Salt-tolerant heterotrophic nitrification microbial agent and application thereof

Abstract

The invention discloses a salt-tolerant heterotrophic nitrification microbial agent and an application thereof. The salt-tolerant heterotrophic nitrification microbial agent is prepared by compounding seed solutions of a halomonas campparagenia strain LJK10, a marenobacter LJK14 and a halomonas basophilic strain LJK7, and the seed solutions of the halomonas campparagenia strain LJK10, the marenobacter LJK14 and the halomonas basophilic strain LJK7 are mixed to form the salt-tolerant heterotrophic nitrification microbial agent. The volume ratio of the seed solution of the Halomonas campparaganensis strain LJK10 to the seed solution of the Mariobacter LJK14 to the seed solution of the Halomonas basophila LJK7 is (2.5 to 3): (2.0 to 2.5): (1.5 to 2.0). The heterotrophic nitrification microbial agent provided by the invention has salinity tolerance of up to 20%, and still has nitrogen removal efficiency of 100% when the salinity is up to 12%. Therefore, the heterotrophic nitrification microbial agent disclosed by the invention has a huge application prospect in the aspect of biological denitrification of high-salinity mariculture tail water.

Description

Technical Field

[0001] The invention relates to the technical field of microbial wastewater treatment, and in particular to a salt-tolerant heterotrophic nitrifying microbial agent and application thereof. Background Art

[0002] With the booming development of marine aquaculture, the continuous increase in its output is accompanied by increasingly severe environmental pollution problems. Marine aquaculture tail water, due to its high salinity, has become a major challenge to the environment. In the aquaculture process, in order to maintain the growth environment of organisms, salt-rich seawater must be used, which makes the salinity of aquaculture water usually maintained between 30g / L and 35g / L, or even higher.

[0003] The types and structures of pollutants contained in aquaculture tailwater are quite complex. Due to the residues of organic matter such as excrement and feed residues of farmed organisms, as well as possible chemical pollution, the ammonia nitrogen, nitrite, nitrogen, phosphorus content and biological oxygen demand (BOD) index in the water body have increased significantly. It is worth noting that only one-quarter to one-half of the nutrients such as phosphorus and nitrogen put into the aquaculture water body can be absorbed, utilized and assimilated by farmed animals, and most of the rest are dissolved in the water, causing eutrophication of the aquaculture tailwater, which in turn has an adverse effect on the normal growth of farmed organisms.

[0004] Heterotrophic nitrifying bacteria are bacteria that can oxidize nitrogen-containing substances and release energy. They have efficient conversion capabilities and can oxidize ammonia nitrogen to produce products such as hydroxylamine, nitrite and nitrate. Even most heterotrophic nitrifying bacteria have aerobic denitrification properties, and then finally convert ammonia nitrogen into gaseous nitrogen (N2), thereby effectively removing nitrogen from the water body, reducing the nitrogen content in the water body, and improving the water quality environment. This biological process has the characteristics of low energy consumption, safe and stable operation, and excellent denitrification effect. It is an efficient and environmentally friendly denitrification method in marine aquaculture tailwater treatment. However, due to the limited salt tolerance of single bacteria, this has limited the widespread application of this technology to a certain extent. In addition, the high-salt and high-nitrogen concentration tailwater produced by marine aquaculture will significantly inhibit the enzyme activity of microorganisms, destroy the balance of their flora structure, and then cause the denitrification performance of microorganisms to decline significantly during the tailwater treatment process. This phenomenon has largely restricted the widespread application of microbial treatment technology in marine aquaculture tailwater treatment. Therefore, seeking a high-salt tailwater denitrification microbial treatment technology that is both energy-saving and environmentally friendly requires us to find an effective solution. Summary of the invention

[0005] In view of the problems in the background technology, the present invention proposes a method for treating high-salinity seawater aquaculture tail water and NH4 + The invention discloses a salt-tolerant heterotrophic nitrifying microbial agent with high -N removal rate, and also provides its application in reducing the ammonia nitrogen content in high-salt water bodies.

[0006] The present invention adopts the following technical solutions:

[0007] A salt-tolerant heterotrophic nitrifying microbial agent is compounded from the seed liquid of Campania Halomonas strain LJK10, Marinobacterium LJK14 and alkaliphilic Halomonas LJK7, wherein the Campania Halomonas strain LJK10 is deposited in the Guangdong Microbial Culture Collection Center on December 23, 2021, with a deposit number of GDMCC NO: 62158, the Marinobacterium LJK14 is deposited in the Guangdong Microbial Culture Collection Center on December 23, 2021, with a deposit number of GDMCC No: 62156, and the alkaliphilic Halomonas LJK7 is deposited in the Guangdong Microbial Culture Collection Center on May 17, 2022, with a deposit number of GDMCC No: 62477; the volume ratio of the seed liquid of the Campanian Halomonas strain LJK10, the Marinobacterium LJK14 and the alkaliphilic Halomonas LJK7 is 2.5-3: 2.0-2.5: 1.5-2.0.

[0008] Preferably, the volume ratio of the seed solutions of the campanian Halomonas strain LJK10, the Marinobacterium LJK14 and the alkaliphilic Halomonas LJK7 is 2.7-2.9:2.3-2.5:1.5-1.7.

[0009] Preferably, the volume ratio of the seed solutions of the Halomonas campanianus strain LJK10, the Bacillus marinoi strain LJK14 and the Halomonas alkaliphilus strain LJK7 is 2.8:2.4:1.6.

[0010] Preferably, the preparation method of the seed solution of each strain is as follows: the corresponding strain is inoculated into LB medium, cultured in a shaking table at 25-35°C and 100-200rpm for 20-30h, centrifuged the bacterial suspension in a refrigerated centrifuge at 3000-5000rpm for 5-15min, removed the supernatant, precipitated, washed the bacteria with sterile water, centrifuged again to remove the supernatant, repeated 2-3 times, and the OD value of the bacterial suspension was adjusted with sterile water. 600 The concentration was adjusted to 0.6-0.8 to obtain the seed solution of the strain.

[0011] As a general inventive concept, the present invention also provides a use of the above-mentioned salt-tolerant heterotrophic nitrifying microbial agent in reducing the ammonia nitrogen content in a high-salt water body.

[0012] Preferably, the application comprises the following steps: adding a salt-tolerant heterotrophic nitrifying microbial agent and a carbon source to a high-salt water body with a pH of 6 to 10, wherein the volume ratio of the salt-tolerant heterotrophic nitrifying microbial agent to the water body is 3% to 15%, and culturing for 45 to 55 hours.

[0013] Preferably, the ammonia nitrogen concentration in the high-salt water body is 50 to 200 mg·L-1 .

[0014] Preferably, the carbon source is added to the high-salinity water body so that the C / N ratio is 10-100.

[0015] Preferably, the carbon source is sodium citrate.

[0016] Preferably, the salinity of the high-salt water body is 1% to 12%.

[0017] Compared with the prior art, the advantages of the present invention are:

[0018] The heterotrophic nitrifying microbial agent of the present invention can tolerate salinity up to 20%, and still has a 100% denitrification efficiency when the salinity is up to 12%, indicating that the heterotrophic nitrifying microbial agent of the present invention has great application prospects in biological denitrification of high-salinity seawater aquaculture tail water.

[0019] Halomonas campisalis LJK10 was deposited in Guangdong Microbiological Culture Collection Center (GDMCC), Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, China. The deposit number is GDMCC NO: 62158 and the deposit date is December 23, 2021.

[0020] Marinobacter sp. LJK14 is deposited in Guangdong Microbiological Culture Collection Center (GDMCC), Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with the deposit number GDMCC No: 62156 and the deposit date is December 23, 2021;

[0021] 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

[0022] In order to make the present invention more easily understood, the present invention will be described in more detail by referring to the specific embodiments shown in the accompanying drawings. These drawings only depict typical embodiments of the present invention and should not be considered as limiting the scope of protection of the present invention.

[0023] Figure 1 This is the colony picture of strain LJK10.

[0024] Figure 2This is the Gram staining result of strain LJK10.

[0025] Figure 3 This is a transmission electron microscopy scan of strain LJK10.

[0026] Figure 4 Agarose gel electrophoresis results of PCR amplification products.

[0027] Figure 5 This is a phylogenetic tree of strain LJK-10 and other related bacteria with similar homology constructed based on 16S rDNA sequence homology using the Maximum Likelihood method.

[0028] Figure 6 This is the colony morphology of strain LJK14.

[0029] Figure 7 This is the bacterial morphology of strain LJK14.

[0030] Figure 8 This is the Gram staining result of strain LJK14.

[0031] Fig. 9 Phylogenetic tree of LJK14 constructed based on 16S rDNA sequence homology.

[0032] Fig.10 This is the colony picture of strain LJK7.

[0033] Fig.11 This is a diagram of the bacterial morphology of strain LJK7.

[0034] Fig.12 This is the phylogenetic tree of LJK7 constructed based on 16S rDNA sequence homology.

[0035] Fig.13 3D response surface plot for mixture designs.

[0036] Fig.14 This graph shows the effects of different carbon sources on the growth and denitrification efficiency of the composite bacterial agent.

[0037] Fig.15 This graph shows the effects of different carbon-nitrogen ratios on the growth and denitrification efficiency of the composite bacterial agent.

[0038] Fig.16 This graph shows the effect of different salinities on the growth and denitrification efficiency of the composite bacterial agent.

[0039] Fig.17 This graph shows the effect of different pH on the growth and denitrification efficiency of the composite bacterial agent.

[0040] Fig.18This is a graph showing the effect of different NH4+-N concentrations on the growth and denitrification efficiency of the composite bacterial agent (C / N=10).

[0041] Fig.19 This is a graph showing the effect of different NH4+-N concentrations on the growth and denitrification efficiency of the composite bacterial agent (C / N=30). DETAILED DESCRIPTION

[0042] The following describes the implementation modes of the present invention with reference to the accompanying drawings so that those skilled in the art can better understand the present invention and implement it. However, the enumerated embodiments are not intended to limit the present invention. In the absence of conflict, the following embodiments and the technical features in the embodiments may be combined with each other, wherein the same components are represented by the same figure marks.

[0043] Seed liquid preparation Luria-Bertani medium (1L): 5g yeast extract, 10g peptone, 33g sodium chloride. Inoculate the strain into LB medium, culture at 25-35°C, 100-200rpm shaking for 20-30h, centrifuge the bacterial suspension in a 3000-5000rpm refrigerated centrifuge for 5-15min, remove the supernatant, precipitate, wash the bacteria with sterile water, centrifuge again to remove the supernatant, repeat 2-3 times, and use sterile water to adjust the OD of the bacterial suspension. 600 The concentration was adjusted to 0.6-0.8 to obtain the seed solution of the strain.

[0044] Heterotrophic nitrification medium (1L): (NH4)2SO40.47g, (CH2COONa)2·6H2O5.63g, NaCl33g, Weiss salt solution 50mL·L -1 .

[0045] Weiss salt solution (1L): K2HPO4·3H2O 6.54g, MgSO4·7H2O 2.5g, NaCl 2.5g, FeSO4·7H2O 0.05g, MnSO4·H2O 0.04g.

[0046] 1. Source of strains:

[0047] The present invention collected the tail water sediment of shrimp cultured in seawater from Pushu Village, Dianbai District, Maoming City, Guangdong Province, and screened out three salt-tolerant HN-AD bacteria through enrichment culture, separation and screening, namely, Campania Halomonas strain LJK10, Marinobacterium LJK14 and Alkaliphilic Halomonas LJK7. The above three salt-tolerant HN-AD bacteria were used to construct a composite heterotrophic nitrifying agent, and the Design-Expert.V8.0.6.1 software was used to perform mixing design and optimize the ratio of the agent, and the influencing factors of the ammonia nitrogen removal performance of the above heterotrophic nitrifying agent were explored through experiments and the NH4 tolerance of the agent was determined. + -N concentration.

[0048] 2. Strain Identification

[0049] 2.1. Identification of LJK10 strain

[0050] Morphological identification

[0051] Take the bacterial slant, pick bacteria under sterile conditions, use the partitioning and streaking method to obtain bacterial single colonies, and observe the colony morphology, color, and texture. Use a sterile inoculation loop to pick colonies, use a Gram staining box to stain, and after staining, place them under an optical microscope to observe the Gram staining results and bacterial morphology. Send the bacterial slant and seed liquid to the Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, and use a transmission electron microscope to observe the basic structure and morphology of bacteria.

[0052] The colonies of strain LJK10 were Figure 1 The Gram staining results are shown in Figure 2 As shown in the figure: The colonies of LJK10 are round, milky white, opaque, with clear edges and a protrusion in the middle. The overall texture is relatively moist and sticky. The Gram staining results are red, indicating that LJK10 is a Gram-negative bacterium. The transmission electron microscopy scanning results are shown in the figure. Figure 3 As shown: The bacteria are short rod-shaped, have capsules and flagella, and are arranged in single (Figure a), double (Figure b) or multiple (Figure c) clusters.

[0053] Physiological and biochemical identification

[0054] Physiological and biochemical identification experiments were carried out in accordance with the Manual of Identification of Common Bacterial Systems. The experimental items are shown in Table 1:

[0055] Table 1 Physiological and biochemical identification indicators

[0056]

[0057] The results of physiological and biochemical identification of strain LJK10 are shown in Table 2: the catalase test was negative, and the oxidase test was positive, indicating that strain LJK10 was an aerobic bacterium; in the oxidation fermentation experiments of glucose, lactose, sucrose and mannitol, acid was produced in both open and closed tubes, indicating that strain LJK10 can utilize these four sugars under aerobic and anaerobic conditions and belongs to the fermentation type; both MR and VP tests were negative, indicating that the decomposition products of the strain were not acidic and did not produce pyruvate; the citrate test was positive, indicating that the strain could utilize citric acid during growth; the indole test was negative, indicating that there was no indole in the product and the strain did not have tryptophanase; the starch hydrolysis was negative, indicating that the strain did not have amylase and did not decompose starch; the results of the motility test showed that the strain was not motile; it could grow in 5%, 7% and 10% NaCl, indicating that the strain had a wide range of adaptability to salinity and could tolerate higher concentrations of salt. Based on the morphological characteristics of bacterial colonies and cells combined with physiological and biochemical tests, and with reference to the Manual of Systematic Identification of Common Bacteria, it was preliminarily inferred that strain LJK10 was Halomonas sp.

[0058] Table 2 Physiological and biochemical characteristics of strain LJK10

[0059]

[0060]

[0061] Note: “﹢” indicates positive, “﹣” indicates negative

[0062] Molecular Biology Identification

[0063] The bacterial DNA was extracted using the Ezup column-type bacterial genomic DNA extraction kit from Sango Biotech. After the genomic DNA was extracted, the genomic DNA was used as a template to amplify the bacterial 16S rDNA using primers for PCR. The primers used the universal primer sequence of bacterial 16S rDNA: 27f: AGAGTT TGATCC TGG CTC AG; 1492r: TAC GGC TAC CTTGTTACGACT T. The PCR amplification reaction system is shown in Table 3:

[0064] Table 3 PCR amplification reaction system

[0065] system drug Dosage / μL PremixTaq 25 27f 2 50μL system 1492r 2 template 20 <![CDATA[H2O]]> 1

[0066] The PCR amplification reaction process was as follows: 95°C pre-denaturation for 3 min before entering the cycle, 95°C denaturation for 45 s, 55°C annealing for 45 s, 72°C extension for 40 s, 35 cycles, and 72°C extension for 10 min. The PCR product was subjected to 1.3% agarose gel electrophoresis, and the electrophoresis results were photographed using a gel imager. Finally, the product was sent to Genewise Biotechnology Co., Ltd. for sequencing. The sequencing results were aligned on the NCBI official website, and the alignment results were used to construct a phylogenetic tree using the Maximum Likelihood software of MEGA7.

[0067] The extracted strain LJK10 genomic gene was amplified by PCR using bacterial 16SrDNA universal primers 27f and 1492r. The agarose gel electrophoresis results of the amplified product are as follows: Figure 4 As shown, it is not difficult to see from the electrophoresis results that the PCR amplification product band of 16SrDNA is bright and clear, without any mixed bands, and the band size is about 1500bp, indicating that the correct target fragment has been obtained.

[0068] The PCR amplification product of LJK10 was sent to Jinweizhi Biotechnology Co., Ltd. for sequencing, and a 16SrDNA sequence with a fragment size of 1416bp was obtained. According to the comparison results of strain LJK10 and other related bacteria on the NCBI website, the phylogenetic tree constructed based on the homology of strain LJK10 is shown in Figure 5: strain LJK10 has a high homology with most of the Halomonas genus. The 16SrDNA sequence similarity between LJK10 and Halo-monas sp.SSL3 is 99.72%, and the similarity with Halomonas sp.SSL14 is 99.65%. It is most closely related to Halomonas cf.campisalis'campaniae'HKS3, with a sequence similarity of 100%, and it is determined that the bacterium is Halomonas cf.campisalis.

[0069] 2.2. Identification of LJK14 strain

[0070] Morphological identification

[0071] 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.

[0072] The morphological characteristics of strain LJK14 are as follows Figure 6As 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 7 As shown: LJK14 bacteria are short rod-shaped, have flagella, no pili, no capsule, and no motility.

[0073] Physiological and biochemical identification

[0074] 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 4:

[0075] Table 4 Physiological and biochemical identification indicators of strain LJK14

[0076]

[0077] Gram staining results Figure 8 Shown: strain LJK14 is a Gram-negative bacterium. The physiological and biochemical characteristics of LJK14 are shown in Table 5: 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.

[0078] 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).

[0079] Table 5 Results of physiological and biochemical identification experiments of strain LJK14

[0080] 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 -

[0081] Note: “+” indicates positive, “-” indicates negative

[0082] Molecular Biology Identification

[0083] 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 6. The PCR amplification reaction conditions are shown in Table 7. 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.

[0084] Table 6 PCR amplification reaction system

[0085]

[0086]

[0087] Table 7 PCR amplification reaction conditions

[0088] PCR conditions parameter Pre-denaturation 95℃3min transsexual 95℃40s annealing 55℃40s extend 72℃40s cycle 30 times extend 72℃7min

[0089] The phylogenetic tree of strain LJK14 was constructed based on the 16S rDNA sequencing results. Fig. 9 As 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.

[0090] 2.3. Identification of LJK7 strain

[0091] Morphological identification

[0092] 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.

[0093] The colony characteristics of strain LJK7 are as follows Fig.10 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 Fig.11 As shown: strain LJK7 is short rod-shaped, straight or curved, with flagella and pili.

[0094] Physiological and biochemical identification

[0095] 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 8:

[0096] Table 8 Physiological and biochemical identification indicators

[0097]

[0098] The results of the physiological and biochemical characteristics test of strain LJK7 are shown in Table 9: 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 pyruvic acid; 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 that strain LJK7 is Halomonas.

[0099] Table 9 Physiological and biochemical characteristics of strain LJK7

[0100] 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 -

[0101] Note: “﹢” indicates positive, and “-” indicates negative.

[0102] Molecular Biology Identification

[0103] The strain LJK7 was inoculated into LB liquid medium and cultured at 30°C for 48h. The bacterial solution was diluted and the 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 the 16S rDNA by PCR according to the PCR reaction system in Table 10 and the reaction conditions in Table 11. The amplified product was verified to be correct by 1% agarose gel electrophoresis and then sequenced by Jinweizhi Biotechnology Co., Ltd. After the sequencing was completed, the NCBI official website was entered, and the sequence was submitted to Blast for comparison analysis. The sequence with higher homology was selected, and the Neighbor-Joining method was used to complete the construction of the phylogenetic tree in MEGA7.0 software.

[0104] Table 10 16S rDNA sequence amplification reaction system

[0105]

[0106] Table 11 PCR reaction conditions

[0107] 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

[0108] The phylogenetic tree of LJK7 was constructed based on 16S rDNA sequence homology. Fig.12 As 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.

[0109] 3. Preliminary study on the mixing of heterotrophic nitrifying bacteria and optimization of the combination ratio

[0110] Preparation of seed solution: LJK10, LJK14, and LJK7 were inoculated into LB medium and cultured in a shaking incubator at 30°C and 150 rpm for 24 h. The bacterial suspension was centrifuged in a refrigerated centrifuge at 4000 rpm for 10 min, the supernatant was removed, the precipitated bacteria were washed with sterile water, and the supernatant was removed by centrifugation. This was repeated 2-3 times. The OD value of the bacterial suspension was adjusted with sterile water. 600 Adjust to 0.6-0.8 as the seed solution for LJK10, LJK14, and LJK7.

[0111] A preliminary study on functional bacteria mixture: NH4 + -N concentration is 100 mg·L -1, C / N is 30, pH is 7, and the preliminary design of mixed strains is shown in Table 11. The seed liquid of each strain was inoculated into 100 mL of heterotrophic nitrification medium according to the ratio in Table 12, and cultured in a shaker at 30°C and 150 rpm for 48 h. The above experimental treatments were repeated 3 times, and the NH4 in the system was measured every 6 h. + -N content.

[0112] Table 12 Preliminary design of mixed strains

[0113]

[0114]

[0115] A preliminary study on NH4 in the culture system of three bacterial strains + -N content changes are as follows: Compared with the strains of the composite bacterial agent, the NH4 + -N removal rate was improved. Each strain combination had 24hNH4 + -N removal rate basically reached the maximum, among which the NH4 + -N removal rate reached 88.3%, LJK10 + LJK7 combination of NH4 + -N removal rate reached 76.2%, and the NH4 + -N removal rate is 90.5%, LJK14 + LJK7 combination of NH4 + -N removal rate was 72.3%. The above results show that the combination of three aerobic denitrifying bacteria expressed good denitrification performance and can be symbiotically cultivated.

[0116] Mixing design determines the compound ratio of microbial agents: NH4 + -N concentration is 200 mg·L -1 , C / N was 15, pH was 7, inoculation volume was 6%, A (LJK10), B (LJK14), C (LJK7) were selected as optimization factors, the boundary values ​​of the inoculation volume of the three strains of seed liquid were set to 0-5mL, the Mixture mixture design experiment in Design-Expert 8.0.6 software was used, and the experiment was arranged according to the mixture design. The seed liquid of each strain was inoculated into a heterotrophic nitrification medium with a volume of 100mL according to the experimental design ratio, and cultured at 30℃, 150rpm shaker for 48h. The above experimental treatments were repeated 3 times, and the NH4 in the system was measured every 6h + -N content.

[0117] Mixture Design Model Analysis:

[0118] Using Design Expert 13 response surface software, a quadratic fit was performed on the data in Table 13 mixture design results to obtain NH4 + -N removal rate (R1) regression equation:

[0119] R1=3.71A+3.53B+3.34C+4.00AB+2.54AC+4.63BC+41.43ABC

[0120] The results of variance analysis of the regression equation are shown in Table 14: The experimental model was extremely significant (p < 0.01), AB (LJK10 + LJK14), BC (LJK14 + LJK7), AC (LJK10 + LJK7) and ABC (LJK10 + LJK14 + LJK7) had a significant effect on the composite bacterial agent NH4 + -N removal rate was significantly affected (p<0.01), while other factors were not significantly affected (p>0.05). Model correlation coefficient R 2 =0.9055, indicating that the model equation can explain 90.55% of the response value (composite bacterial agent NH4 + -N removal rate) changes; Corrected correlation coefficient R 2 (Adj) =0.8539, R 2 With R 2 (Adj) The difference is within 0.2, the correlation between the predicted value and the true value is high, the equation model has high credibility, and the regression equation has good fitting. This model can be used to analyze the relationship between the proportions of different functional bacteria in the composite microbial agent.

[0121]

[0122]

[0123] Results Analysis The mixture design contours and 3D response surface show that the three-dimensional response surface diagram is spherical, such as Fig.13 As shown, the mixing ratio of strains LJK10, LJK14, and LJK7 has an effect on the composite bacterial agent NH4 + -N removal has an interactive effect; the presence of a "red vertex" in the contour map indicates that the proportion of the bacterial population of the composite microbial agent is within the experimental design range. + -N removal rate has a maximum value, A (LJK10) is the dominant factor, when the ratio of strain LJK10 is 45.0%, NH4 + -N removal rate is the largest. The above experimental results show that when the strain LJK10:LJK14:LJK7=2.4∶2.2∶1.4, the denitrification effect of YL bacterial agent is the best.

[0124] 4. Optimization of growth conditions of composite bacterial agents

[0125] In the process of optimizing the heterotrophic nitrification performance of the composite bacterial agent, a single-factor experimental device with different inorganic and organic carbon sources (sodium bicarbonate, sucrose, glucose, sodium citrate, sodium acetate), carbon-nitrogen ratio (0, 10, 30, 50, 80, 100, 150), salinity (1%, 4%, 8%, 12%, 16%, 20%) and pH value (5, 6, 7, 8, 9, 10) was designed to compare and analyze the denitrification effect of the composite bacterial agent under different conditions and determine the optimal growth conditions of the bacterial agent. In addition, the effects of different ammonia nitrogen concentrations on the growth and denitrification effect of the composite bacterial agent under low carbon-nitrogen ratio (10) and high carbon-nitrogen ratio (30) were designed. This series of research and analysis laid a solid foundation for the subsequent use of the bacterial agent in the fermentation tank to more efficiently remove ammonia nitrogen from aquaculture tail water.

[0126] The seed liquid of the three strains was compounded to form a composite bacterial agent, which was then introduced into a fermenter for cultivation. Fermentation was carried out in sequence according to the single factors of carbon source, carbon-nitrogen ratio, salinity, pH, and ammonia nitrogen concentration. The experimenters configured the culture medium according to the designed culture medium ratio. The configured and sterilized culture medium flowed into the fermenter through a sealed pipe. The volume ratio of the heterotrophic nitrifying composite bacterial agent to the high salt water in the fermenter was 3% to 15%. After a set culture time of 45 to 55 hours, the effects of each single factor on the strain LJK10 were detected.

[0127] Carbon source optimization

[0128] Carbon source is a key factor in the growth of microorganisms, providing the carbon skeleton and energy required to build cell structures. Different carbon sources will lead to differences in the metabolic pathways and physiological characteristics of microorganisms. The growth status and denitrification effect of composite bacterial agents under different carbon sources are as follows: Fig.14 As shown: In the carbon-free medium, no growth was found in the composite bacterial agent after 48 hours of cultivation, and NH4 + -N removal rate is 0, almost no NO3 is produced - -N and NO2 - -N, which shows that carbon source plays an indispensable role in the growth and product production of microorganisms; when the composite bacterial agent uses inorganic carbon source sodium bicarbonate as carbon source, its biomass is 0.003 after 48h of cultivation, NH4 + -N removal rate does not exceed 40%, almost no NO3 is produced - -N and NO2 - -N, indicating that the composite bacterial agent showed weak growth under the culture conditions of inorganic carbon source, but the ammonia nitrogen removal rate was better than that of general heterotrophic nitrifying bacteria, indicating that the bacteria may have certain potential in inorganic carbon source culture medium; when organic carbon sources sucrose and glucose were used as carbon sources, the OD of the composite bacterial agent was 600 are 0.014 and 0.050 respectively, and its NH4 +-N removal rates were 14.99% and 24.07% respectively. Compared with the sodium bicarbonate group, this group of data showed that the composite bacterial agent was more inclined to grow in the organic carbon source medium, because heterotrophic bacteria usually use organic matter as a carbon source to support their growth and metabolic activities, but its ammonia nitrogen removal rate was lower than that of the sodium bicarbonate group. It may be that the bacteria are not adapted to grow under sucrose and glucose, two types of organic carbon sources, so that its ammonia nitrogen removal ability is greatly weakened; while in the medium with sodium citrate and sodium acetate as carbon sources, the denitrification efficiency of the composite bacterial agent was significantly improved, and NH4 + The removal rates of -N were 99.96% and 96.92% respectively, indicating that the composite bacterial agent prefers to utilize sodium citrate and sodium acetate, and the composite bacterial agent grows best when sodium citrate is used as the carbon source. In summary, sodium citrate is the best carbon source for the composite bacterial agent.

[0129] Optimization of carbon-nitrogen ratio

[0130] The carbon-nitrogen ratio refers to the ratio of the total content of carbon to nitrogen in an organism or culture medium, which is related to the growth and metabolic activity of microorganisms. Too high a carbon-nitrogen ratio makes it impossible for microorganisms to synthesize enough protein and other nitrogen-containing compounds. Under low carbon-nitrogen ratio conditions, the strain cannot effectively remove nitrogen due to the lack of sufficient electron donors and energy sources, and causes the accumulation of metabolites. The growth status and denitrification effect of the composite bacterial agent under different carbon-nitrogen ratios are shown in Figure 2. Fig.15 As shown: When the carbon-nitrogen ratio of the composite bacterial agent is 0, NH4 + -N removal rate is 0, almost no NO is produced 3- -N and NO 2- -N, the composite agent showed no signs of growth, and the biomass was 0, indicating that the composite agent could not obtain sufficient carbon source and energy to support growth. Therefore, the carbon-nitrogen ratio plays a key role in the growth and metabolism of microorganisms. When the carbon-nitrogen ratio was 10, the biomass of the composite agent was high, 1.277, and the ammonia nitrogen removal rate was as high as 85.28%, indicating that with the increase of the carbon-nitrogen ratio, the carbon and nitrogen sources in the culture medium were fully utilized by the strain, exerting its significant denitrification ability. When the carbon-nitrogen ratio was 150, the denitrification efficiency of the strain decreased significantly, and NH4 + -N removal rate dropped to 55.34%, and almost no NO was generated 3- -N and NO 2- -N, the strain is not growing well, OD 600 The value was 0.082, indicating that the strain showed intolerance to high carbon-nitrogen ratio. Excessively high carbon-nitrogen ratio restricted the strain's utilization of nitrogen, thus affecting its normal physiological function and metabolic pathway. When the carbon-nitrogen ratio was between 30 and 100, the denitrification rate of the composite bacterial agent was high, and NH4 +-N removal rate was as high as 100%, with a small amount of NO3--N accumulation, which was consistent with the growth of the strain, and OD600 was maintained at 1.92-1.99. In summary, the heterotrophic nitrifying bacteria in this study have a wider range of adaptability to the carbon-nitrogen ratio, which is better than most heterotrophic nitrifying bacteria.

[0131] Salinity Optimization

[0132] Appropriate salinity is crucial for the growth of heterotrophic nitrifying bacteria. Excessive salt concentration in the growth environment will make the microorganisms sensitive to the external environment, affect the normal metabolism of the microorganisms, and even cause the death of the microorganisms. Fig.16 As shown: When the salinity of the composite bacteria agent is between 1% and 12%, the growth of the strain is good, and the OD 600 Generally higher than 1.5, when the salinity is 4%, OD 600 The maximum value reached 2.034. In this range (1%-12%), the biomass of the strain showed a trend of first increasing and then decreasing, and NH4 + -N removal rate is as high as 100%, almost no NO3 is produced - -N and NO 2- -N; When the salinity exceeded 16%, the growth of the composite bacterial agent was obviously restricted to a certain extent, and the biomass dropped to 0.18 (16%) and 0.02 (20%), respectively, and NH4 + The removal rate of -N decreased significantly. The ammonia removal rates at high salinity of 16% and 20% were 47.29% and 25.81% respectively, and almost no NO was produced. 3- -N and NO 2- -N, which shows that the composite bacterial agent can adapt to higher salinity conditions and has good salt tolerance. Studies have shown that high-salt environments interfere with the absorption of organic matter by bacteria, induce cytoplasm dissolution, greatly reduce their survival rate, and even cause bacteria to almost die. The three strains in the composite bacterial agent of the present invention are all heterotrophic nitrification-aerobic denitrification bacteria separated and purified from a seawater aquaculture farm in Dianbai District, Maoming City. Their salt tolerance is significantly higher than the above general heterotrophic nitrification strains, and the strains in the bacterial agent may be able to produce and secrete specific metabolites under specific salt concentrations. These substances help them maintain osmotic pressure balance in the salt environment, thereby promoting the growth and denitrification ability of the strains, which just proves that the composite bacterial agent has the ability to treat a certain concentration of seawater aquaculture tail water. The heterotrophic nitrifying bacteria in this study showed a wide range of salt tolerance (1%-16%), which is particularly outstanding compared to most nitrifying bacteria.

[0133] Previous studies have shown that LJK10 tolerates salinity of 6% in ammonia nitrogen water. When the salinity is 5%, the OD 600 and NH4 +-N removal rate decreased; LJK14 tolerated salinity of 7% in ammonia nitrogen water, and when the salinity was 6%, the bacterial OD 600 and NH4 + -N removal rate decreased; LJK7 tolerated salinity of 6% in ammonia nitrogen water, and when the salinity was 5%, the bacterial OD 600 and NH4 + The -N removal rate is reduced.

[0134] pH Optimization

[0135] The enzymatic reaction in the microorganism needs to be carried out at an appropriate pH. The pH can also change the charge distribution on the membrane surface, thereby affecting the absorption and utilization of substances by microorganisms, and further affecting the growth of heterotrophic nitrification-aerobic denitrification bacteria. Fig.17 As shown: The composite bacterial agent showed only weak growth signs at pH 5, with a biomass of 0.023, and did not show denitrification ability, with an ammonia nitrogen removal rate of 0% and almost no NO production 3- -N and NO 2- -N, which may be because most heterotrophic nitrifying bacteria are suitable for growth in a neutral (pH6.0-9.0) environment. The over-acidic environment inhibits the growth of microorganisms and cannot fully exert their growth and denitrification potential. This further shows that the composite bacterial agent is not suitable for growth under over-acidic conditions; when the pH is 6-10, the composite bacterial agent has excellent growth conditions, and OD 600 Generally higher than 1.47, and NH4 + -N removal rate is as high as 100%, with a small amount of NO3 - -N accumulation, but when the pH is 10, the strain biomass is reduced to 1.47, indicating that the overly alkaline environment is also not conducive to the growth of microorganisms. Therefore, neutral to weakly alkaline culture medium is more conducive to the growth of composite bacteria and ammonia nitrogen metabolism. The pH tolerance range of different heterotrophic nitrifying bacteria is different. The pH tolerance range of composite bacteria is wider (6.0-10.0), which is better than that of single bacteria. Studies have shown that a weakly alkaline environment may be more conducive to heterotrophic nitrification-aerobic denitrification. This is because the weakly alkaline environment promotes the partial NH4 + Converted into NH3, the state of free ammonia is more conducive to bacteria's ammonia nitrogen metabolism.

[0136] NH4 + -N concentration tolerance test

[0137] Ammonia nitrogen, as one of the commonly used nitrogen sources in heterotrophic nitrifying microbial culture media, can provide nutrients to microorganisms to maintain their growth. Too high or too low ammonia nitrogen concentrations will have an adverse effect on the metabolism of microorganisms. Too low ammonia nitrogen concentrations will keep microorganisms in a low nutritional state during the culture process, which is not conducive to their growth, while too high concentrations will inhibit microbial growth, resulting in their heterotrophic nitrification performance cannot be fully exerted. This experiment focuses on the growth and denitrification performance of the composite bacterial agent in the carbon-nitrogen ratio optimization experiment. It is designed to test the effect of initial ammonia nitrogen concentration on the growth and denitrification of the composite bacterial agent under carbon-nitrogen ratios of 10 and 30.

[0138] When the carbon-nitrogen ratio of the composite bacterial agent is 10, the growth status and denitrification effect under different ammonia nitrogen concentrations are as follows Fig.18 As shown in the figure, with the increase of initial ammonia nitrogen concentration, the biomass of the composite bacterial agent also increased, and OD 600 The biomass of the composite bacteria agent exceeded 1 when the ammonia nitrogen concentration was 60 mg / L to 120 mg / L, but when the ammonia nitrogen concentration reached 100 mg / L and 120 mg / L, the biomass of the composite bacteria agent grew higher, and there was a small amount of NO3 - -N accumulation. It is worth noting that when the ammonia nitrogen concentration is 60 mg / L, the denitrification efficiency of the composite bacteria is the highest, reaching 94.79%. When the ammonia nitrogen concentration is 80, 100, and 120 mg / L, the denitrification efficiency of the composite bacteria is 91.49%, 93.16%, and 91.09%, respectively. The average denitrification efficiency of this experimental group is as high as 92.63%, indicating that the composite bacteria exhibits excellent denitrification ability when the initial ammonia nitrogen concentration is 60 mg / L to 120 mg / L, indicating that the bacteria has a wide range of ammonia nitrogen tolerance when the carbon-nitrogen ratio is 10, and also has the ability of heterotrophic nitrification under low ammonia nitrogen concentration conditions.

[0139] When the carbon-nitrogen ratio of the composite bacterial agent is 30, the growth status and denitrification effect under different ammonia nitrogen concentrations are as follows Fig.19 As shown in the figure, with the increase of initial ammonia nitrogen concentration, the growth of the composite bacterial agent also increased, and the OD600 values ​​were 1.413, 1.902, 1.995, and 2.039, respectively, indicating that with the increase of ammonia nitrogen concentration in the culture medium, the biomass of the composite bacterial agent increased, while NO3 - -N and NO2 --N accumulation decreased accordingly. When the ammonia nitrogen concentration was 50mg / L to 200mg / L, the composite bacterial agent showed good denitrification ability, and the ammonia nitrogen removal rates were 99.86%, 99.96%, 100%, and 99.82%, respectively. The average denitrification efficiency of the whole group was as high as 99.91%, indicating that the bacteria had a strong tolerance to high ammonia nitrogen concentrations in a culture medium with a carbon-nitrogen ratio of 30. Although the biomass was the lowest when the ammonia nitrogen concentration was 50mg / L, its denitrification rate could be as high as 99.86%, indicating that the bacteria could also efficiently treat ammonia nitrogen at lower ammonia nitrogen concentrations. The performance of the composite bacterial agent at 50-200 mg / L ammonia nitrogen concentration is better than that of general heterotrophic nitrification-aerobic denitrification bacteria. For example, the strain Rhodococcus sp.LS-2 studied by Hou Dongmei et al. and Pseudomonas sp.LLM-5 studied by Cao Xuesong et al. had an average denitrification efficiency of no more than 50% at low ammonia nitrogen concentration (50-80 mg / L) and high ammonia nitrogen concentration (100-200 mg / L). In contrast, the composite bacterial agent showed its wide range of ammonia nitrogen tolerance.

[0140] Optimization and verification conclusion

[0141] After a series of single-factor experiments, the composite bacterial agent has a wide range of adaptability to the carbon-nitrogen ratio (30-100), and exhibits salt tolerance in the salinity range of (1%-12%); at the same time, the pH tolerance range (6.0-10.0) is also relatively wide, and the ammonia nitrogen removal rate of the composite bacterial agent is as high as 100% within the above tolerance range. In addition, the composite bacterial agent exhibits a wide range of ammonia nitrogen tolerance (50-200 mg / L) under high and low carbon-nitrogen ratios, and in this range, the ammonia nitrogen removal rate exceeds 90% on average.

[0142] After comprehensive consideration of the cost issue, sodium citrate was selected as the best carbon source, and the carbon-nitrogen ratio was set to 30, the salinity was 4%, and the pH was 8 as the optimal growth conditions for the bacterial agent. Through the above experimental results, it can be found that the composite bacterial agent has a wide tolerance range for carbon-nitrogen ratio, salinity, pH and ammonia nitrogen concentration. Therefore, the application of the composite bacterial agent in the treatment process of marine aquaculture tail water has certain feasibility and application prospects.

[0143] The embodiments described above are only preferred specific implementations of the present invention. This specification uses the phrases "in one embodiment", "in another embodiment", "in yet another embodiment" or "in other embodiments" to refer to one or more of the same or different embodiments according to the present disclosure. Common changes and substitutions made by those skilled in the art within the scope of the technical solution of the present invention should be included in the protection scope of the present invention.

Claims

1. A salt-tolerant heterotrophic nitrifying microbial agent, characterized in that: It is compounded by the seed liquid of Campanian Halomonas strain LJK10, Marinobacterium LJK14 and alkaliphilic Halomonas LJK7. The Campanian Halomonas strain LJK10 is deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 23, 2021, and the preservation number is GDMCC NO: 62158. The Marinobacterium LJK14 is deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 23, 2021, and the preservation number is GDMCC No: 62156. The alkaliphilic Halomonas LJK7 is deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 17, 2022, and the preservation number is GDMCC No: 62477; the volume ratio of the seed liquid of the Campanian Halomonas strain LJK10, the Marinobacterium LJK14 and the alkaliphilic Halomonas LJK7 is 2.5-3: 2.0-2.5: 1.5-2.

0.

2. The salt-tolerant heterotrophic nitrifying microbial agent according to claim 1, characterized in that: The volume ratio of the seed liquid of the campanian salt mononas strain LJK10, the marinobacterium LJK14 and the alkaliphilic salt mononas LJK7 is 2.7-2.9:2.3-2.5:1.5-1.

7.

3. The salt-tolerant heterotrophic nitrifying microbial agent according to claim 2, characterized in that: The volume ratio of the seed liquid of the campanian Halomonas strain LJK10, the Marinobacterium LJK14 and the alkaliphilic Halomonas LJK7 is 2.8:2.4:1.

6.

4. The salt-tolerant heterotrophic nitrifying microbial agent according to any one of claims 1 to 3, characterized in that: The preparation method of the seed solution of each strain is as follows: the corresponding strain is inoculated into LB medium, cultured in a shaking table at 25-35°C and 100-200 rpm for 20-30 h, the bacterial suspension is centrifuged in a refrigerated centrifuge at 3000-5000 rpm for 5-15 min, the supernatant is removed, the sediment is washed with sterile water, and the supernatant is removed by centrifugation again, and the OD of the bacterial suspension is adjusted with sterile water. 600 The concentration was adjusted to 0.6-0.8 to obtain the seed solution of the strain.

5. Use of the salt-tolerant heterotrophic nitrifying microbial agent as claimed in any one of claims 1 to 4 in reducing the ammonia nitrogen content in high-salt water bodies.

6. The use according to claim 5, characterized in that: The application comprises the following steps: adding a salt-tolerant heterotrophic nitrifying microbial agent and a carbon source into a high-salt water body with a pH of 6 to 10, wherein the volume ratio of the salt-tolerant heterotrophic nitrifying microbial agent to the water body is 3% to 15%, and culturing for 45 to 55 hours.

7. The use according to claim 6, characterized in that: Ammonia nitrogen concentration in high-salt water is 50-200 mg·L -1 .

8. The use according to claim 6, characterized in that: When a carbon source is added to a high-salinity water body, the C / N ratio in the water body is 10 to 100.

9. The use according to claim 6, characterized in that: The carbon source is sodium citrate.

10. The use according to claim 6, characterized in that: The salinity of the high-salinity water body is 1% to 12%.

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