Salt-tolerant aerobic denitrification basophilic halomonas, seed solution and application

Abstract

The invention discloses a salt-tolerant aerobic denitrification basophilic halomonas, a seed solution and application, the basophilic halomonas is a basophilic halomonas LJK7, and is preserved in the Guangdong Microbial Culture Collection Center, the preservation time is May 17, 2022, and the preservation number is GDMCC No: 62477. The invention further discloses a preparation method of the basophilic halomonas. The basophilic halomonas LJK7 is a multi-tolerance denitrifying bacterium with efficient denitrification capacity, can provide an excellent strain resource for research, development and improvement of a sewage biological denitrification process, can cope with complex environmental changes of actual wastewater, and has a wide application prospect in actual salt-containing wastewater denitrification.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a salt-tolerant aerobic denitrifying alkaliphilic halomonas bacterium, a seed liquid and an application thereof. Background Art

[0002] With the development of industry and the intensification of human activities, more and more nitrogen elements are transported into water bodies, causing excessive nitrogen content in water bodies. The nitrogen pollution in seawater quality monitored is mainly inorganic nitrogen, and the monitoring of 24 typical marine ecosystems found that 6 were in a healthy state and 18 were in a sub-healthy state. Nitrogen elements mainly enter water bodies through industrial sewage, domestic sewage, agriculture and other channels. Once excessive nitrogen is transported into the water body, it will not only damage the water environment, but also endanger aquatic animals and human health. It can be seen that it is particularly important to control excessive nitrogen in water bodies.

[0003] At present, biological denitrification technology is the main method for treating nitrogen pollution in wastewater. Compared with physical and chemical methods of nitrogen removal, biological denitrification has the advantages of low investment cost, simple operation and no secondary pollution. However, traditional biological denitrification is mainly divided into two independent processes: aerobic nitrification and anaerobic denitrification, which results in the disadvantages of large floor space, complex process, long time and high operating cost. Since 1983, Robertson and Kuenen have isolated a new strain Thiosphaerapantotropha GB17 in the denitrification water treatment system, realizing simultaneous nitrification and denitrification (SND), breaking the concept of autotrophic nitrification and anaerobic denitrification in traditional biological denitrification, overcoming the defects of traditional biological denitrification technology, and providing new ideas for biological denitrification technology.

[0004] With the discovery of heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria, more and more studies have shown that HN-AD bacteria have excellent heterotrophic nitrification and aerobic denitrification characteristics and can achieve efficient nitrogen removal. For example, strains such as Pseudomonas stutzeri LJ-1 have excellent denitrification performance. Therefore, screening HN-AD strains with efficient nitrogen removal has become one of the hot research directions in the field of wastewater treatment.

[0005] In recent years, with the continuous development of new biological denitrification technologies, heterotrophic nitrification-aerobic denitrification bacteria have made great progress. Many researchers at home and abroad have begun to explore this field in depth and continuously produce relevant research results. So far, many species of HN-AD bacteria have been isolated, such as Pseudomonas, Halomonas, Alcaligenes, Acinetobacter and Bacillus, all of which have HN-AD functions. Compared with autotrophic nitrifying bacteria and anaerobic denitrifying bacteria, HN-AD bacteria not only have the advantages of fast reproduction and high denitrification efficiency, but also can adapt to various extreme environmental conditions (such as low C / N, high ammonia nitrogen, high salt, low temperature, high temperature, etc.). The actual wastewater composition conditions are complex, which also makes the screening of strains that tolerate extreme environments and the exploration of their denitrification characteristics a hot topic in the field of HN-AD bacteria research. Summary of the invention

[0006] 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 alkaliphilic halomonas with a wide tolerance range for C / N, salinity, pH and temperature and capable of coping with the complex environmental changes of actual wastewater. The present invention also provides a seed solution of the alkaliphilic halomonas and its application in treating nitrogen-containing wastewater.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A salt-tolerant aerobic denitrifying alkaliphilic halomonas, wherein the alkaliphilic halomonas is Halomonas salifodinae LJK7, which is deposited in Guangdong Microbial Culture Collection Center on May 17, 2022, and the deposit number is GDMCC No: 62477.

[0009] As a general inventive concept, the present invention also provides a seed solution of salt-tolerant aerobic denitrifying alkaliphilic halomonas, wherein the seed solution is prepared by activating and culturing the above-mentioned alkaliphilic halomonas LJK7.

[0010] The above-mentioned seed solution, preferably, the activation culture comprises the following process:

[0011] The alkaliphilic Halomonas LJK7 was inoculated into LB medium and cultured to the logarithmic phase. The obtained bacterial solution was centrifuged to remove the supernatant, and sterilized saline was added after washing to obtain the seed solution of the alkaliphilic Halomonas.

[0012] 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 12 to 18 hours.

[0013] The above-mentioned seed solution, preferably, has a centrifugal speed of 3000-5000 rpm and a centrifugal time of 5-15 min.

[0014] The above-mentioned seed solution, preferably, has a salinity of 3.3% of the sterilized salt water, and the biomass OD600 of the diluted bacterial solution is 0.6-0.8.

[0015] The above-mentioned seed solution, preferably, the culture medium is LB culture medium.

[0016] 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.

[0017] As a general inventive concept, the present invention also provides a use of the above-mentioned salt-tolerant aerobic denitrifying alkaliphilic halomonas in treating nitrogen-containing wastewater.

[0018] In the above application, preferably, the nitrogen-containing wastewater contains NH 4 + -N, NO 3 - -N and NO 2 - -One or more of N.

[0019] In the above application, preferably, the nitrogen-containing wastewater is NO 3 - -N wastewater, the NO 3 - -N wastewater has a C / N ratio of 20 to 80. 3 - -N The salinity of wastewater is 1 to 7%.

[0020] In the above application, preferably, the NO-containing 3 - The pH of the wastewater containing NO is 7 to 11. 3 - -N wastewater temperature is 25℃~40℃.

[0021] In the above application, preferably, the NO-containing 3 - -N The wastewater contains a carbon source.

[0022] In the above application, preferably, the carbon source is one or more of sodium succinate, sodium citrate and sodium acetate.

[0023] Compared with the prior art, the advantages of the present invention are:

[0024] 1. The results of factors affecting the denitrification of aerobic denitrification by strain LJK7 showed that strain LJK7 had a wide tolerance range for C / N, salinity, pH, and temperature: it could grow well when C / N was 20-80, and could remove almost all NO in the range of C / N=20-40. 3 - -N; it can grow well in the salinity range of 1-7%, NO 3 - -N denitrification rate reached more than 81.00%; at pH = 7-11, strain LJK7 could grow normally, and NO 3 - -N removal rate is higher than 95.00%; at a temperature of 25℃~40℃, the strain grows well, at 25~35℃, NO 3 - -N removal rate was higher than 92.00%. At 40℃, the NO 3 - -N removal rate is still 81.42%. Halomonas salifodinae LJK7 is a multi-tolerant denitrifying bacterium with high efficiency of nitrogen removal, which can provide excellent strain resources for the research and development and improvement of sewage biological denitrification process, can cope with the complex environmental changes of actual wastewater, and has broad application prospects in the denitrification of actual saline wastewater.

[0025] 2. Utilization of different nitrogen sources by strain LJK7: 4 + -N, NO 3 - -N, NO 2 - When -N was the only nitrogen source, the nitrogen removal rates of strain LJK7 reached 100%, 84.82% and 77.96% respectively; under mixed nitrogen source culture conditions, strain LJK7 had the ability to simultaneously remove NH 4 + -N, NO 2 - -N and NO 3 - -N capability, NH 4 + -N, NO 2 - -N and NO 3 - -N removal rates were as high as 100%, 94.18% and 96.70% respectively. The above results showed that strain LJK7 is a highly efficient aerobic denitrification strain.

[0026] Halomonas salifodinae LJK7 is deposited in Guangdong Microbiological Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, with the deposit number GDMCC No: 62477 and the deposit date May 17, 2022. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the colony picture of strain LJK7.

[0028] Figure 2 This is the bacterial morphological characteristics of strain LJK7.

[0029] Figure 3 Phylogenetic tree of LJK7 constructed based on 16S rDNA sequence homology.

[0030] Figure 4 For strain LJK7, NH 4 + -N is a diagram of the growth and denitrification rules of a single nitrogen source, where the left diagram is the growth diagram and the right diagram is the denitrification diagram.

[0031] Figure 5 For strain LJK7 with NO 2 - -N is a diagram of the growth and denitrification rules of a single nitrogen source, where the left diagram is the growth diagram and the right diagram is the denitrification diagram.

[0032] Figure 6 For strain LJK7 with NO 3 - -N is a diagram of the growth and denitrification rules of a single nitrogen source, where the left diagram is the growth diagram and the right diagram is the denitrification diagram.

[0033] Figure 7 This is a diagram of the growth and denitrification rules of strain LJK7 using mixed nitrogen sources, where the left figure is the growth diagram and the right figure is the denitrification diagram.

[0034] Figure 8 This graph shows the effects of different carbon sources on the growth and aerobic denitrification performance of strain LJK7.

[0035] Fig. 9 This is a graph showing the effects of different C / N ratios on the growth and aerobic denitrification performance of strain LJK7.

[0036] Fig.10 This graph shows the effects of different salinities on the growth and aerobic denitrification performance of strain LJK7.

[0037] Fig.11This graph shows the effects of different pH on the growth and aerobic denitrification performance of strain LJK7.

[0038] Fig.12 This graph shows the effects of different temperatures on the growth and aerobic denitrification performance of strain LJK7. DETAILED DESCRIPTION

[0039] 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.

[0040] Source of strain: The present invention collected seawater aquaculture sludge from Pushu Village, Dianbai District, Maoming City, Guangdong Province, and obtained a salt-tolerant HN-AD strain named LJK7 through enrichment culture, separation and purification.

[0041] Culture medium and use

[0042] (1) Denitrification medium 1 (DM1): Its formula is shown in Table 1, and it is used to explore the ability of strain LJK7 to remove nitrate nitrogen by denitrification under aerobic conditions;

[0043] (2) Denitrification medium 2 (DM2): Its formula is shown in Table 1 and is used to explore the ability of strain LJK7 to remove nitrite nitrogen by denitrification under aerobic conditions;

[0044] (3) Nitrification medium (NM): Its formula is shown in Table 1, and it is used to test the ability of strain LJK7 to remove ammonia nitrogen through nitrification under aerobic conditions;

[0045] (4) Simultaneous nitrification and denitrification medium (SND): Its formula is shown in Table 1, and it is used to test the simultaneous nitrification and denitrification performance of strain LJK7;

[0046] (5) Weiss salt solution: its formula is shown in Table 1, which provides trace elements for the growth of strain LJK7;

[0047] (6) LB liquid medium: its formula is as shown in Table 1, basic nutrient medium of strain LJK7;

[0048] (7) LB solid medium: Its formula is shown in Table 1, used for morphological observation and preservation of strain LJK7 colonies.

[0049] Table 1 Culture medium types and formulations

[0050]

[0051] Experimental instruments and equipment

[0052] The main experimental instruments and equipment used in this study are shown in Table 2.

[0053] Table 2 Testing instruments and equipment

[0054]

[0055] Experimental methods

[0056] Morphological identification

[0057] The purified target strain LJK7 was streaked onto LB solid culture medium and cultured at 30°C for 36 h to observe the colony morphology. The microscopic morphological characteristics of single colonies were observed using a transmission electron microscope (completed by the Institute of Urban Environment, Chinese Academy of Sciences, Xiamen). A small amount of bacteria was picked and identified by Gram staining to determine whether they were Gram-negative or Gram-positive bacteria.

[0058] The colony characteristics of strain LJK7 are as follows Figure 1 As shown: After the strain LJK7 was cultured on a solid plate for 2 days, round, raised, neatly edged and smooth pale yellow colonies were observed; the bacterial morphology results were as follows Figure 2 As shown: strain LJK7 is short rod-shaped, straight or curved, with flagella and pili.

[0059] Physiological and biochemical identification

[0060] Refer to the Bergey's Bacterial Identification Manual and the Common Bacterial System Identification Manual for physiological and biochemical identification tests. The test items are shown in Table 3:

[0061] Table 3 Physiological and biochemical identification indicators

[0062]

[0063]

[0064] The results of the physiological and biochemical characteristics test of strain LJK7 are shown in Table 4: the results of the catalase and oxidase tests of strain LJK7 are positive; the results of the MR and VP tests are negative, indicating that the decomposition products of the strain are non-acidic substances and do not produce pyruvate; the indole test is negative, indicating that there is no tryptophanase; the citrate test is positive; the starch hydrolysis test is positive, indicating that there is amylase; strain LJK7 can decompose glucose, lactose, sucrose, sucrose and mannitol; the results of the salt tolerance test show that the results are positive under the conditions of 5% and 7% NaCl mass fraction, which indicates that strain LJK7 has good salt tolerance. According to the above characteristics, the Bergey Bacteria Identification Manual and the Common Bacteria System Identification Manual were consulted to judge strain LJK7 as Halomonas.

[0065] Table 4 Physiological and biochemical characteristics of strain LJK7

[0066] Test items result Test items result Oxidase + Citrate + Catalase + Starch hydrolysis + Glucose oxidative fermentation Produces acid but not gas Indole assay - Lactose oxidative fermentation Produces acid but not gas Sportiness - Sucrose oxidative fermentation Produces acid but not gas 5% NaCl + Mannitol oxidative fermentation Produces acid but not gas 7% NaCl + MRI testing - VP test -

[0067] Note: “﹢” indicates positive, and “-” indicates negative.

[0068] Molecular Biology Identification

[0069] The strain LJK7 was inoculated into LB liquid medium and cultured at 30°C for 48h. The bacterial solution was diluted and genomic DNA was extracted using a DNA extraction kit. This was used as a template, and the bacterial 16S rDNA universal primers (27F, 1492R) were used to amplify 16S rDNA by PCR according to the PCR reaction system in Table 5 and the reaction conditions in Table 6. The amplified product was sequenced by Jinweizhi Biotechnology Co., Ltd. after being verified by 1% agarose gel electrophoresis. After sequencing, the NCBI official website was entered, and the sequence was submitted to Blast for comparison analysis. Sequences with higher homology were selected, and the Neighbor-Joining method was used to complete the construction of the phylogenetic tree in MEGA 7.0 software.

[0070] Table 5 16S rDNA sequence amplification reaction system

[0071]

[0072]

[0073] Table 6 PCR reaction conditions

[0074] step Temperature and time cycle Pre-denaturation 95℃3min 1 transsexual 95℃45s 30 annealing 55℃45s 30 extend 72℃45s 30 Final extension 72℃7min 1

[0075] The phylogenetic tree of LJK7 was constructed based on 16S rDNA sequence homology. Figure 3As shown in the figure, the 16S rDNA of LJK7 strain is clustered with Halomonas salifodinae strain ZSH30, and the relationship is the closest. Combining the morphological characteristics of LJK7 with physiological and biochemical tests, the strain was identified as Halomonas salifodinae and named Halomonas salifodinae LJK7.

[0076] Water quality measurement methods

[0077] The various detection indicators in water quality and their corresponding analysis methods are shown in Table 7. 2 - -N, NO 3 - -N and NH 4 + The determination principle and method of -N refer to the national standard "Marine Survey Specification Part 4: Seawater Chemical Element Survey".

[0078] Table 7 Main detection indicators and analysis methods

[0079] Detection indicators Analytical methods <![CDATA[Nitrate nitrogen (NO 3 - -N)]]> Zinc-cadmium reduction method <![CDATA[Nitrite nitrogen (NO 2 - -N)]]> Diazo-azo method <![CDATA[Ammonia nitrogen (NH 4 + -N)]]> Sodium hypobromite oxidation method <![CDATA[Biomass (OD 600 )]]> UV spectrophotometry pH pH Meter

[0080] Study on the Denitrification Performance of Strain LJK7

[0081] Seed solution preparation

[0082] The strain LJK7 was inoculated into LB liquid medium, cultured at 30°C and 150 rpm for 12-18 h, then the bacterial solution was taken out, centrifuged at 4000 rpm for 10 min, the supernatant was removed and the bacterial cells were left. The cells were washed 2-3 times with 3.3% saline (sterilized) and the bacterial concentration in the bacterial solution was diluted to control the biomass OD of the diluted cells. 600 =0.6~0.8.

[0083] Utilization of different inorganic nitrogen species by strain LJK7

[0084] To investigate the ability of strain LJK7 to utilize NH under aerobic conditions 4 + -N, NO 2 - -N and NO 3 - -N capacity, with ammonium sulfate, sodium nitrite, and potassium nitrate as the sole nitrogen source and mixed nitrogen source (ammonium sulfate + potassium nitrate + sodium nitrite), the strain seed liquid with a volume percentage of 3% and in the dry logarithmic phase was inoculated into 70mL of the above culture medium (the culture medium was placed in a 150mL conical flask) containing different nitrogen sources, and cultured at 30℃ and 150rpm for 54h. Samples were taken every 6h to measure the biomass OD of the strain 600 , pH and trinitrogen (NH4 + -N, NO 2 - -N, NO 3 - -N) concentration, and each experiment was repeated three times.

[0085] Ability of strain LJK7 to utilize ammonia nitrogen

[0086] strain LJK7 with NH 4 + -N is the only nitrogen source and the growth and denitrification rules are as follows Figure 4 As shown: strain LJK7 grew slowly within 0 to 6 hours and was in the adaptation period, but NH 4 + -N concentration decreased significantly from 97.05 mg / L to 44.88 mg / L, and NH 4 + The maximum removal rate of -N was 8.70 mg / L / h; the logarithmic growth phase began after 6 to 18 hours, and the biomass OD 600 Rapidly increased from 0.195 to 1.34, 6-12h, NH 4 + -N concentration dropped to 4.53 mg / L, NH 4 + -N removal rate is as high as 95.33%, and the removal rate is 6.73mg / L / h. Within 18h, NH 4 + -N was completely removed; strain LJK7 entered a stable growth phase after 18-54h. 4 + During the culture process with -N as the only nitrogen source, the pH value gradually increased, indicating that the strain LJK7 produced alkali during its growth, and no NO was detected. 2 - -N, NO 3 - -N accumulation, and the results showed that strain LJK7 could exert efficient NH 4 + -N removes capability.

[0087] Ability of strain LJK7 to utilize nitrite nitrogen

[0088] strain LJK7 with NO 2 - -N is the only nitrogen source and the growth and denitrification rules are as follows Figure 5 As shown: strain LJK7 can directly utilize NO 2 - -N is the only nitrogen source for growth metabolism. 600 The results showed that the adaptation period was 0 to 6 hours, and the NH4 + -N is the only nitrogen source growth. The same is that it takes 6h adaptation period to enter the logarithmic growth phase. The difference is that the 6h biomass OD6 00 Only 0.06, NO 2 - The removal rate of -N was much lower than that of NH 4 + -N removal rate; 6-24h strain LJK7 entered the logarithmic growth phase, biomass OD 600 From 0.06 to 1.37, NO 2 - -N concentration decreased significantly from 92.33 mg / L to 25.49 mg / L, and NO 2 - The removal rate of -N reached a maximum of 3.71 mg / L / h, indicating that strain LJK7 could effectively remove NO 2 - -N degradation mainly occurs in the logarithmic growth stage; 24h to 54h enters the stable growth stage, and the final biomass OD 600 1.16, good growth, 24h~30h, NO 2 - -N concentration slowly decreased at a rate of 0.58 mg / L / h, and in the subsequent growth phase, NO 2 - -N concentration tends to be stable. 2 - -N was the sole nitrogen source during the entire growth phase, and the pH rose slowly, indicating that strain LJK7 produced alkali during its growth. 2 - -N is the only nitrogen source, and NO 2 - The removal rate of -N is higher than 77.00%, and there is no NO 3 - -N, NH 4 + -N accumulation, indicating that strain LJK7 is sensitive to NO 2 - -N has good degradation ability and has certain application value in the removal of nitrogen from wastewater containing nitrite nitrogen.

[0089] Ability of strain LJK7 to utilize nitrate nitrogen

[0090] strain LJK7 with NO 3 - -N is the only nitrogen source and the growth and denitrification rules are as follows Figure 6As shown: strain LJK7 grew slowly within 0 to 18 hours and was in the adaptation period. Compared with strain LJK7, 4 + -N and NO 2 - -N as nitrogen source, strain LJK7 3 - -N showed a longer adaptation period when it was the only nitrogen source, and no NH 4 + -N accumulation, NO 2 - -N Almost no accumulation, NO 3 - -N concentration decreased from 103.51 mg / L to 91.18 mg / L, and its removal rate was only 0.89 mg / L / h; strain LJK7 entered the logarithmic growth phase at 18-30h, and the biomass OD 600 The maximum value reached 1.22 at 30h, NO 3 - -N content dropped sharply, and by 30h, NO 3 - -N concentration dropped to 12.39 mg / L, NO 3 - -N removal rate was as high as 6.57 mg / L / h; strain LJK7 was in a stable growth period from 30 to 54 h, OD 600 Around 1.00, the final OD 600 The value is 0.98, NH 4 + The accumulation of a small amount of -N may be due to the fact that strain LJK7 has the function of nitrate dissimilatory reduction to ammonium, or the release of NH4+ during bacterial cell death and lysis. 4 + -N. During the entire growth process, the pH value gradually increased, indicating that strain LJK7 produced alkali during growth and denitrification. At the same time, strain LJK7 was sensitive to NO 3 - -N removal rate exceeded 84.00%, showing good NO 3 - -N removes capability.

[0091] Ability of strain LJK7 to utilize mixed nitrogen sources

[0092] strain LJK7 with NH 4 + -N, NO 3 - -N, NO 2 - -N is a mixed nitrogen source and the growth and denitrification rules are as follows Figure 7As shown in the figure, when the mixed nitrogen source was used for culture, the strain LJK7 had almost no adaptation period and directly entered the logarithmic growth phase, which was different from the culture with NH 4 + The growth of the culture medium with -N as the only nitrogen source was similar to that of the culture medium with 24h biomass OD 600 Reached a maximum value of 1.57; entered a stable growth phase after 24 hours, and the final biomass OD 600 The results showed that the growth of the strain in mixed nitrogen source medium was better than that in single nitrogen source medium. The utilization of different inorganic nitrogen by strain LJK7 was as follows: ammonium sulfate > potassium nitrate > sodium nitrite. 4 + -N concentration dropped sharply, NH 4 + -N concentration decreased significantly from 33.42 mg / L to 2.53 mg / L, with a maximum removal rate of 5.29 mg / L / h. 4 + -N was completely removed, probably because ammonia nitrogen is more easily absorbed, transformed and utilized by cells, and participates in the synthesis of cellular biomacromolecules, or it may be because the enzyme activity of strain LJK7 to oxidize ammonia nitrogen is higher than that of nitrate reductase, which is also the reason why strain LJK7 can be used in NH 4 + -N is the only nitrogen source in the culture medium, which can grow rapidly and remove NH efficiently. 4 + -N reason; NO 3 - -N content changes showed that strain LJK7 degraded NO 3 - -N rate is 3.31mg / L / h, NO 3 - -N and NH 4 + -N was efficiently utilized by strain LJK7, proving that strain LJK7 can simultaneously utilize NH 4 + -N and NO 3 - -N; 6h~12h, NO 3 - -N content keeps decreasing rapidly, 18h~24h, NO 3 - -N content increased slightly, and after 24 hours, NO 3 - -N content is basically stable; NO 2 - -N content determination results show that: 0~6h NO 2 --N is hardly used by strain LJK7, 6h~30h, NO 2 - -N is rapidly degraded, NO 2 - -N concentration decreased significantly from 33.86 mg / L to 2.73 mg / L, and the removal rate was 1.30 mg / L / h. After 30 h, strain LJK7 degraded NO 2 - -N is very slow, up to 54h, NO 2 - The -N removal rate reached 94.18%.

[0093] Factors affecting aerobic denitrification performance of strain LJK7

[0094] Effect of carbon sources on aerobic denitrification performance of LJK7

[0095] To investigate the effect of different carbon sources on the denitrification ability of strain LJK7, the other components of DM1 medium remained unchanged, and the carbon sources were replaced with anhydrous sodium acetate, sodium citrate, sucrose, sodium succinate, glucose and sodium bicarbonate for experiments. The medium without added carbon source was used as a control. 3% (volume fraction) strain LJK7 seed liquid was inoculated into 50 mL of DM1 medium with different carbon sources and cultured at 30°C, 150 rpm for 48 h.

[0096] Carbon sources provide energy and electron donors for denitrifying microorganisms, which is crucial for the growth and denitrification of microorganisms. The effects of different carbon sources on the growth metabolism and denitrification capacity of strain LJK7 are shown in the following table. Figure 8 As shown in the figure, strain LJK7 did not grow when sodium bicarbonate, an inorganic carbon source, was used as the sole carbon source, indicating that strain LJK7 was a non-autotrophic strain; when sodium succinate, sodium citrate, and sodium acetate, small molecular organic carbon, were used as carbon sources, strain LJK7 had good growth and denitrification effects, indicating that strain LJK7 was a heterotrophic strain, and when sodium succinate and sodium citrate were used as carbon sources, strain LJK7 had good NO 3 - -N removal rates were 71.84% and 83.69% respectively, but NH 4 + -N accumulation was obvious. When sodium acetate was used as the carbon source, strain LJK7 grew best, and NO 3 - -N removal rate reached 85.20%, NH 4 + -N only accumulated in small amounts; strain LJK7 could not utilize glucose for growth and denitrification, but had good utilization of sucrose, NO 3 - -N removal rate reached more than 98.00%, but NH 4 +-N accumulation is obvious, causing secondary pollution. In summary, considering that strain LJK7 grows best when sodium acetate is used as the carbon source, NO 3 - -N removal effect is good, and there is almost no accumulation of NO 2 - -N, NH 4 + -N, therefore, sodium acetate was selected as the carbon source in the subsequent experiment to explore the influencing factors of aerobic denitrification of strain LJK7.

[0097] Effect of C / N ratio on aerobic denitrification performance of LJK7

[0098] To investigate the effect of different carbon-nitrogen ratios (C / N) on the denitrification characteristics of strain LJK7, following the aforementioned method, a suitable carbon source was comprehensively selected as the only carbon source for DM1 culture medium, and the other components remained unchanged. The carbon source content was changed, and the culture medium was prepared according to C / N of 10, 20, 40, 60, 80, and 100. 3% (volume fraction) of strain LJK7 seed liquid was inoculated into 50 mL of culture medium with different carbon-nitrogen ratios, and cultured at 30°C, 150 rpm for 48 h.

[0099] The growth, reproduction and denitrification pathway of HN-AD bacteria are closely related to the C / N ratio. The results of the effects of different C / N ratios on strain LJK7 are shown in the figure below. Fig. 9 As shown in the figure: when C / N is 0, that is, when no carbon source is added, strain LJK7 cannot grow, and can only grow and metabolize normally when sufficient carbon source is added; the biomass OD of strain LJK7 in the range of C / N = 10-80 600 The growth rate was good between 0.86 and 2.00, but the aerobic denitrification ability of strain LJK7 was different due to different C / N ratios. When the C / N ratio was 10, NO 3 - -N removal rate was above 60.00%. With the increase of C / N, strain LJK7 3 - -N removal rate also increased. When C / N increased to 20, NO 3 - -N is completely removed, within the range of C / N=20~40, all NO can be removed 3 - -N, when C / N rises to 60, NO 3 - -N removal rate was 84.79%, indicating that strain LJK7 is a strain with a wide tolerance to C / N range and strong denitrification ability. When C / N=80, NO 3 --N removal rate dropped to 51.23%, and denitrification performance was significantly inhibited. When C / N = 100, the strain hardly grew. It may be that the excessive C / N caused excess nutrients, resulting in severe inhibition of strain growth and denitrification. In summary, too low or too high C / N limited the growth rate and denitrification performance of strain LJK7. When C / N = 20-40, strain LJK7 could grow and metabolize normally and completely remove NO 3 - -N, following the principles of low experimental cost and avoiding environmental pollution caused by excess nutrients, C / N=20 was selected in subsequent experiments to study the influencing factors of aerobic denitrification performance.

[0100] Effect of salinity on aerobic denitrification performance of LJK7

[0101] To investigate the effect of different salinities on the denitrification characteristics of strain LJK7, the above method was followed to comprehensively select the optimal C / N. The other components of DM1 culture medium remained unchanged, the NaCl content was changed, and the salinity of the culture medium was set to 0, 1%, 3%, 5%, 7%, 9%, and 12%, respectively. 3% (volume fraction) of strain LJK7 seed liquid was inoculated into 50 mL of culture medium with different salinities and cultured at 30°C and 150 rpm for 48 h.

[0102] The strain LJK7 was isolated and screened from a high-salinity environment. Its growth metabolism and denitrification effects varied with different salinities. Fig.10 As shown: The salinity is 0, the strain cannot grow and no NO is removed 3 - -N; when the salinity was 1-7%, strain LJK7 grew vigorously, OD 600 The values ​​were all greater than 1.60. When the salinity increased from 0 to 1%, the strain LJK7 grew rapidly, and the biomass OD 600 1.70, NO 3 - The removal rate of NO-N was 88.69%, indicating that the growth and denitrification performance of strain LJK7 were very sensitive to salt content; when the salinity increased to 3%, the denitrification ability of strain LJK7 reached the best, and NO 3 - -N removal rate was as high as 98.59%; at a salinity of 5%, strain LJK7 had a significant effect on NO 3 - -N removal can still reach 93.55%; when the salinity increased to 7%, the strain grew well, NO 3 -N removal rate decreased to 81.01%, and denitrification capacity decreased; salinity increased to 9%, the growth and denitrification pathway of the strain were significantly inhibited, OD 600 Reduced to 0.89, NO 3 --N removal rate dropped to 59.56%; at a salinity of 12%, strain LJK7 barely grew and only removed trace amounts of NO 3 - -N.

[0103] In summary, too low or too high salinity limits the growth and reproduction of strain LJK7 and its aerobic denitrification and denitrification capacity. When the salinity is 0, strain LJK7 does not grow, but when the salinity is in the range of 1% to 7%, it grows vigorously, indicating that strain LJK7 is a moderate halophile, NO 3 - -N removal was higher than 80%, indicating that denitrification-related enzymes still maintained high catalytic activity in a wide range of salinity, and strain LJK7 tolerated a wide range of salinity. Considering the growth and denitrification of strain LJK7 under different salinities, 3% salinity was selected for subsequent experiments to explore the influencing factors of aerobic denitrification characteristics of strain LJK7.

[0104] Effect of pH on aerobic denitrification performance of LJK7

[0105] To investigate the effect of different pH values ​​on the denitrification capacity of strain LJK7, the above method was followed to select the optimal salinity. The other components of the DM1 medium remained unchanged, and the pH of the medium was adjusted to 6, 7, 8, 9, 10, and 11, respectively. 3% (volume fraction) of strain LJK7 seed solution was inoculated into 50 mL of culture medium with different pH values, and cultured at 30°C and 150 rpm for 48 h.

[0106] pH is crucial to the growth of microorganisms. It can affect the growth and denitrification of HN-AD bacteria by changing the activity of intracellular enzymes. Fig.11 The results of the effects of different pH on the growth and denitrification of strain LJK7 show that the strain can hardly grow at pH = 6. 600 =0.02, NO 3 - The removal rate of -N was only 0.90%; the strain grew well when the pH was 7, 8, 9, 10, and 11, and the OD 600 When the pH value was equal to or greater than 9, the biomass decreased slightly, but had no effect on the denitrification performance of the strain, and NO 3 - The removal rate of -N was higher than 95.00%. However, when pH = 11, there was a high concentration of NO 2 - -N accumulation, causing serious secondary pollution, may be due to excessively high pH inhibiting NO 2 - -N reductase activity.

[0107] The above results show that strain LJK7 cannot grow and reproduce in a weakly acidic environment, but can exert good denitrification performance in neutral and alkaline environments. Compared with most strains that can only grow in neutral or weakly alkaline conditions, strain LJK7 has the advantage of tolerating a higher alkaline environment. 2 - Taking into account the accumulation of -N, pH = 7-8 was selected in the subsequent experiments to explore the influencing factors of aerobic denitrification characteristics of strain LJK7.

[0108] Effect of temperature on aerobic denitrification performance of LJK7

[0109] To investigate the effect of different temperatures on the denitrification effect of strain LJK7, the above method was followed, the composition of DM1 medium remained unchanged, the medium was adjusted to an appropriate pH, 3% (volume fraction) strain LJK7 seed liquid was inoculated into 50 mL of medium, and the medium was placed at 25°C, 30°C, 35°C, 40°C, and 45°C, respectively, and cultured at 150rpm for 48h.

[0110] All the above influencing factors were tested in triplicate. After 48 h, samples were taken to detect the biomass OD of strain LJK7. 600 , pH and trinitrogen (NH 4 + -N, NO 2 - -N, NO 3 - -N) concentration.

[0111] Temperature has an important influence on the growth and metabolism of HN-AD bacteria. High temperature will change the structure of nucleic acids or proteins and denature them, while low temperature will limit the catalytic activity of enzymes. Fig.12 As shown: When the temperature is 25-40℃, strain LJK7 grows well, OD 600 The lowest was 1.49; when the temperature was 25℃, 30℃, and 35℃, the denitrification rate of strain LJK7 was as high as 92.91%, 95.10%, and 98.66%, respectively; when the temperature was 40℃, the denitrification capacity was slightly reduced, but NO 3 - -N removal rate still reached 81.42%, with very little NO 2 - -N (0.94 mg / L) and NH 4 + -N (0.65 mg / L) accumulation; when the temperature was 45°C, the biomass OD of strain LJK7 600 0.32, NO 3 --N removal rate was reduced to 14.96%, and more NH 4 + -N and NO 2 - -N accumulation, which indicates that the growth and development of strain LJK7 and the denitrification pathway are inhibited by high temperature. In summary, strain LJK7 grows well at 25-40℃ and has a high denitrification rate. It can be concluded that strain LJK7 is an efficient aerobic denitrification bacterium with a wide temperature tolerance range. Since the denitrification rate of strain LJK7 is the highest at 35℃, the optimal culture temperature of strain LJK7 is 35℃.

[0112] in conclusion

[0113] In this study, a salt-tolerant aerobic denitrifying bacterium LJK7 was isolated from marine aquaculture sediments in Dianbai District, Maoming City, Guangdong Province. The strain LJK7 was identified, its utilization of different nitrogen sources was explored, and the factors affecting its aerobic denitrification performance were studied. The following conclusions were drawn:

[0114] (1) Strain LJK7 was identified as Halomonas salifodinae LJK7 through morphological observation, physiological and biochemical tests, and 16S rDNA sequence homology analysis and was named Halomonas salifodinae LJK7.

[0115] (2) Utilization of different nitrogen sources by strain LJK7: When NH 4 + -N, NO 3 - -N, NO 2 - When -N was the only nitrogen source, the nitrogen removal rates of strain LJK7 reached 100%, 84.82% and 77.96% respectively; under mixed nitrogen source culture conditions, strain LJK7 had the ability to simultaneously remove NH 4 + -N, NO 2 - -N and NO 3 - -N capability, NH 4 + -N, NO 2 - -N and NO 3 - -N removal rates were as high as 100%, 94.18% and 96.70% respectively. The above results showed that strain LJK7 is a highly efficient aerobic denitrification strain.

[0116] (3) The results of the factors affecting the denitrification of aerobic denitrification by strain LJK7 showed that strain LJK7 had a wide tolerance range for C / N, salinity, pH, and temperature: it could grow well when C / N was 20-80, and could remove almost all NO in the range of C / N = 20-40. 3 - -N; it can grow well in the salinity range of 1-7%, NO 3 - -N denitrification rate reached more than 81.00%; at pH = 7-11, strain LJK7 could grow normally, and NO 3 - -N removal rate is higher than 95.00%; at a temperature of 25℃~40℃, the strain grows well, at 25~35℃, NO 3 - -N removal rate was higher than 92.00%. At 40℃, the NO 3 - The -N removal rate was still 81.42%. In summary, strain LJK7 is a multi-tolerant strain that can cope with the complex environmental changes of actual wastewater, greatly improving the application range of this strain.

[0117] 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 alkaliphilic halomonas, It is characterized in that The alkaliphilic halomonas is Halomonas salifodinae LJK7, which is deposited in Guangdong Provincial Microbiological Culture Collection Center on May 17, 2022, and the deposit number is GDMCC No: 62477.

2. A seed solution of a salt-tolerant aerobic denitrifying alkaliphilic halomonas, It is characterized in that The seed solution is prepared by activating and culturing the alkaliphilic halomonas LJK7 according to claim 1.

3. The seed solution according to claim 2, It is characterized in that The activation culture comprises the following processes: The alkaliphilic Halomonas LJK7 was inoculated into LB medium and cultured to the logarithmic phase. The obtained bacterial solution was centrifuged to remove the supernatant, and sterilized saline was added after washing to obtain the seed solution of the alkaliphilic Halomonas.

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 12-18 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 sterilized 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 alkaliphilic halomonas 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, NO 3 - -N and NO 2 - -One or more of 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 wastewater has a C / N ratio of 20 to 80. 3 - -N The salinity of wastewater is 1 to 7%.

12. The use according to claim 11, It is characterized in that The NO-containing 3 - The pH of the wastewater containing NO is 7 to 11. 3 - -N wastewater temperature is 25℃~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 and sodium acetate.

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