Salt-tolerant heterotrophic nitrifying microbial agent and application thereof

Abstract

The application discloses a kind of salt-tolerant heterotrophic nitrification microbial inoculant and application thereof, the salt-tolerant heterotrophic nitrification microbial inoculant is compounded by seed liquid of halomonas campaniensis strain LJK10, Marinobacterium LJK14 and halomonas alkaliphila LJK7, the volume ratio of the seed liquid of halomonas campaniensis strain LJK10, Marinobacterium LJK14 and halomonas alkaliphila LJK7 is 2.5-3:2.0-2.5:1.5-2.0.The heterotrophic nitrification microbial inoculant of the application tolerates salinity up to 20%, and when salinity is up to 12%, there is still 100% denitrification efficiency.It is shown that the heterotrophic nitrification microbial inoculant of the application has great application prospect in the aspect of biological denitrification of high salinity mariculture tail water.

Description

Technical Field

[0001] The present 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] The booming marine aquaculture industry, coupled with its continued growth, is facing increasingly severe environmental pollution challenges. Marine aquaculture tailwater, due to its high salinity, poses a significant environmental challenge. During the aquaculture process, salt-rich seawater is essential to maintain a viable growth environment for the organisms. This typically results in a salinity level between 30g / L and 35g / L, or even higher.

[0003] The types and structures of pollutants in aquaculture tailwater are quite complex. Due to the residual organic matter such as excrement and feed residues from farmed organisms, as well as possible chemical contamination, the levels of ammonia nitrogen, nitrite, nitrogen, phosphorus, and biological oxygen demand (BOD) 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 added to aquaculture water are actually absorbed, utilized, and assimilated by farmed animals. The majority of the remaining nutrients dissolve in the water, causing eutrophication of the tailwater, which in turn has a negative impact on the normal growth of farmed organisms.

[0004] Heterotrophic nitrifying bacteria are bacteria that oxidize nitrogen-containing substances and release energy. They possess efficient conversion capabilities, converting ammonia nitrogen into products such as hydroxylamine, nitrite, and nitrate. Most heterotrophic nitrifying bacteria also possess aerobic denitrification capabilities, ultimately converting ammonia nitrogen into gaseous nitrogen (N2). This effectively removes nitrogen from water, reducing nitrogen content and improving water quality. This biological process offers low energy consumption, safe and stable operation, and excellent denitrification results, making it an efficient and environmentally friendly denitrification method for treating marine aquaculture tailwater. However, the limited salt tolerance of individual bacteria has limited its widespread application. Furthermore, the high salt and nitrogen concentrations in tailwater produced by marine aquaculture significantly inhibit microbial enzyme activity, disrupting the balance of their bacterial communities and leading to a significant decline in microbial denitrification performance during tailwater treatment. This phenomenon has significantly hindered the widespread application of microbial treatment technologies in marine aquaculture tailwater treatment. Therefore, the search for an energy-efficient and environmentally friendly microbial denitrification technology for high-salinity tailwater is urgent. 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 by the seed liquid of Campanian Halomonas strain LJK10, Marinobacterium LJK14 and alkaliphilic Halomonas LJK7, wherein the Campanian Halomonas strain LJK10 is deposited in the Guangdong Provincial Microbial Culture Collection Center on December 23, 2021, with a deposit number of GDMCC NO: 62158, the Marinobacterium LJK14 is deposited in the Guangdong Provincial 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 Provincial 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 Halomonas campania strain LJK10, the Bacillus marinoi strain LJK14 and the alkaliphilic Halomonas strain 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 campania strain LJK10, the Marinobacterium LJK14 and the alkaliphilic Halomonas LJK7 is 2.8:2.4:1.6.

[0010] Preferably, the preparation method of the seed liquid 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 hours, the bacterial suspension is centrifuged in a refrigerated centrifuge at 3000-5000 rpm for 5-15 minutes, the supernatant is removed, the sediment is precipitated, the bacteria are washed with sterile water, and the supernatant is removed by centrifugation again, and the OD value 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.

[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-salt 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 as high as 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 Provincial Microbiological Culture Collection Center (GDMCC), Guangdong Institute of Microbiology, Building 59, 5th Floor, 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 was deposited in Guangdong Provincial Microbial Culture Collection Center (GDMCC), Guangdong Institute of Microbiology, Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou, China. The deposit number is GDMCC No: 62156 and the deposit date is December 23, 2021.

[0021] Halomonas salifodinae LJK7 is deposited in Guangdong Provincial 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 is 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 with reference 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 image of strain LJK10.

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

[0025] Figure 3 This is a transmission electron microscopy image 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 Figure 2 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] Figure 9 This is the phylogenetic tree of LJK14 constructed based on 16S rDNA sequence homology.

[0032] Figure 10 This is the colony image of strain LJK7.

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

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

[0035] Figure 13 Design a 3D response surface plot for a mixture.

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

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

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

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

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

[0041] Figure 19 This is a graph showing the effects 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 embodiments 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. Unless there is a conflict, the following embodiments and the technical features in the embodiments can 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 and culture at 25-35°C, 100-200rpm shaking for 20-30h. Centrifuge the bacterial suspension in a refrigerated centrifuge at 3000-5000rpm for 5-15min, remove the supernatant, precipitate, wash the bacteria with sterile water, centrifuge again and remove the supernatant. Repeat 2-3 times. Use sterile water to adjust the OD value 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 (1 L): (NH4)2SO4 0.47 g, (CH2COONa)2·6H2O 5.63 g, NaCl 33 g, Weiss salt solution 50 mL·L -1 .

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

[0046] 1. Source of strains:

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

[0048] 2. Strain Identification

[0049] 2.1. Identification of LJK10 strain

[0050] Morphological identification

[0051] Bacteria were picked from a bacterial slant under aseptic conditions. Single bacterial colonies were obtained using the streak method and observed for morphology, color, and texture. Colonies were picked using a sterile inoculating loop and stained using a Gram stain kit. After staining, the slant and seed solution were observed under an optical microscope for Gram stain results and bacterial morphology. The bacterial slant and seed solution were sent to the Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, where the basic structure and morphology of the bacteria were observed using a transmission electron microscope.

[0052] The colonies of strain LJK10 were Figure 1 The Gram staining results are shown in Figure 2 As shown: 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 result is red, indicating that LJK10 is a Gram-negative bacterium. The transmission electron microscopy results are as follows Figure 3 As shown: The bacterial body is short rod-shaped, with capsule and flagella, and is 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 conducted 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] Table 2 shows the physiological and biochemical identification results of strain LJK10. The catalase test was negative and the oxidase test was positive, indicating that strain LJK10 is an aerobic bacterium. In the oxidative 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 both aerobic and anaerobic conditions and is a fermentative bacterium. Both the MR and VP tests were negative, indicating that the decomposition products of the strain are not acidic and no pyruvate is produced. The citrate test was positive, indicating that the strain can utilize citric acid during growth. The indole test was negative, indicating that the product contained no indole and that the strain does not possess tryptophanase. The starch hydrolysis test was negative, indicating that the strain does not possess amylase and does not decompose starch. The motility test results showed that the strain was non-motile. The strain was able to grow in 5%, 7%, and 10% NaCl, indicating that the strain has a wide range of adaptability to salinity and can tolerate higher salt concentrations. 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 belonged to the genus Halomonas.

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

[0059]

[0060]

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

[0062] Molecular biology identification

[0063] Bacterial DNA was extracted using the Sango Biotech Ezup column-based bacterial genomic DNA extraction kit. After genomic DNA extraction, PCR amplification of bacterial 16S rDNA was performed using the genomic DNA as a template. Primers used the following universal bacterial 16S rDNA primer sequences: 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 procedure was as follows: pre-denaturation at 95°C for 3 minutes followed by cycling, denaturation at 95°C for 45 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 40 seconds. After 35 cycles, the PCR products were electrophoresed on a 1.3% agarose gel, photographed using a gel imager, and sequenced at GeneWeiZhi Biotechnology Co., Ltd. Sequence alignment was performed on the NCBI website, and a phylogenetic tree was constructed using the Maximum Likelihood function in MEGA7 software.

[0067] The genomic gene of strain LJK10 was extracted and amplified by PCR using bacterial 16SrDNA universal primers 27f and 1492r. The agarose gel electrophoresis results of the amplified product were as follows: Figure 4 As shown in the figure, it is not difficult to see from the electrophoresis results that the PCR amplification product band of 16S rDNA is bright and clear, without any mixed bands, and the band size is about 1500 bp, 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, resulting in a 1416-bp 16S rDNA sequence. Based on the NCBI website's alignment of strain LJK10 with other related bacteria, a phylogenetic tree constructed based on the homology of strain LJK10 is shown in Figure 5. Strain LJK10 shares high homology with most species of the genus Halomonas. The 16S rDNA sequence of LJK10 shows 99.72% similarity to Halomonas sp.SSL3 and 99.65% similarity to Halomonas sp.SSL14. The closest relative to Halomonas cf.campisalis 'campaniae' HKS3 is 100% similarity, confirming that the strain 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, cultured for 12 h, and the bacteria were picked up. 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, cultured for 12 h, and sent to the Institute of Microbiology, Guangdong Academy of Sciences, where 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, 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 according to the Bergey's Manual of Bacterial Identification and the Manual of Identification of Common Bacterial Systems. 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 grew without paraffin sealing but did not grow with paraffin sealing, indicating that it can adapt to aerobic environments but cannot grow in anaerobic environments; the gas production test was negative, indicating that strain LJK14 does not produce gas; the catalase and oxidase tests were both negative, indicating that strain LJK14 does not contain catalase and oxidase; the MR and VP tests were negative, indicating that the decomposition products of strain LJK14 are non-acidic substances and that it does not produce pyruvate; the indole test was negative, indicating that strain LJK14 does not have tryptophanase; the citrate test was negative, indicating that strain LJK14 cannot utilize citrate; the starch hydrolysis test was positive, indicating that strain LJK14 has amylase; the results of the glucose, lactose, sucrose and mannitol oxidative fermentation tests showed that it cannot decompose glucose, lactose, sucrose and mannitol; the salinity test showed that strain LJK14 can grow at 5% and 10% salinity but not at 15% salinity, indicating that it 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 (Marine Bacteria), and Marinobacter (Marinobacter).

[0079] Table 5 Physiological and biochemical identification results of strain LJK14

[0080] project result project result No paraffin sealing + MR measurement - With paraffin seal - VP determination - Gas production experiment - Citrate - Oxidase - starch hydrolysis + Catalase - Indole determination - Glucose oxidative fermentation No acid or gas production Motility - Lactose oxidative fermentation No acid or gas production 5% salinity + Sucrose oxidative fermentation No acid or gas production 10% salinity + Mannitol oxidative fermentation No acid or gas production 15% salinity -

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

[0082] Molecular biology identification

[0083] Strain LJK14 was inoculated onto LB medium and cultured for 48 hours. DNA was then extracted using an Ezup column-based bacterial genomic DNA extraction kit and amplified by PCR. The PCR amplification reaction system is shown in Table 6. The PCR amplification conditions are shown in Table 7. The PCR products were electrophoresed on a 1% agarose gel, photographed using a gel imaging system, and sent to the Institute of Microbiology, Guangdong Academy of Sciences, for 16S rDNA analysis and species confirmation. Sequencing results were then analyzed by blast analysis on the NCBI website. A phylogenetic tree was constructed using the MEGA 7.0 neighbor-joining method to complete 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. Figure 9 As shown, the 16S rDNA of strain LJK14 clustered closely with Marinobacter sp. U13690101122-SW176 (JQ082151.1:16-1435), with a similarity of 99.93%. Based on its morphological, physiological, and biochemical characteristics, strain LJK14 was identified as a member of the genus Marinobacter and named Marinobacter sp. LJK14. Strain LJK14 has been deposited with the Institute of Microbiology, Guangdong Academy of Sciences, under the patent number GDMCC No. 62156.

[0090] 2.3 Identification of LJK7 strain

[0091] Morphological identification

[0092] The purified target strain LJK7 was streaked onto LB solid medium and cultured at 30°C for 36 h. The colony morphology was observed. 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 Gram staining was used to identify whether they were Gram-negative or Gram-positive bacteria.

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

[0094] Physiological and biochemical identification

[0095] Physiological and biochemical identification tests were performed according to Bergey's Bacterial Identification Manual and Common Bacterial System Identification Manual. The test items are shown in Table 8:

[0096] Table 8 Physiological and biochemical identification indicators

[0097]

[0098] Table 9 shows the results of physiological and biochemical characterization tests for strain LJK7. The catalase and oxidase tests for strain LJK7 were both positive; the MR and VP tests were negative, indicating that the strain's decomposition products are non-acidic and that it does not produce pyruvate. The indole test was negative, indicating the absence of tryptophanase; the citrate test was positive; and the starch hydrolysis test was positive, indicating the presence of amylase. Strain LJK7 was able to decompose glucose, lactose, sucrose, sucrose, and mannitol. The salt tolerance test was positive at both 5% and 7% NaCl concentrations, indicating that strain LJK7 has good salt tolerance. Based on these characteristics, and after consulting the Bergey's Manual of Bacterial Identification and the Manual of Systematic Identification of Common Bacteria, strain LJK7 was identified as 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 determination - Lactose oxidative fermentation Produces acid but not gas Motility - Sucrose oxidative fermentation Produces acid but not gas 5% NaCl + Mannitol oxidative fermentation Produces acid but not gas 7% NaCl + MRI test - VP test -

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

[0102] Molecular biology identification

[0103] The strain LJK7 was inoculated into LB liquid medium and cultured at 30°C for 48 hours. 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 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 sequencing was completed, the NCBI official website was accessed 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 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. Figure 12 As shown, the 16S rDNA of strain LJK7 clustered with that of Halomonas salifodinae strain ZSH30, showing the closest relationship. 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 again. Repeat 2-3 times and adjust the OD value of the bacterial suspension 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 the mixed strain 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 mixtures + -N content changes are as follows: Compared with the strains of composite bacteria, the NH4 + -N removal rate has been improved. Each strain combination has a 24hNH4 + -N removal rate basically reached the maximum, among which the NH4 + -N removal rate reached 88.3%, and the NH4 + -N removal rate reached 76.2%, and the NH4 + -N removal rate is 90.5%, LJK14 + LJK7 combination of NH4 + The -N removal rate was 72.3%. The above results show that the combination of three aerobic denitrifying bacteria has good denitrification performance and can be symbiotically cultivated.

[0116] Mixing design determines the compound ratio of microbial agent: NH4 + -N concentration is 200 mg·L -1 , C / N was 15, pH was 7, inoculation size was 6%, A (LJK10), B (LJK14), and C (LJK7) were selected as optimization factors, and 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. 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℃ and 150rpm 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, the data in Table 13 mixture design results were fitted quadratically to obtain NH4 + Regression equation of -N removal rate (R1):

[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 is extremely significant (p < 0.01), AB (LJK10 + LJK14), BC (LJK14 + LJK7), AC (LJK10 + LJK7) and ABC (LJK10 + LJK14 + LJK7) have a significant effect on the compound bacterial agent NH4 + The effect of -N removal rate was extremely significant (p<0.01), while the effects of other items were not significant (p>0.05). 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 is highly credible, and the regression equation has a good fit. 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 contour lines and 3D response surface show that the three-dimensional response surface diagram is spherical, such as Figure 13 As shown, it shows that the mixing ratio of strains LJK10, LJK14, and LJK7 has an effect on the composite bacterial agent NH4 + -N removal has an interactive effect; there is a "red vertex" in the contour map, indicating that the bacterial population ratio of the composite microbial agent is within the experimental design range of NH4 + -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 highest. 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 ratios (0, 10, 30, 50, 80, 100, 150), salinity (1%, 4%, 8%, 12%, 16%, 20%) and pH values ​​(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 ratios (10) and high carbon-nitrogen ratios (30) were also designed. This series of research and analysis laid a solid foundation for the subsequent use of the bacterial agent in fermentation tanks 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 fermentation tank 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 prepared the culture medium according to the designed culture medium ratio. The prepared and sterilized culture medium flowed into the fermentation tank through a sealed pipe. The volume ratio of the heterotrophic nitrifying composite bacterial agent to the high salt water in the fermentation tank 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 microbial growth, providing the carbon skeleton and energy required to build cell structure. Different carbon sources will lead to differences in the metabolic pathways and physiological characteristics of microorganisms. The growth status and denitrification effect of the composite microbial agent under different carbon sources are as follows: Figure 14 As shown: In the carbon-free medium, no growth signs were found in the composite bacterial agent culture for 48 hours, 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 ability 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 0.014 and 0.050 respectively, and the 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 their ammonia nitrogen removal ability is greatly weakened. In the culture medium with sodium citrate and sodium acetate as carbon sources, the denitrification efficiency of the composite bacterial agent was significantly improved, and NH4 + The -N removal rates were 99.96% and 96.92%, respectively, indicating that the composite inoculum prefers to utilize sodium citrate and sodium acetate, and the composite inoculum grows best when sodium citrate is used as the carbon source. In summary, sodium citrate is the best carbon source for the composite inoculum.

[0129] Carbon-nitrogen ratio optimization

[0130] The carbon-nitrogen ratio refers to the ratio of the total carbon to nitrogen content in an organism or culture medium, which is related to the growth and metabolic activity of microorganisms. Too high a carbon-nitrogen ratio prevents microorganisms from synthesizing 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, resulting in the accumulation of metabolites. Figure 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 is 10, the biomass of the composite agent is high, which is 1.277, and the ammonia nitrogen removal rate is as high as 85.28%, indicating that with the increase of the carbon-nitrogen ratio, the carbon and nitrogen sources in the culture medium are fully utilized by the strain, and its significant denitrification ability is exerted. When the carbon-nitrogen ratio is 150, the denitrification efficiency of the strain decreases 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 in poor growth condition, 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 rates were as high as 100%, with a small amount of NO3--N accumulation, which was consistent with the growth of the strain, and the OD600 was maintained at 1.92-1.99. In summary, the heterotrophic nitrifying bacteria in this study have a wide range of adaptability to carbon-nitrogen ratios, 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. The growth status and denitrification effect of the composite bacterial agent at different salinities are as follows: Figure 16 As shown: When the salinity of the composite bacteria agent is between 1% and 12%, the strain grows well, 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, and the biomass dropped to 0.18 (16%) and 0.02 (20%), respectively. + The -N removal rate dropped 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 isolated and purified from a seawater aquaculture farm in Dianbai District, Maoming City. Their salt tolerance is significantly higher than that of the general heterotrophic nitrification strains mentioned above, and the strains in the bacterial agent may be able to produce and secrete specific metabolites at 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. This just proves that the composite bacterial agent has the ability to treat a certain concentration of seawater aquaculture tail water. The heterotrophic nitrifying bacterial agent in this study exhibited 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 bacterial OD 600 and NH4 +-N removal rate is reduced; LJK14 tolerates salinity of 7% in ammonia nitrogen water. When the salinity is 6%, the bacterial OD 600 and NH4 + -N removal rate is reduced; LJK7 tolerates salinity of 6% in ammonia nitrogen water, and when the salinity is 5%, the bacterial OD 600 and NH4 + -N removal rate is reduced.

[0134] pH optimization

[0135] The enzymatic reaction in microorganisms 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. Figure 17 As shown: The composite bacterial agent showed only weak signs of growth at a pH of 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 (pH 6.0-9.0) 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 ammonia nitrogen metabolism by bacteria.

[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. Excessively high or low ammonia nitrogen concentrations will have an adverse effect on microbial metabolism. Excessively low ammonia nitrogen concentrations will keep microorganisms in a low nutritional state during cultivation, which is not conducive to their growth, while excessively high concentrations will inhibit microbial growth, resulting in their heterotrophic nitrification performance not being fully utilized. This experiment focuses on the growth and denitrification performance of the composite bacterial agent in the carbon-nitrogen ratio optimization experiment. The design 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: Figure 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 They were 1.035, 1.266, 1.465, and 1.582, respectively. Although the biomass of the composite bacteria agent exceeded 1 when the ammonia nitrogen concentration was 60 mg / L to 120 mg / L, 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. Notably, the composite inoculant achieved the highest denitrification efficiency at an ammonia nitrogen concentration of 60 mg / L, reaching 94.79%. At concentrations of 80, 100, and 120 mg / L, the composite inoculant achieved denitrification efficiencies of 91.49%, 93.16%, and 91.09%, respectively. The average denitrification efficiency for this experimental group reached 92.63%, demonstrating that the composite inoculant exhibited excellent denitrification capabilities at initial ammonia nitrogen concentrations ranging from 60 mg / L to 120 mg / L. This suggests that the bacteria has a wide ammonia nitrogen tolerance range at a carbon-nitrogen ratio of 10 and possesses heterotrophic nitrification capabilities even under low ammonia nitrogen concentrations.

[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: Figure 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. This shows that with the increase of ammonia nitrogen concentration in the culture medium, the biomass of the composite bacterial agent showed an increasing trend, while NO3 - -N and NO2 --N accumulation decreased accordingly. When the ammonia nitrogen concentration was between 50 mg / L and 200 mg / L, the composite bacterial agent showed good denitrification ability, with ammonia nitrogen removal rates of 99.86%, 99.96%, 100%, and 99.82%, respectively. The average denitrification efficiency of the entire 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 lowest when the ammonia nitrogen concentration was 50 mg / L, its denitrification rate was still as high as 99.86%, indicating that the bacteria could efficiently treat ammonia nitrogen even at lower ammonia nitrogen concentrations. The composite bacterial agent outperformed standard heterotrophic nitrifying and aerobic denitrifying bacteria at ammonia nitrogen concentrations of 50-200 mg / L. For example, the strains Rhodococcus sp. LS-2 studied by Hou Dongmei et al. and Pseudomonas sp. LLM-5 studied by Cao Xuesong et al. had average denitrification efficiencies of less than 50% at low ammonia nitrogen concentrations (50-80 mg / L) and high ammonia nitrogen concentrations (100-200 mg / L). In contrast, the composite bacterial agent demonstrated a broad ammonia nitrogen tolerance range.

[0140] Optimization and verification conclusions

[0141] A series of single-factor experiments revealed that the composite bacterial agent has a wide adaptability to carbon-nitrogen ratios (30-100), exhibiting salt tolerance within a salinity range of 1% to 12%. It also has a broad pH tolerance range (6.0-10.0), achieving a 100% ammonia-nitrogen removal rate within these tolerances. Furthermore, the composite bacterial agent exhibited a broad ammonia-nitrogen tolerance range (50-200 mg / L) at both high and low carbon-nitrogen ratios, with an average ammonia-nitrogen removal rate exceeding 90%.

[0142] After comprehensive consideration of cost, sodium citrate was selected as the optimal carbon source, and the optimal growth conditions for the inoculant were set at a carbon-nitrogen ratio of 30, a salinity of 4%, and a pH of 8. The above experimental results indicate that the composite inoculant exhibits a wide tolerance range for carbon-nitrogen ratio, salinity, pH, and ammonia nitrogen concentration. Therefore, its application in marine aquaculture tailwater treatment processes has certain feasibility and application prospects.

[0143] The embodiments described above are merely preferred embodiments of the present invention. The phrases "in one embodiment," "in another embodiment," "in yet another embodiment," or "in other embodiments" used in this specification may refer to one or more of the same or different embodiments of the present disclosure. Any common changes and substitutions made by those skilled in the art within the scope of the present invention are intended to be encompassed within the scope of protection of the present invention.

Claims

1. A salt-tolerant heterotrophic nitrifying microbial agent, characterized in that: It is compounded by the seed liquid of Campania Halomonas strain LJK10, Marinobacterium LJK14 and alkaliphilic Halomonas LJK7. The Campania Halomonas strain LJK10 is deposited in the Guangdong Provincial Microbial Culture Collection Center on December 23, 2021, and the preservation number is GDMCC NO: 62158. The Marinobacterium LJK14 is deposited in the Guangdong Provincial Microbial 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 Microbial 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 liquids 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.

3. The salt-tolerant heterotrophic nitrifying microbial agent according to claim 2, characterized in that The volume ratio of the seed liquids 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 value 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 according to 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 6-10, wherein the volume ratio of the salt-tolerant heterotrophic nitrifying microbial agent to the water body is 3%-15%, and culturing for 45-55 hours.

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

8. The use according to claim 6, characterized in that A carbon source is added to a high-salinity water body to achieve a C / N ratio of 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-salt water body is 1% to 12%.

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