown in Table 3.

[0055] Table 3 OD of Paracoccus mutans cultured under different salinity conditions 600 Value and viable count

[0056]

[0057] From the results in Table 3, it can be seen that Paracoccus variabilis can reproduce and grow normally at salinity below 5%, and its growth condition is best under salinity below 4%. This strain is moderately salt-tolerant.

[0058] Example 4. Experiment on degradation of ammonia nitrogen in actual wastewater by Paracoccus mutans

[0059] Under sterile conditions, Paracoccus mutans was inoculated into a 250mL conical flask containing 100mL of activation medium, the composition of the activation medium was: 10g / L tryptone, 5g / L yeast extract powder, 10g / L sodium chloride, the solvent was water and the pH was 6.5-7, and the strain was activated by shaking and culturing in a shaker at 30°C and 200rpm for 24h to obtain an activation solution of Paracoccus mutans. After testing, the number of viable bacteria in the activation solution was 6.5 billion cfu / mL.

[0060] Take the biochemical wastewater from Shandong Kangdien Biotechnology Co., Ltd., adjust the pH to 7.8, and then divide it into a conical bottle with a volume of 250 mL and a liquid volume of 80 mL, and sterilize it at 121°C for 20 min for use.

[0061] Add 80 μl of the activation solution of Paracoccus mutans to the prepared biochemical section wastewater. Three parallel experiments were set up. The biochemical section wastewater without the activation solution of Paracoccus mutans was used as the blank control. The ammonia nitrogen content in the wastewater was tested every 24 hours.

[0062] The ammonia nitrogen detection method was carried out in accordance with “HJ535-2009 Determination of Ammonia Nitrogen-Nessler’s Reagent Spectrophotometry”, and the results are shown in Table 4.

[0063] Table 4 Degradation results of ammonia nitrogen in biochemical wastewater by Paracoccus mutans

[0064]

[0065] According to the data in Table 4, when the inoculation amount of Paracoccus mutans is 1000ppm, the degradation efficiency of ammonia nitrogen in the biochemical section wastewater is very high, the ammonia nitrogen degradation efficiency is about 60% in 24 hours, the ammonia nitrogen degradation efficiency is about 75% in 48 hours, and the ammonia nitrogen degradation efficiency in 72 hours can reach more than 90%.

[0066] Example 5. Fermentation process of Paracoccus mutans

[0067] The fermentation method of Paracoccus mutans comprises the following steps:

[0068] (1) Primary seed culture: Under sterile conditions, Paracoccus variabilis was inoculated into a nutrient medium, the composition of which was: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, the solvent was water, pH = 7-7.5, and cultured at 30°C and 200 rpm for 24 h to obtain a primary seed culture solution;

[0069] (2) Secondary seed culture: Under sterile conditions, the primary seed culture solution was inoculated into a nutrient medium at an inoculum rate of 10 vol%, and cultured at 30° C. and 200 rpm for 18 h to obtain a secondary seed culture solution;

[0070] (3) Fermentation: After the fermentation medium in the fermentation tank is disinfected, the secondary seed culture solution is inoculated into the fermentation medium at an inoculum rate of 1-10 vol%. The fermentation medium composition is: 11 g / L glucose, 2 g / L yeast extract powder, 2 g / L ammonium sulfate, 0.5 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate, 5×10 -3 g / L, fermentation was carried out under the conditions of controlling the temperature to 25-35°C, the rotation speed to 150-300rpm, and the ventilation ratio to 1:(1-2). The fermentation was stopped when the dissolved oxygen began to rise, and the fermentation liquid of Paracoccus mutans was obtained. After testing, the number of viable bacteria in the fermentation liquid was 35 billion cfu / mL.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Paracoccus mutans Paracoccus versutus ) and a microbial agent whose active ingredient comprises Paracoccus variabilis for degrading ammonia nitrogen in sewage and wastewater, wherein the Paracoccus variabilis is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee with the deposit number: CGMCC No. 23528, and the salinity of the sewage and wastewater is 2%-5%.

2. The use according to claim 1, characterized in that: The ammonia nitrogen concentration in the wastewater is below 400 mg / L.

3. The use according to claim 2, characterized in that: The ammonia nitrogen concentration in the wastewater is 100-200 mg / L.

4. The use according to any one of claims 1 to 3, characterized in that: The inoculation amount of the activation solution or microbial agent of the Paracoccus mutans is above 100 ppm.

5. The use according to claim 4, characterized in that: The inoculation amount of the activation solution or microbial inoculant of Paracoccus mutans is 100-2000ppm.

6. The use according to claim 5, characterized in that: The inoculation amount of the activation solution or microbial inoculant of Paracoccus mutans is 100-1000 ppm.

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