Salt-tolerant aerobic denitrification Marenobacter spp., seed solution and application of Marenobacter spp.
By screening and identifying the aerobic denitrification-resistant Marinobacter sp. LJK14, the problem of low denitrification efficiency in high-salt wastewater in the prior art was solved, and efficient response and efficient denitrification effect were achieved for complex environmental changes.
Patent Information
- Application Number
- CN202311655163.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-05
- Publication Date
- 2025-06-06
AI Technical Summary
The existing heterotrophic nitrification-aerobic denitrification bacteria have nitrogen removal effects in high-salt wastewater due to salinity limitations and are difficult to cope with complex environmental changes.
Marinobacter sp. LJK14, a salt-resistant aerobic denitrification-resistant strain, was screened and identified. This strain had a large tolerance range to salinity, pH and temperature, and its seed liquid was prepared for treating nitrogen-containing wastewater.
The strain LJK14 can grow in the salinity range of 0 to 9%, and the NO3-N removal rate reaches more than 44.00%, and it shows efficient nitrogen removal ability under pH 6 to 10 and temperatures of 20 to 40°C.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, and in particular to a salt-tolerant aerobic denitrifying Marinobacterium, a seed liquid and an application thereof. Background Art
[0002] In recent years, water pollution has become increasingly serious. Nitrogen is an important pollutant in water, and it mainly exists in four forms: organic nitrogen, ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen. Excessive ammonia nitrogen concentration in water will cause eutrophication of water bodies; excessive nitrate nitrogen concentration will cause human diseases such as hypertension and methemoglobin; nitrate can be converted into nitrite in the body, which can easily induce various diseases and even cause cancer. Therefore, solving the problem of nitrogen pollution in water bodies and studying nitrogen treatment have become one of the hot topics in the current sewage treatment field.
[0003] Heterotrophic nitrification-aerobic denitrification bacteria can simultaneously perform nitrification and denitrification in high-salinity wastewater, reducing nitrogen pollution in wastewater. The denitrification of nitrogen-containing wastewater by heterotrophic nitrification-aerobic denitrification bacteria avoids the shortcomings of traditional processes such as separation of aerobic nitrification and anoxic denitrification processes, large investment, and low efficiency. In nitrogen wastewater treatment applications, it has the characteristics of rapid growth, resistance to organic load, and high denitrification efficiency.
[0004] In recent years, a large amount of high-salt wastewater, that is, wastewater with a total salt content of more than 1%, has been generated in many links of social operation, such as aquaculture effluent, medicine, shipping, chemical industry, papermaking, and seawater utilization. The denitrification effect of heterotrophic nitrification-aerobic denitrification bacteria in high-salt wastewater is limited by salinity. Therefore, screening out a strain of highly efficient and salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria for high-salt wastewater treatment has important application value. Summary of the invention
[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a salt-tolerant aerobic denitrifying Marinobacterium that has a wide tolerance range for salinity, pH and temperature and can cope with the complex environmental changes of actual wastewater. Correspondingly, a seed liquid of the Marinobacterium is also provided, as well as its application in treating nitrogen-containing wastewater.
[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:
[0007] A salt-tolerant aerobic denitrifying Marinobacter, wherein the Marinobacter is Marinobacter sp. LJK14, which is preserved in the Guangdong Provincial Microbial Culture Collection Center on December 23, 2021, and the preservation number is GDMCC No: 62156.
[0008] As a general inventive concept, the present invention also provides a seed solution of salt-tolerant aerobic denitrifying Marinobacterium, wherein the seed solution is prepared by activating and culturing the above-mentioned Marinobacterium LJK14.
[0009] The above-mentioned seed solution, preferably, the activation culture comprises the following process:
[0010] Marinobacterium LJK14 was inoculated into LB medium and cultured to the logarithmic phase. The obtained bacterial solution was centrifuged to remove the supernatant, and sterile saline was added after washing to obtain the Marinobacterium seed solution.
[0011] The above-mentioned seed solution is preferably cultured in a process of shaking at a temperature of 25 to 35° C. and a rotation speed of 100 to 200 rpm for 10 to 15 hours.
[0012] The above-mentioned seed solution, preferably, has a centrifugal speed of 3000-5000 rpm and a centrifugal time of 5-15 min.
[0013] The above-mentioned seed solution, preferably, the salinity of the sterile salt water is 3.3%, and the biomass OD600 of the diluted bacterial solution is 0.6-0.8.
[0014] The above-mentioned seed solution, preferably, the culture medium is LB culture medium.
[0015] The above-mentioned seed solution, preferably, the composition of the LB medium is: tryptone 10g·L -1 , yeast extract 5g·L -1 , NaCl 10g·L -1 , pH 7.0.
[0016] As a general inventive concept, the present invention also provides a use of the above-mentioned salt-tolerant aerobic denitrifying Bacillus marinoi in treating nitrogen-containing wastewater.
[0017] In the above application, preferably, the nitrogen-containing wastewater contains NH 4 + -N and / or NO 3 - -N.
[0018] In the above application, preferably, the nitrogen-containing wastewater is NO 3 - -N wastewater, the NO 3 - -N The salinity of wastewater is 0-9%.
[0019] In the above application, preferably, the NO-containing 3 - The pH of the wastewater containing NO is 6 to 10.3 - -N The temperature of wastewater is 20℃~40℃.
[0020] In the above application, preferably, the NO-containing 3 - -N The wastewater contains a carbon source.
[0021] In the above application, preferably, the carbon source is one or more of sodium succinate, sodium citrate, sodium acetate and sucrose.
[0022] Compared with the prior art, the advantages of the present invention are:
[0023] 1. The results of factors affecting the denitrification of aerobic denitrification by strain LJK14 showed that strain LJK14 had a wide tolerance range for salinity, pH and temperature: it could grow in a salinity range of 0-9%, and NO 3 - -N denitrification rates were all above 44.00%. When the salinity was 3%, 6% and 9%, strain LJK14 had a significant effect on NO 3 - -N removal rates were 99.97%, 99.99% and 96.45% respectively; at pH = 6-10, strain LJK14 could grow normally, and NO 3 - -N removal rate was higher than 60.00%, and when pH value was 9 and 10, strain LJK14 had a significant effect on NO 3 - -N removal rates were 99.99% and 98.80% respectively; at 20℃~40℃, the strain grew well, at 20~30℃, NO 3 - -N removal rate can reach more than 99%. When the temperature rises to 40℃, NO 3 - The -N removal rate was still 31.48%. In summary, strain LJK14 is a multi-tolerant strain that can cope with the complex environmental changes of actual wastewater and has important application value.
[0024] 2. Utilization of different nitrogen sources by strain LJK14: Under aerobic conditions, strain LJK14 used NH 4 + -N, NO 3 - When -N is the only nitrogen source, the denitrification rate can reach about 98%. 4 + When -N was used as the nitrogen source, the denitrification rate was as high as 0.86 mg / L·h; when NH 4 + -N, NO 2 --N, NO 3 - -N mixed nitrogen source NH 4 + -N, NO 3 - The removal rates of -N were 98% and 100%, respectively, indicating that strain LJK14 could easily utilize NH 4 + -N and NO 3 - -N.
[0025] Marinobacter sp. LJK14 is preserved in the Guangdong Microbial Culture Collection Center (abbreviated as: GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, with the preservation number GDMCC No: 62156 and the preservation date of December 23, 2021. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is the colony morphology of strain LJK14.
[0027] Figure 2 This is the bacterial morphology of strain LJK14.
[0028] Figure 3 This is the Gram staining result of strain LJK14.
[0029] Figure 4 Phylogenetic tree of LJK14 constructed based on 16S rDNA sequence homology.
[0030] Figure 5 The growth and denitrification characteristics of strain LJK14 using different nitrogen sources, where (a) is based on NO 3 - -N is the nitrogen source; (b) NO 2 - -N is the nitrogen source; (c) NH 4 + -N is the nitrogen source; (d) NH 4 + -N, NO 2 - -N and NO 3 - -N is a mixed nitrogen source.
[0031] Figure 6 This graph shows the effect of different carbon sources on the aerobic denitrification performance of strain LJK14.
[0032] Figure 7This graph shows the effect of different salinities on the aerobic denitrification performance of strain LJK14.
[0033] Figure 8 This graph shows the effect of different pH values on the aerobic denitrification performance of strain LJK14.
[0034] Fig. 9 This graph shows the effect of different temperatures on the aerobic denitrification performance of strain LJK14. DETAILED DESCRIPTION
[0035] The present invention is further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby.
[0036] The invention obtains a salt-tolerant aerobic denitrifying bacterium labeled as LJK14 from a shrimp tail pond sediment sample in Pushu Village, Dianbai District, Maoming City, after enrichment culture in a salt-tolerant aerobic denitrification medium, screening of target strains, and separation and purification culture.
[0037] Culture medium
[0038] The culture medium types, formulations and sterilization conditions used in the experiment are shown in Table 1.
[0039] Table 1 Culture medium types, formulations and sterilization conditions
[0040]
[0041]
[0042]
[0043] Experimental instruments
[0044] The instruments used in the experiment are shown in Table 2.
[0045] Table 2 Experimental instruments
[0046]
[0047]
[0048] Strain identification
[0049] Morphological identification
[0050] The strain LJK14 was inoculated into beef extract peptone medium, and the bacteria were picked after culturing for 12 hours. LJK14 was Gram-stained using the Gram kit of Changde Beekman Biotechnology Co., Ltd., and the strain morphology and Gram staining results were observed under a microscope. The strain LJK14 was inoculated into beef extract peptone medium and LB medium, and after culturing for 12 hours, it was sent to the Institute of Microbiology, Guangdong Academy of Sciences, and the morphological characteristics and motility of the strain were observed using a transmission electron microscope.
[0051] The morphological characteristics of strain LJK14 are as follows Figure 1 As shown: The results of colony observation showed that LJK14 was flesh-pink, raised, irregularly round colonies with smooth surface, irregular edges, moist and sticky bacteria that were difficult to pick up and had a special smell. Figure 2 As shown: LJK14 bacteria are short rod-shaped, have flagella, no pili, no capsule, and no motility.
[0052] Physiological and biochemical identification
[0053] Physiological and biochemical identification tests were performed on strain LJK14 with reference to the Bergey's Bacteria Identification Manual and the Common Bacteria System Identification Manual. The test items are shown in Table 3:
[0054] Table 3 Physiological and biochemical identification indicators of strain LJK14
[0055]
[0056] Gram staining results Figure 3 Shown: strain LJK14 is a Gram-negative bacterium. The physiological and biochemical characteristics of LJK14 are shown in Table 4: strain LJK14 grows without paraffin sealing, but does not grow with paraffin sealing, indicating that it can adapt to aerobic environment but cannot grow in anaerobic environment; the gas production test is negative, indicating that strain LJK14 does not produce gas; the catalase and oxidase tests are both negative, indicating that strain LJK14 does not contain catalase and oxidase; the MR and VP tests are negative, indicating that the decomposition products of strain LJK14 are non-acidic substances and do not produce pyruvate; the indole test is negative, indicating that strain LJK14 does not have tryptophanase; the citrate test is negative, indicating that strain LJK14 cannot utilize citrate; the starch hydrolysis test is positive, indicating that strain LJK14 has amylase; the results of glucose, lactose sucrose and mannitol oxidation fermentation tests indicate that glucose, lactose, sucrose and mannitol cannot be decomposed; the salinity experiment shows that strain LJK14 can grow at 5% and 10% salinity, but does not grow at 15% salinity, and is a salt-tolerant bacterium.
[0057] After consulting Bergey's Manual of Bacterial Identification and Manual of Systematic Identification of Common Bacteria, it can be preliminarily inferred that strain LJK14 belongs to Kingdom Monera (Prokaryotes), Pseudomonadota (Pseudomonas), Gammaproteobacteria (γ-Proteobacteria), Pseudomonadales (Pseudomonadales), Marinobacteraceae (Marinobacteraceae), and Marinobacter (Marinobacilus).
[0058] Table 4 Results of physiological and biochemical identification experiments of strain LJK14
[0059] project result project result No paraffin sealing + MR determination - With paraffin wax seal - VP determination - Gas production experiment - Citrate - Oxidase - Starch hydrolysis + Catalase - Indole assay - Glucose oxidative fermentation No acid or gas generation Sportiness - Lactose oxidative fermentation No acid or gas generation 5% salinity + Sucrose oxidative fermentation No acid or gas generation 10% salinity + Mannitol oxidative fermentation No acid or gas generation 15% salinity -
[0060] Note: “+” indicates positive, “-” indicates negative
[0061] Molecular Biology Identification
[0062] The strain LJK14 was inoculated into LB medium and cultured for 48 hours. The bacterial liquid DNA was extracted using the Ezup column bacterial genomic DNA extraction kit and PCR amplification was performed. The PCR amplification reaction system is shown in Table 5. The PCR amplification reaction conditions are shown in Table 6. After 1% agarose gel electrophoresis, the PCR amplification product was photographed using a gel imaging system and sent to the Institute of Microbiology of Guangdong Academy of Sciences for 16S rDNA detection to determine the bacterial species. The sequencing results were analyzed by Blast alignment on the NCBI official website, and the phylogenetic tree was constructed using the MEGA7.0 neighbor-joining method to complete the homology analysis of strain LJK14.
[0063] Table 5 PCR amplification reaction system
[0064]
[0065]
[0066] Table 6 PCR amplification reaction conditions
[0067] PCR conditions parameter Pre-denaturation 95℃3min transsexual 95℃40s annealing 55℃40s extend 72℃40s cycle 30 times extend 72℃7min
[0068] The phylogenetic tree of strain LJK14 was constructed based on the 16S rDNA sequencing results. Figure 4As shown: The 16SrDNA of strain LJK14 and Marinobacter sp.U13690101122-SW176 (JQ082151.1:16-1435) are clustered into one branch, with the closest relationship and a similarity of 99.93%. Combined with the morphological characteristics and physiological and biochemical characteristics of strain LJK14, the strain was identified as Marinobacter sp.LJK14. Strain LJK14 was deposited in the Institute of Microbiology, Guangdong Academy of Sciences for patent preservation of strains, and the strain number is GDMCC No.62156.
[0069] The water quality measurement method refers to the measurement principles and methods of each water quality item in the national standard "Marine Survey Specification Part 4: Seawater Chemical Element Survey", and the volume is reduced to measure the denitrification kinetics of strain LJK14. 3 - -N, NO 2 - -N, NH 4 + -N、OD 600 , pH value and total nitrogen (Total nitrogen, TN) are tested. The water quality test methods are shown in Table 7:
[0070] Table 7 Water quality measurement methods
[0071]
[0072]
[0073] Study on the denitrification performance of bacterial strains
[0074] Preparation of LJK14 seed solution
[0075] The strain LJK14 was inoculated into LB medium at 30°C and 150rpm. After culturing for 12h, samples were taken into sterile centrifuge tubes and centrifuged at 4000rpm for 10min, and the supernatant was discarded. After multiple centrifugations to accumulate sufficient bacteria, an appropriate amount of sterile saline (salinity of 3.3%) was added and pipetted evenly. After centrifugation at 4000rpm for 10min, the supernatant was discarded and the bacteria were retained. After repeating this three times, an appropriate amount of sterile saline was added to dilute the bacterial solution, and the bacterial biomass of the bacterial solution was detected using a UV-visible spectrophotometer, and the OD value was controlled at about 0.7 at 600nm.
[0076] The denitrification law of LJK14 was studied by using salt-tolerant aerobic denitrification culture medium with potassium nitrate (0.72 g / L), ammonium sulfate (0.472 g / L), sodium nitrite (0.49 g / L) and mixed nitrogen sources (KNO 3 0.2407 g / L, (NH 4) 2 SO 4 0.1573 g / L, NaNO 2 0.1643 g / L) were nitrogen sources, the initial nitrogen content was unified as 100 mg / L, and the culture medium of each nitrogen source was not inoculated as a blank control. According to the volume of the salt-tolerant aerobic denitrification culture medium, the LJK14 bacterial solution was inoculated into the salt-tolerant aerobic denitrification culture medium at a 3% inoculation amount. The culture conditions were 30°C, 150 rpm, and cultured for 48 h. Samples were taken every 6 h to detect OD 600 , pH value, NO 3 - -N, NO 2 - -N and NH 4 + -H content, calculate the nitrogen removal rate, and set up three parallel experiments in each group. The calculation formula for nitrogen removal rate is as follows:
[0077]
[0078] Nitrogen is an essential element for microbial growth and metabolism. It is also a substrate and performance indicator for aerobic denitrification. Different nitrogen sources have different effects on strains, and strains have different utilization rates of different nitrogen sources. Figure 5 As shown: When strain LJK14 was treated with NO 3 - -N as nitrogen source (such as Figure 5 (a)), the biomass of strain LJK14 can reach a maximum of 1.157 at 48h, and NO 2 - -N and NH 4 + -N has a small amount of accumulation, 42~48hNO 3 - The fastest removal rate of -N was 0.92 mg / L·h, and the 48h NO 3 - -N removal rate can reach up to 97.98%; when strain LJK14 NO 2 - -N as nitrogen source (such as Figure 5 (b) The biomass of strain LJK14 was the highest at 48h, with a biomass of 0.227. 2 - The highest removal rate of -N was 0.24 mg / L·h, and the 36h NO 2 - The highest removal rate of -N was 55.78%, and the NO in the culture medium was 3 - -N and NH 4+ -N accumulation is extremely low, NO 2 - -N removal rate was less than 50%; when strain LJK14 was treated with NH 4 + -N as nitrogen source (such as Figure 5 (c) Figure), the biomass of strain LJK14 can reach a maximum of 1.302 in 36h, and NH 4 + -N removal efficiency can reach up to 97.86%, 30h NH 4 + The fastest removal rate of -N is 0.86mg / L·h, and 48hNO 3 - -N and NO 2 - -N accumulation was extremely low; when strain LJK14 was treated with NH 4 + -NNO 2 - -N and NO 3 - -N is a mixed nitrogen source (such as Figure 5 (d) ): The biomass of strain LJK14 was the highest at 0.302 at 48h, and NH 4 + -N removal rate is up to 100%, 12~24hNH 4 + -N removal rate gradually decreased to 25%, and strain LJK14 removed NO in 24-36h. 3 - -N, generate NH 4 + -N, 36hNO 3 - The removal rate of -N is up to 98%, and the removal rate of NO 3 - The maximum removal rate of -N was 0.92 mg / L·h, and the removal rate of NH 4 + -N removal rate recovered to 100%, and NO in the culture medium was 3 - -N and NH 4 + -N accumulation is extremely low, NO 2 - -N accumulation is relatively large. 2 - When -N and mixed nitrogen sources were used as nitrogen sources, the biomass of LJK14 was low, and the nitrogen removal performance and nitrogen removal rate were not ideal, which may be due to NO 2 --N has an inhibitory effect on the growth and denitrification of LJK14; when potassium nitrate and ammonium sulfate are used as nitrogen sources, strain LJK14 grows well and has a high nitrogen removal efficiency. Therefore, strain LJK14 has a strong NO 3 - -N and NH 4 + -N removal capability, with priority given to removing NH 4 + -N.
[0079] Factors affecting aerobic denitrification performance
[0080] Effect of carbon source on aerobic denitrification performance
[0081] According to the above, the best nitrogen source was selected, and glucose (2.734 g / L), sucrose (2.361 g / L), sodium citrate (4.057 g / L), sodium acetate (3.4197 g / L), sodium bicarbonate (6.954 g / L) and no carbon (0 g / L) were used as carbon sources, respectively. The C / N ratio of different carbon source culture media was kept equal. The culture medium of each carbon source was not inoculated as a blank control. According to the volume of salt-tolerant aerobic denitrification culture medium, the inoculation amount was 3%, and the OD 600 The LJK14 bacterial solution with a concentration of about 0.7 was inoculated into the salt-tolerant aerobic denitrification medium and cultured at 30°C and 150rpm for 48h. Samples were taken to test the OD of each culture medium. 600 , pH value, NO 3 - -N, NO 2 - -N and NH 4 + -H content, calculation of NO 3 - -N removal rate, three parallel experiments were set for each group of experiments.
[0082] Carbon source is an essential nutrient for the growth and metabolism of microorganisms. Different carbon sources have different effects on the growth and performance of strains. 3 - -N removal performance affects the results such as Figure 6 As shown: When glucose was used as the carbon source, the biomass of strain LJK14 was 0.0025, NO 3 - -N removal rate was 20.72%; when sodium bicarbonate was used as carbon source, the biomass of strain LJK14 was 0.002, NO 3 - -N removal rate was 7.83%; when sodium succinate was used as carbon source, the biomass of strain LJK14 could reach 0.717, NO 3 --N removal rate can reach 97.99%; when sucrose is used as carbon source, the biomass of strain LJK14 is 0.024, NO 3 - -N removal rate was 49%; when sodium citrate was used as carbon source, the biomass of strain LJK14 was 0.121, NO 3 - -N removal rate was 28%; when sodium acetate was used as carbon source, the biomass of strain LJK14 was 0.244, NO 3 - -N removal rate was 52%; when no carbon was used as the carbon source, the biomass of strain LJK14 was 0, NO 3 - The above results show that when glucose, sodium bicarbonate, sucrose, sodium citrate, sodium acetate and no carbon are used as carbon sources, the biomass of LJK14 is low, and NO 3 - -N removal performance is not ideal; strain LJK14 grows well when sodium succinate is used as carbon source, and NO 3 - -N removal rate is high. Therefore, the strain LJK14 was determined to use sodium succinate as the carbon source to explore its subsequent aerobic denitrification performance.
[0083] Effect of salinity on aerobic denitrification performance
[0084] According to the above selection of the best nitrogen source and the best carbon source, the salinity gradient was set to 0%, 1%, 3%, 6%, 9% and 12%. The culture medium of each salinity gradient was not inoculated as a control. According to the volume of salt-tolerant aerobic denitrification culture medium, the OD 600 The LJK14 bacterial solution with a concentration of about 0.7 was inoculated into the salt-tolerant aerobic denitrification medium and cultured at 30°C and 150rpm for 48h. Samples were taken to test the OD of each culture medium. 600 , pH value, NO 3 - -N and NO 2 - -N content, calculation of NO 3 - -N removal rate, three parallel experiments were set for each group of experiments.
[0085] Effect of salinity on NO production by salt-tolerant aerobic denitrifying bacteria 3 - -N removal performance is crucial. Different salinities have an important influence on the NO removal performance of strain LJK14 3 - -N removal performance affects the results such as Figure 7 As shown: salinity of 0% and 1%, the biomass of strain LJK14 was 0.036 and 0.081, NO 3- -N removal efficiencies were 44% and 63% with 0% salinity and no NO 2 - -N accumulation. NO is present at 1% salinity 2 - -N accumulation reached 15mg / L; when the salinity was 3%, 6% and 9%, the biomass of strain LJK14 could reach 0.683, 0.749 and 0.796, NO 3 - -N removal rates were 99.97%, 99.99% and 96.45% respectively. 2 - -N accumulation, 43mg / L, 33mg / L and 29mg / L respectively, indicating that a certain salinity promotes the growth of strain LJK14 and NO 3 - -N is removed, but more NO is present 2 - -N accumulation. When the salinity was 12%, the biomass of strain LJK14 was 0.104, NO 3 - -N removal rate was 7.22%, no NO 2 - In summary, the biomass of strain LJK14 was the highest at 9% salinity, but at 6% salinity, strain LJK14 NO 3 - -N removal rate was the highest, therefore, the comprehensive strain LJK14NO 3 - Considering the N removal rate and experimental cost factors, a salinity of 6% was selected to explore the factors affecting the subsequent denitrification of strain LJK14.
[0086] Effect of pH value on aerobic denitrification performance
[0087] According to the above selection of the best nitrogen source, the best carbon source and the best salinity, the pH gradient was set to 6, 7, 8, 9, and 10. The culture medium of each pH gradient was not inoculated as a control. According to the volume of the salt-tolerant aerobic denitrification culture medium, the OD 600 The LJK14 bacterial solution with a concentration of about 0.7 was inoculated into the salt-tolerant aerobic denitrification medium and cultured at 30°C and 150rpm for 48h. Samples were taken to test the OD of each culture medium. 600 , pH value, NO 3 - -N and NO 2 - -N content, calculation of NO 3 - -N removal rate, three parallel experiments were set for each group of experiments.
[0088] pH value can affect the growth state and performance of bacteria. The optimal pH value is conducive to the optimal growth of bacteria and the best performance. 3 - -N removal performance affects the results such as Figure 8 As shown: When the pH values were 6, 7 and 8, the biomass of strain LJK14 was low, which were 0.563, 0.52 and 0.408 respectively, and NO 3 - -N removal rates were 69%, 60% and 69% respectively, with a small amount of NO 2 - -N accumulation; at pH 9 and 10, the biomass of strain LJK14 was 0.872 and 0.421, NO 3 - -N removal rates were 99.99% and 98.80%, respectively, and NO 2 - -N accumulation was very small. In summary, when the pH values were 6, 7 and 8, the biomass of LJK14, NO 3 - -N removal performance is not ideal. Since strain LJK14 produces alkaline substances during growth, which increases the pH value of the culture medium, when the pH is 10, the culture medium environment has reached an alkaline environment, which inhibits the production of alkaline substances by strain LJK14, thereby inhibiting the growth of strain LJK14. Therefore, considering the growth of strain LJK14 and experimental cost factors, pH 9 was selected as the pH value for the subsequent denitrification influencing factor exploration experiment.
[0089] Effect of temperature on aerobic denitrification performance
[0090] According to the above selection of the best nitrogen source, the best carbon source, the best salinity and the best pH value, the temperature gradient was set to 20℃, 25℃, 30℃, 35℃, and 40℃. The culture medium of each temperature gradient was not inoculated as a control. According to the volume of the salt-tolerant aerobic denitrification culture medium, the OD 600 The LJK14 bacterial solution with a concentration of about 0.7 was inoculated into the salt-tolerant aerobic denitrification medium and cultured at 30°C and 150rpm for 48h. Samples were taken to test the OD of each culture medium. 600 , pH value, NO 3 - -N and NO 2 - -N content, calculation of NO 3 - -N removal rate, three parallel experiments were set for each group of experiments.
[0091] Temperature can directly affect the growth state of bacteria, thereby affecting the performance of the strain. The optimal temperature is conducive to the growth of bacteria to the best state and to the best performance. 3 - -N removal performance affects the results such as Fig. 9 As shown: When the temperature is 20℃, 25℃ and 30℃, the biomass of strain LJK14 is 0.859, 0.997 and 0.898, NO 3 - -N removal rates were 73%, 78% and 99.11% respectively; at temperatures of 35℃ and 40℃, the biomass of strain LJK14 was 0.211 and 0.259, and NO 3 - The removal rates of -N were 44.65% and 31.48% respectively. The growth of strain LJK14 and the generation of NO in LJK14 were inhibited at 35℃ and 40℃. 3 - -N removal enzyme activity, thereby inhibiting NO 3 - In summary, when the temperature was 30°C, the biomass of LJK14 was high and NO 3 - -N removal rate is high, and NO 2 - -N accumulation is the least. Therefore, considering the growth condition of strain LJK14 and the growth environment of the strain, 30℃ was selected as the temperature for the subsequent denitrification influencing factor exploration experiment.
[0092] Nitrogen balance analysis According to the above selection of the optimal carbon source, optimal salinity, optimal pH value and optimal temperature, a salt-tolerant aerobic denitrification medium with potassium nitrate as the only nitrogen source was set, and the initial nitrogen content was 100 mg / L. According to the volume of the salt-tolerant aerobic denitrification medium, the inoculation amount was 3%, and the OD 600 The LJK14 bacterial solution with a concentration of about 0.7 was inoculated into the salt-tolerant aerobic denitrification medium and cultured at 30°C and 150 rpm for 48 h. The NO content of each medium was tested every 12 h. 3 - -N, NO 2 - -N, NH 4 + -H and total nitrogen (TN) content, calculate the intracellular nitrogen content and TN removal rate, and set up three parallel experiments for each group of experiments. The formulas for intracellular nitrogen content and TN removal rate are as follows:
[0093] N 胞内氮 (mg / L)=TN 未离心 -TN 已离心
[0094]
[0095] The strain LJK14 was used to generate NO in the salt-tolerant aerobic denitrification medium. 3 - The changes in various nitrogen contents during the -N removal process are shown in Table 8: The nitrogen element in the initial culture medium is NO 3 - -N form, with a total nitrogen content of 94.36 mg / L. In 0-24h, NO 3 --N content decreased rapidly, with the accumulated intracellular nitrogen reaching 40.1 mg / L in 24 h. NO in the culture medium was 3 - -N was 0 mg / L, and the TN removal rate was 20.18%, indicating that strain LJK14 used NO 3 - When -N is used as the nitrogen source, part of it is converted into biomass in the form of assimilation, and the other part is converted into N by aerobic denitrification. 2 In the future, strain LJK14 will use NO 3 - The exact metabolic pathway of NO-N needs to be further analyzed through the metagenomic data of the strain, and isotope tracing technology should be used to accurately verify how strain LJK14 utilizes NO 3 - -N, thus laying a theoretical foundation for its efficient treatment of saline wastewater.
[0096] Table 8 Nitrogen balance analysis
[0097]
[0098]
[0099] in conclusion:
[0100] In this experiment, a salt-tolerant aerobic denitrifying bacterium, LJK14, was obtained from sediment samples of shrimp tail ponds in Pushu Village, Dianbai District, Maoming City, after enrichment culture in salt-tolerant aerobic denitrification medium, screening of target strains, and separation and purification. The results were taxonomic identification, denitrification kinetics, factors affecting denitrification performance, and preliminary exploration of denitrification mechanisms, and the following conclusions were drawn:
[0101] (1) A highly efficient and salt-tolerant aerobic denitrifying bacterium, LJK14, was isolated from the shrimp tailwater sediments in Pushu Village, Dianbai District, Maoming City. It was identified as Marinobacter sp. LJK14 through morphological observation, physiological and biochemical identification and 16S rDNA sequence analysis.
[0102] (2) The utilization of different nitrogen sources by strain LJK14 showed that strain LJK14 had a strong NO 3 --N and NH 4 + -N utilization capacity, and priority is given to NH 4 + -N,NO 2 - -N inhibits the growth and nitrogen removal of strain LJK14, NO 3 - -N utilization ≥ NH 4 + -N utilization > mixed nitrogen source utilization > NO 2 - -N utilization.
[0103] (3)Strain LJK14 NO 3 - The optimized conditions for -N removal were: sodium succinate as carbon source, salinity tolerance range of 3-9%, pH 9, and growth temperature range of 20℃-30℃. The strain LJK14 grew well and the denitrification rate could reach more than 99%.
[0104] (4) NO removal by strain LJK14 3 - -N nitrogen balance analysis showed that strain LJK14 utilized NO 3 - -N is converted into biomass in the form of assimilation, and the other part is converted into N by aerobic denitrification. 2 or N 2 O is released into the atmosphere.
[0105] The above is only a preferred embodiment of the present application and does not constitute any form of limitation to the present application. Although the present application is disclosed as a preferred embodiment as above, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.
Claims
1. A salt-tolerant aerobic denitrifying strain of Marinobacterium, It is characterized in that The Marinobacter sp. is Marinobacter sp. LJK14, which is preserved in the Guangdong Provincial Microbiological Culture Collection Center on December 23, 2021, and the preservation number is GDMCC No: 62156.
2. A seed solution of a salt-tolerant aerobic denitrifying Bacillus marinoi, It is characterized in that The seed liquid is prepared by activating and culturing the Marinobacterium LJK14 described in claim 1.
3. The seed solution according to claim 2, It is characterized in that The activation culture comprises the following processes: Marinobacterium LJK14 was inoculated into LB medium and cultured to the logarithmic phase. The obtained bacterial solution was centrifuged to remove the supernatant, and sterile saline was added after washing to obtain the Marinobacterium seed solution.
4. The seed solution according to claim 3, It is characterized in that The culture process is: shaking culture at a temperature of 25-35° C. and a rotation speed of 100-200 rpm for 10-15 hours.
5. The seed solution according to claim 3, It is characterized in that The centrifugal speed of the bacterial liquid is 3000-5000 rpm, and the centrifugal time is 5-15 min.
6. The seed solution according to claim 3, It is characterized in that The salinity of the sterile saline solution is 3.3%, and the biomass OD600 of the diluted bacterial solution is 0.6-0.
8.
7. The seed solution according to claim 3, It is characterized in that The culture medium is LB culture medium.
8. The seed solution according to claim 7, It is characterized in that The composition of the LB medium is: tryptone 10g·L -1 , yeast extract 5g·L -1 , NaCl 10g·L -1 , pH 7.
0.
9. Use of the salt-tolerant aerobic denitrifying Bacillus marinoi as claimed in claim 1 in treating nitrogen-containing wastewater.
10. The use according to claim 9, It is characterized in that The nitrogen-containing wastewater contains NH 4 + -N and / or NO 3 - -N.
11. The use according to claim 10, It is characterized in that The nitrogen-containing wastewater contains NO 3 - -N wastewater, the NO 3 - -N The salinity of wastewater is 0-9%.
12. The use according to claim 11, It is characterized in that The NO-containing 3 - The pH of the wastewater containing NO is 6 to 10. 3 - -N The temperature of wastewater is 20℃~40℃.
13. The use according to claim 10, It is characterized in that The NO-containing 3 - -N The wastewater contains a carbon source.
14. The use according to claim 13, It is characterized in that The carbon source is one or more of sodium succinate, sodium citrate, sodium acetate and sucrose.