Salt-tolerant heterotrophic nitrification composite microbial agent and application thereof

Abstract

The application discloses a kind of salt-tolerant heterotrophic nitrification composite microbial inoculant and application thereof, the salt-tolerant heterotrophic nitrification composite microbial inoculant is by alkali salt Pseudomonas LJK2, Marinobacterium LJK14 and alkali salt Pseudomonas LJK7 seed liquid compound, the volume ratio of alkali salt Pseudomonas LJK2, Marinobacterium LJK14 and alkali salt Pseudomonas LJK7 seed liquid is 2.0~2.5:1.5~2.0:1.0~1.5.The heterotrophic nitrification composite microbial inoculant of the application tolerates salinity up to 20%, and when salinity is up to 12%, still has more than 98% denitrification efficiency.It is shown that the heterotrophic nitrification composite microbial inoculant of the application has great application prospect in high salinity seawater aquaculture tail water biological denitrification.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial wastewater treatment, and particularly relates to a salt-tolerant heterotrophic nitrification composite microbial agent and application thereof. BACKGROUND

[0002] At present, the research focus of domestic seawater aquaculture tail water treatment technology is to explore and develop new technologies that not only can efficiently remove various pollutants in seawater aquaculture tail water, but also can reduce treatment cost. Microbial treatment technology is considered as one of the important means for seawater aquaculture tail water treatment. Traditional microbial denitrification technology mainly relies on the action of autotrophic nitrifying bacteria and anaerobic denitrifying bacteria, and is divided into two stages of aerobic and anaerobic, which converts nitrogen-containing organic matter into N2 through ammonia and nitrification and denitrification, so as to achieve the purpose of removing nitrogen. Because the growth rate of autotrophic nitrifying bacteria is relatively slow, a long cultivation time is required, which limits the wide application of this technology to some extent. In addition, the high-salt and high-nitrogen concentration tail water produced by seawater aquaculture can significantly inhibit the enzyme activity of microorganisms and destroy the balance of the microbial community structure, thereby causing the denitrification performance of microorganisms to decrease significantly during the tail water treatment process. To a great extent, this phenomenon restricts the wide application of microbial treatment technology in seawater aquaculture tail water treatment. Therefore, it is urgent for us to find an effective solution to seek a high-salt tail water denitrification microbial treatment technology that is energy-saving and environmentally friendly. SUMMARY

[0003] In view of the problems in the background art, the present application provides a salt-tolerant heterotrophic nitrification composite microbial agent which can adapt to high-salinity seawater aquaculture tail water treatment and has a high NH4 + -N removal rate, and also provides the application of the salt-tolerant heterotrophic nitrification composite microbial agent in reducing the ammonia nitrogen content in high-salt water bodies.

[0004] The present application adopts the following technical solutions:

[0005] The salt-tolerant heterotrophic nitrification composite microbial agent is compounded by seed liquids of Halomonas alkaliphila LJK2, Marinobacterium LJK14 and Halomonas alkaliphila LJK7, the Halomonas alkaliphila LJK2 is preserved in the Guangdong Microbial Culture Collection Center, the preservation time is December 23, 2021, and the preservation number is GDMCC NO: 62157, the Marinobacterium LJK14 is preserved in the Guangdong Microbial Culture Collection Center, the preservation time is December 23, 2021, and the preservation number is GDMCC No: 62156, the Halomonas alkaliphila LJK7 is preserved in the Guangdong Microbial Culture Collection Center, the preservation time is May 17, 2022, and the preservation number is GDMCC No: 62477, and the volume ratio of the seed liquids of the Halomonas alkaliphila LJK2, the Marinobacterium LJK14 and the Halomonas alkaliphila LJK7 is 2.0-2.5:1.5-2.0:1.0-1.5.

[0006] Preferably, the volume ratio of the seed liquid of Halomonas sp. LJK2, Marinobacterium LJK14 and Halomonas sp. LJK7 is 2.1-2.3: 1.7-1.9: 1.1-1.3.

[0007] Preferably, the volume ratio of the seed liquid of Halomonas sp. LJK2, Marinobacterium LJK14 and Halomonas sp. LJK7 is 2.2: 1.8: 1.2.

[0008] Preferably, the preparation method of the seed liquid of each strain is as follows: the corresponding strain is inoculated into LB medium, and cultured at 25-35℃ and 100-200rpm for 20-30h; the bacterial suspension is centrifuged in a refrigerated centrifuge at 3000-5000rpm for 5-15min; the supernatant is removed, the precipitate is washed with sterile water, and the supernatant is removed again after centrifugation; the above steps are repeated 2-3 times; and the OD of the bacterial suspension is adjusted to 0.6-0.8 with sterile water to obtain the seed liquid of the strain. 600 Preferably, the preparation method of the seed liquid of each strain is as follows: the corresponding strain is inoculated into LB medium, and cultured at 25-35℃ and 100-200rpm for 20-30h; the bacterial suspension is centrifuged in a refrigerated centrifuge at 3000-5000rpm for 5-15min; the supernatant is removed, the precipitate is washed with sterile water, and the supernatant is removed again after centrifugation; the above steps are repeated 2-3 times; and the OD of the bacterial suspension is adjusted to 0.6-0.8 with sterile water to obtain the seed liquid of the strain.

[0009] As a general inventive concept, the application also provides a use of the above-mentioned salt-tolerant heterotrophic nitrification composite microbial agent in reducing the ammonia nitrogen content in a high-salinity water body.

[0010] Preferably, the use comprises the following steps: adding the salt-tolerant heterotrophic nitrification composite microbial agent and a carbon source into a high-salinity water body with a pH of 6-8, the volume ratio of the salt-tolerant heterotrophic nitrification composite microbial agent to the water body being 3%-15%, and culturing for 45-55h.

[0011] Preferably, the ammonia nitrogen concentration in the high-salinity water body is 50-200mg·L -1 .

[0012] Preferably, the carbon source is added into the high-salinity water body to make the C / N ratio in the water body be 30-100.

[0013] Preferably, the carbon source is sodium citrate.

[0014] Preferably, the salinity in the high-salinity water body is 1%-12%.

[0015] Compared with the prior art, the application has the following advantages:

[0016] The heterotrophic nitrification composite microbial agent of the application tolerates a salinity of up to 20%, and still has a denitrification efficiency of more than 98% when the salinity is up to 12%. This shows that the heterotrophic nitrification composite microbial agent of the application has great application prospects in the biological denitrification of tail water of high-salinity seawater aquaculture.

[0017] Halomonas alkaliphila LJK2, deposited in Guangdong Microbial Culture Collection Center (GDMCC) located at 5th Floor, Building 59, 100 Martyrs' Road, Guangzhou, Guangdong Microorganism Institute, with the accession number of GDMCC NO: 62157, on December 23, 2021;

[0018] Marinobacter sp. LJK14, deposited in Guangdong Microbial Culture Collection Center (GDMCC) located at 5th Floor, Building 59, 100 Martyrs' Road, Guangzhou, Guangdong Microorganism Institute, with the accession number of GDMCC No: 62156, on December 23, 2021;

[0019] Halomonas salifodinae LJK7, deposited in Guangdong Microbial Culture Collection Center (GDMCC) located at 5th Floor, Building 59, 100 Martyrs' Road, Guangzhou, Guangdong Microorganism Institute, with the accession number of GDMCC No: 62477, on May 17, 2022. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to make the present application more easily understood, the present application will be described in more detail by referring to the specific embodiments shown in the drawings. These drawings only depict typical embodiments of the present application and should not be considered as limiting the scope of the present application.

[0021] Figure 1 Figure 1 is a colony morphology diagram of LJK2 strain.

[0022] Figure 2 Figure 2 is a transmission electron scanning microscope diagram of LJK2 strain.

[0023] Figure 3 Figure 3 is a phylogenetic tree of LJK2 constructed based on 16S rDNA sequence homology.

[0024] Figure 4 Figure 4 is a colony morphology diagram of LJK14 strain.

[0025] Figure 5 Figure 5 is a cell morphology diagram of LJK14 strain.

[0026] Figure 6 Figure 6 is a gram staining result diagram of LJK14 strain.

[0027] Figure 7 Figure 7 is a phylogenetic tree of LJK14 constructed based on 16S rDNA sequence homology.

[0028] Figure 8 Colony map of strain LJK7.

[0029] Figure 9 Cell morphology feature map of strain LJK7.

[0030] Figure 10 Phylogenetic tree of LJK7 constructed based on 16S rDNA sequence homology.

[0031] Figure 11 3D response surface map of mixture design.

[0032] Figure 12 Figure of effect of different carbon sources on heterotrophic nitrification and denitrification performance of the compound microbial agent.

[0033] Figure 13 Figure of effect of different C / N on heterotrophic nitrification and denitrification performance of the compound microbial agent.

[0034] Figure 14 Figure of effect of different NH4 + - concentrations on heterotrophic nitrification and denitrification performance of the compound microbial agent (C / N = 10).

[0035] Figure 15 Figure of effect of different NH4 + - concentrations on heterotrophic nitrification and denitrification performance of the compound microbial agent (C / N = 30).

[0036] Figure 16 Figure of effect of different salinity on heterotrophic nitrification and denitrification performance of the compound microbial agent.

[0037] Figure 17 Figure of effect of different pH on heterotrophic nitrification and denitrification performance of the compound microbial agent. DETAILED DESCRIPTION

[0038] Embodiments of the present application will be described below with reference to the accompanying drawings, so that those skilled in the art can better understand the present application and implement it, but the listed examples are not intended to limit the present application, and the following examples and technical features in the examples can be combined with each other without conflict, wherein the same components are denoted by the same reference numerals.

[0039] Seed liquid preparation Luria-Bertani medium (1 L): yeast extract 5 g, peptone 10 g, sodium chloride 33 g. The strain is inoculated into the LB medium, and cultured at 25-35 °C, 100-200 rpm on a shaker for 20-30 h. The bacterial suspension is centrifuged at 3000-5000 rpm in a refrigerated centrifuge for 5-15 min, the supernatant is removed, the precipitate is washed with sterile water, and the supernatant is removed again after centrifugation, and the process is repeated 2-3 times. The OD 600Adjust to 0.6-0.8, obtain the seed liquid of the strain.

[0040] Heterotrophic nitrification medium (1L): (NH4)2SO40.47g, (CH2COONa)2·6H2O 5.63g, NaCl 33g, Wilkins salt solution 50mL·L -1 .

[0041] Wilkins salt solution (1L): K2HPO4·3H2O 6.54g, MgSO4·7H2O 2.5g, NaCl 2.5g, FeSO4·7H2O 0.05g, MnSO4·H2O 0.04g.

[0042] I. Strain source:

[0043] The present application collects seawater shrimp tail water sediments from Poshu Village in Liuhua District of Maoming City, Guangdong Province, and three strains of salt-tolerant HN-AD bacteria, namely Alcaligenes sp. LJK2, Marinobacter sp. LJK14 and Alcaligenes sp. LJK7, are screened out through enrichment culture and separation. The above-mentioned three salt-tolerant HN-AD bacteria are used to construct a complex heterotrophic nitrification bacterial agent, the ratio of the agent is optimized by using Design-Expert.V8.0.6.1 software for mixing design, and the influencing factors of the above-mentioned heterotrophic nitrification bacterial agent on deamination and nitrogen removal performance are explored through experiments, and the NH4 + -N concentration tolerated by the bacterial agent is determined.

[0044] II. Strain identification

[0045] 2.1. LJK2 strain identification

[0046] Morphological identification

[0047] The strain after isolation and purification is inoculated on solid culture medium, and the colony characteristics of the strain are observed after 36h of culture. Single colonies are picked and sent to the Institute of Urban Environment, Chinese Academy of Sciences (Xiamen City) for observation of bacterial morphology and characteristics by transmission electron microscope.

[0048] Figure 1 The colony observation figure result shows that LJK2 is a light yellow, convex round small colony with smooth and complete edge, wet and viscous; it is a gram-negative bacterium;The transmission electron microscope result shows that the LJK2 bacterial body is fusiform, straight or curved, with flagella and pilus, and no capsule. Figure 2

[0049] Physiological and biochemical identification

[0050] According to the "Common Bacteria System Identification Manual" and "Berger's Bacteria Identification Manual", physiological and biochemical identification test is carried out. The test items are shown in Table 1:

[0051] Table 1 Physiological and biochemical identification index

[0052]

[0053] The LJK2 strain is a gram-negative bacterium, and its physiological and biochemical characteristics are shown in Table 2: the LJK2 strain can adapt to anaerobic and aerobic environments; the contact enzyme and oxidase tests are positive; 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 it does not have tryptophanase; the citrate test is positive; the starch hydrolysis test is negative, indicating that it does not have amylase; and the glucose, lactose, sucrose and mannitol oxidation fermentation test results indicate that it can decompose glucose, lactose and sucrose, but not mannitol.

[0054] Table 2 Physiological and biochemical characteristics of the LJK2 strain

[0055] Test item Result Test item Result Test item Result Contact enzyme ﹢ No paraffin seal ﹢ Indole assay ﹣ Oxidase ﹢ With paraffin seal ﹢ Motility ﹣ Glucose oxidation fermentation Acid produced, no gas produced MR test ﹣ 5% NaCl ﹢ Lactose oxidation fermentation Acid produced, no gas produced VP test ﹣ 7% NaCl ﹢ Sucrose oxidation fermentation Acid produced, no gas produced Citrate ﹢ 10% NaCl ﹢ Mannitol oxidation fermentation No acid produced, no gas produced Starch hydrolysis ﹣

[0056] Note: "+" indicates positive, and "-" indicates negative

[0057] Molecular biology identification

[0058] The 16S rDNA of the bacterial solution was PCR amplified after dilution of the bacterial solution cultured for 48 h. The PCR reaction system was 25 μL: template DNA 1 μL; primer 27F ((5'-AGAGTTTGATCCTGGCTCAG-3') 1 μL; primer 1492R (5'-TACGACTTAACCCCAATCGC-3') 1 μL; Premix Taq enzyme 12.5 μL; sterile water 9.5 μL. The reaction conditions were: pre-denaturation 95°C for 3 min; denaturation process 95°C for 45 s; annealing process 55°C for 45 s; extension process 72°C for 45 s; repeat the second step for 30 cycles; final extension 72°C for 7 min. The product was electrophoresed on a 1% agarose gel, and the gel imaging system was photographed and sent to Jinweizhi Biotechnology Co., Ltd. for sequencing. The sequence of the sequencing result was analyzed by Blast comparison on the NCBI website, and the phylogenetic tree was constructed by MEGA7.0 adjacency method to complete the homology analysis of the strain.

[0059] The LJK2 phylogenetic tree based on 16S rDNA sequence homology is as follows: Figure 3The 16S rDNA of the LJK2 strain is clustered with Halomonas alkaliphila X3, and the genetic relationship is closest. Combined with the morphological characteristics and physiological and biochemical characteristics of LJK2, according to the Common Bacteria System Identification Manual and the Bergey's Bacteria Identification Manual, the strain is identified as Halomonas alkaliphila LJK2. On December 23, 2021, the strain was preserved in the Guangdong Academy of Microbiology Institute for strain patent preservation, and the strain number was GDMCC No.62157.

[0060] 2.2, LJK14 strain identification

[0061] Morphological identification

[0062] Strain LJK14 was inoculated into beef extract peptone medium, and after 12h of culture, the bacterial cells were picked and subjected to gram staining using the gram stain kit of Changde Bikeman Biotechnology Co., Ltd. The strain morphology and gram staining results were observed under a microscope. Strain LJK14 was inoculated into beef extract peptone medium and LB medium, and after 12h of culture, it was sent to the Guangdong Academy of Microbiology Institute for observation of strain morphological characteristics and motility using a transmission electron microscope.

[0063] The morphological characteristics of strain LJK14 are shown in Figure 4 The colony observation results show that LJK14 is meat pink, convex, irregular round colony, smooth surface, irregular edge, wet and sticky bacterial cells, not easy to pick, and has a special smell. The bacterial cell morphology is shown in Figure 5 The LJK14 bacterial cells are short rod-shaped, have flagella, no pilus, no capsule, and no motility.

[0064] Physiological and biochemical identification

[0065] The physiological and biochemical identification test of strain LJK14 was carried out according to the Bergey's Bacteria Identification Manual and the Common Bacteria System Identification Manual. The test items are shown in Table 3:

[0066] Table 3 Physiological and biochemical identification indexes of strain LJK14

[0067]

[0068] The gram staining results are shown in Figure 6As shown: strain LJK14 is a gram-negative bacteria. The physiological and biochemical characteristics of LJK14 are shown in Table 4: strain LJK14 grows without paraffin sealing and does not grow with paraffin sealing, indicating that it can adapt to aerobic environment and cannot grow in anaerobic environment; the gas production experiment is negative, indicating that the strain LJK14 does not produce gas; the contact enzyme and oxidase tests are negative, indicating that the strain LJK14 does not contain contact enzyme and oxidase; MR and VP tests are negative, indicating that the decomposition products of the strain LJK14 are non-acidic substances and do not produce pyruvic acid; the indole experiment is negative, indicating that the strain LJK14 does not have tryptophanase; the citrate test is negative, indicating that the strain LJK14 cannot utilize citrate; the starch hydrolysis test is positive, indicating that the strain LJK14 has amylase; the glucose, lactose, sucrose and mannitol oxidation fermentation test results indicate that it does not decompose glucose, lactose, sucrose and mannitol; the salinity experiment indicates that the strain LJK14 can grow at 5% and 10% salinity, and does not grow at 15% salinity, which is a salt-tolerant bacteria.

[0069] After querying the "Berger's Bacteria Identification Manual" and "Common Bacteria System Identification Manual", it can be preliminarily inferred that the strain LJK14 is Kingdom Monera (prokaryotic kingdom), Pseudomonadota (Pseudomonas door), Gammaproteobacteria (gamma-protozoan class), Pseudomonadales (Pseudomonas door), Marinobacteraceae (Marinobacteraceae), Marinobacter (Marinobacter).

[0070] Table 4 Physiological and biochemical identification experiment results of strain LJK14

[0071] Item Result Item Result No paraffin seal + M-R assay - With paraffin seal - V-P assay - Gas production test - Citrate - Oxidase - Starch hydrolysis + Contact enzyme - Indole assay - Glucose oxidation fermentation No acid produced, no gas produced Motility - Lactose oxidation fermentation No acid produced, no gas produced 5% salinity + Sucrose oxidation fermentation No acid produced, no gas produced 10% salinity + Mannitol oxidation fermentation No acid produced, no gas produced 15% salinity -

[0072] Note: "+" indicates positive, "-" indicates negative

[0073] Molecular biology identification

[0074] Strain LJK14 was inoculated into LB medium, and after 48h of culture, the Ezup column type bacterial genomic DNA extraction kit was used to extract the bacterial liquid DNA and perform PCR amplification. The PCR amplification reaction system is shown in Table 5. The PCR amplification reaction conditions are shown in Table 6. After 1% agarose gel electrophoresis of the PCR amplification product, a gel imaging system was used to take a picture, and the Guangdong Provincial Academy of Sciences Institute of Microbiology was sent for 16S rDNA detection to determine the bacterial species. The sequencing result sequence was analyzed by Blast comparison on the NCBI website, and the MEGA7.0 neighbor-joining method was used to construct a phylogenetic tree to complete the homology analysis of strain LJK14.

[0075] Table 5 PCR amplification reaction system

[0076]

[0077]

[0078] Table 6 PCR amplification reaction conditions

[0079] PCR conditions Parameters Pre-denaturation 95°C 3 min Denaturation 95℃40s Annealing 55℃40s Extension 72℃40s Cycles 30 times Extension 72°C 7 min

[0080] The phylogenetic tree of strain LJK14 was constructed according to the 16S rDNA sequencing results as shown in Figure 7 : the 16S rDNA of strain LJK14 was clustered with Marinobacter sp. U13690101122-SW176 (JQ082151.1: 16-1435) with the closest genetic relationship and the similarity of 99.93%. In combination with the morphological characteristics and physiological and biochemical characteristics of strain LJK14, the strain was identified as Marinobacter sp., and named as Marinobacter sp. LJK14. Strain LJK14 was preserved in the Guangdong Provincial Academy of Microbiology for the patent preservation of the strain, and the strain number was GDMCC No. 62156.

[0081] 2.3, LJK7 strain identification

[0082] Morphological identification

[0083] The purified target strain LJK7 was streaked on LB solid medium, and incubated at 30°C for 36 h, and the colony characterization morphology was observed; the single colony microscopic morphological characteristics were observed by transmission electron microscope (completed by Xiamen Institute of City Environment, Chinese Academy of Sciences); a small amount of bacterial cells were picked and observed under a microscope to identify whether they were gram-negative or gram-positive bacteria by Gram staining method.

[0084] The colony characteristics of strain LJK7 are shown in Figure 8 : after 2 d of incubation on solid plate, round and raised, smooth-edged and light yellow colonies of strain LJK7 were observed; the cell morphology results are shown in Figure 9 : strain LJK7 was short rod-shaped, straight or curved, with flagella and fimbriae.

[0085] Physiological and biochemical identification

[0086] The physiological and biochemical identification test was carried out according to the "Berger's Bacterial Identification Manual" and "Common Bacterial System Identification Manual". The test items are shown in Table 7:

[0087] Table 7 Physiological and biochemical identification indexes

[0088]

[0089] The physiological and biochemical characteristics test results of the strain LJK7 are shown in Table 8: the contact enzyme and oxidase test results of the strain LJK7 are positive; the MR and VP test results are negative, indicating that the decomposition product of the strain is a non-acidic substance and no pyruvic acid is produced; the indole experiment is negative, indicating that the strain does not have tryptophanase; the citrate test is positive; the starch hydrolysis test is positive, indicating that the strain has amylase; the strain LJK7 can decompose glucose, lactose, sucrose, sucrose and mannitol; the salt tolerance experiment results show that the results are positive under the conditions of 5% and 7% NaCl mass fraction, which indicates that the strain LJK7 has good salt tolerance. According to the above characteristics, referring to the "Berger Bacterium Identification Manual" and "Common Bacterium System Identification Manual", it is judged that the strain LJK7 is Halomonas.

[0090] Table 8 Physiological and biochemical characteristics of strain LJK7

[0091] Test item Result Test item Result Oxidase + Citrate + Contact enzyme + Starch hydrolysis + Glucose oxidation fermentation Acid produced, no gas produced Indole assay - Lactose oxidation fermentation Acid produced, no gas produced Motility - Sucrose oxidation fermentation Acid produced, no gas produced 5% NaCl + Mannitol oxidation fermentation Acid produced, no gas produced 7% NaCl + MR test - VP test -

[0092] Note: "+" indicates positive, "-" indicates negative.

[0093] Molecular biology identification

[0094] The strain LJK7 was inoculated into LB liquid medium and cultured at 30°C for 48h, and the bacterial liquid was diluted and the genomic DNA was extracted by using a DNA extraction kit. The genomic DNA was used as a template, and the 16S rDNA was amplified by PCR using bacterial 16S rDNA universal primers (27F, 1492R) according to the PCR reaction system in Table 9 and the reaction conditions in Table 10. After the amplified product was verified correct by 1% agarose gel electrophoresis, the sequencing was completed by Goldengene Biotechnology Co., Ltd. After sequencing, the sequencing sequence was submitted to the NCBI website for comparison and analysis by Blast, and the sequences with higher homology were selected. The Neighbor-Joining method was used to construct the phylogenetic tree in MEGA 7.0 software.

[0095] Table 9 16S rDNA sequence amplification reaction system

[0096]

[0097] Table 10 PCR reaction conditions

[0098] Step Temperature and time Cycles Pre-denaturation 95°C 3 min 1 Denaturation 95℃45s 30 Annealing 55℃45s 30 Extension 72℃45s 30 Final extension 72°C 7 min 1

[0099] The LJK7 phylogenetic tree based on 16S rDNA sequence homology is as follows: Figure 10The 16S rDNA of LJK7 strain was clustered with Halomonas salifodinae strain ZSH30, and the genetic relationship was closest. Combined with the morphological characteristics and physiological and biochemical tests of LJK7, the strain was identified as Halomonas salifodinae LJK7.

[0100] III. Preliminary exploration of heterotrophic nitrifying bacteria agent mixture and optimization of combination ratio

[0101] Seed liquid preparation: LJK2, LJK14, LJK7 were inoculated into LB medium, 30℃, 150rpm shaker for 24h, 4000rpm centrifuge for 10min, supernatant was removed, and the precipitated bacteria were washed with sterile water, then centrifuged to remove the supernatant, repeated 2-3 times, and the OD of the bacterial suspension was adjusted to 0.6-0.8 with sterile water as the seed liquid of LJK2, LJK14, LJK7. 600

[0102] Preliminary exploration of functional bacteria mixture: NH4 + -N concentration was 100mg·L -1 , C / N was 30, pH was 7, and the mixed strain preliminary exploration design was shown in Table 11. The seed liquid of each strain was inoculated into 100mL heterotrophic nitrification medium in the proportion of Table 11, and cultured at 30℃, 150rpm in a shaker for 48h. Each experimental treatment was repeated 3 times, and the NH4 + -N content in the system was measured every 6h.

[0103] Table 11 Mixed strain preliminary exploration design

[0104]

[0105] The NH4 + -N content change in the culture system of the preliminary exploration of the three strains was as follows: compared with the strains of the composite bacteria agent, the NH4 + -N removal rate of the composite bacteria agent was improved. The NH4 + -N removal rate of each strain combination basically reached the maximum, among which the NH4 + -N removal rate of LJK2+LJK14 combination reached 92.2%, the NH4 + -N removal rate of LJK2+LJK7 combination reached 85.2%, and the NH4 + -N removal rate of LJK2+LJK14+LJK7 combination was 93.4%, and the NH4 + -N removal rate of LJK14+LJK7 combination was 75.1%. The above results showed that the combination of the three strains of aerobic denitrifying bacteria expressed good denitrification performance and could be symbiotic cultured.​

[0106] The mixture design determines the compound ratio of the bacterial agent: NH4 + -N concentration is 200 mg·L -1 , C / N is 15, pH is 7, inoculation amount is 6%, selects A (LJK2), B (LJK14), C (LJK7) as optimization factors, the boundary value of the inoculation volume of the seed liquid of the three strains is set to 0-5 mL, adopts Mixture design experiment in Design-Expert 13 software, arranges the experiment according to the mixture design, pours the seed liquid of each strain into the heterotrophic nitrification medium with a capacity of 100 mL according to the ratio of the experimental design, and cultures at 30°C, 150 rpm on a shaking table for 48 h, repeats each experimental treatment 3 times, and measures the NH4 + -N content in the system every 6 h.

[0107] Mixture design model analysis:

[0108] Using Design Expert 13 response surface software, the data in Table 12 mixture design results are twice fitted to obtain the regression equation of NH4 + -N removal rate (R1):

[0109] R1 = 2.84A + 3.11B + 3.07C + 1.72AB + 1.74AC + 2.17BC + 25.37ABC

[0110] The variance analysis result of the regression equation is shown in Table 12: the experimental model is extremely significant (p<0.01), AB (LJK2+LJK14), BC (LJK14+LJK7) and its ABC (LJK2+LJK14+LJK7) have extremely significant influence (p<0.01) on the NH4 + -N removal rate of the compound microbial agent, and the influence of other items is not significant (p>0.05). The model correlation coefficient R 2 =0.94, which indicates that the model equation can explain 94.7% of the change of the response value (NH4 + -N removal rate of the compound microbial agent); the corrected correlation coefficient R 2 (Adj) =0.90, the difference between R 2 and R 2 (Adj) is less than 0.1, the correlation between the predicted value and the true value is high, the equation model has high reliability, the regression equation fitting degree is good, and the model can be used to analyze the mixed design ratio of different functional bacteria in the compound microbial agent.

[0111]

[0112]

[0113]

[0114] Results Analysis The mixture design contour lines and 3D response surface show that the three-dimensional response surface diagram is spherical, such as Figure 11 As shown, the mixing ratio of strains LJK2, 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 (LJK2) is the dominant factor, when the ratio of strain LJK2 is 42.0%, NH4 + -N removal rate is the highest. The above experimental results show that when the strain LJK2:LJK14:LJK7=2.2∶1.8∶1.2, the denitrification effect of YL bacterial agent is the best.

[0115] 4. Optimization of growth conditions of composite bacterial agents

[0116] The other conditions remained unchanged, and carbon-free (no carbon source added), sodium bicarbonate, glucose, sucrose, sodium citrate, and sodium acetate were used as carbon sources, respectively, and inorganic carbon source sodium bicarbonate and carbon-free culture medium were used as controls; C / N was set to 0, 10, 30, 50, 80, 100, and 150, respectively; when C / N=10, the nitrogen source concentration was set to 60 mg / L, 80 mg / L, 100 mg / L, and 120 mg / L, respectively; when C / N=30, the nitrogen source concentration was set to 50 mg / L, 100 mg / L, 150 mg / L, and 200 mg / L, respectively; the salinity was set to 1%, 4%, 8%, 12%, 16%, 18%, and 20%, respectively; and the pH was set to 5, 6, 7, 8, 9, and 10, respectively.

[0117] The seed liquid of the three strains was compounded to form a composite agent and then inoculated into the heterotrophic nitrification medium. The volume ratio of the composite agent to the heterotrophic nitrification medium was 3% to 15%. After culturing for 45 to 55 hours, samples were taken to detect OD 600 , pH, NH4 + -N, NO2 - -N and NO3 - -N content.

[0118] Effect of carbon source on the treatment of tail water by composite bacterial agent

[0119] like Figure 12As shown in Figure 1, the effects of different carbon sources on the growth of the composite microbial agent, when no carbon was added as the carbon source, the biomass of the composite microbial agent hardly grew and the denitrification rate was extremely low, indicating that the growth of the bacteria required the addition of a certain carbon source; when sodium bicarbonate (inorganic carbon) was used as the carbon source, the growth of the composite microbial agent was poor and the denitrification performance was not ideal, indicating that the bacteria could be autotrophic using this carbon source but the denitrification effect was poor; when glucose was used as the carbon source, the biomass of the strain was 0.343, the biomass, NH4 + -N removal rate were all lower than those of other carbon sources; when sucrose was used as the carbon source, the NH4 + -N removal rate of the composite microbial agent reached 84.47%, but the biomass OD 600 was only 0.378, and the growth of the composite microbial agent was poor; when sodium citrate and sodium acetate were used as the carbon sources, the biomass of the composite microbial agent reached 1.362 and 1.255, respectively, and the NH4 + -N removal rates were as high as 98.06% and 99.46%, respectively, indicating that both sodium citrate and sodium acetate were conducive to the growth of the composite microbial agent and could effectively utilize these two carbon sources for heterotrophic nitrification. However, when both of these two carbon sources were used, the biomass of the strain, the NH4 + -N removal rate, and the accumulation of nitrite and nitrate were analyzed, and it was found that sodium citrate was selected as the carbon source for the subsequent nitrification influencing factor exploration experiment. In summary, the utilization of different carbon sources by the composite microbial agent was as follows: sodium citrate > sodium acetate > sucrose > sodium bicarbonate > glucose > no carbon.

[0120] Effect of C / N on the treatment of tail water by the composite microbial agent

[0121] Effect of different C / N on the growth of the composite microbial agent Figure 13 As shown in Figure 2, when C / N was 0, i.e., no sufficient carbon source was provided, the NH4 + -N removal rate of the composite microbial agent was extremely low and the strain hardly grew, which might be due to the lack of carbon source leading to the inability of the composite microbial agent to carry out normal metabolic activities; when C / N was 150, although the carbon source was sufficient, the NH4 + -N removal rate was low and the biomass was low, which might be because the excessively high C / N led to the inability of the composite microbial agent to effectively utilize the carbon source during nitrification, or the excessive carbon source inhibited the strain, indicating that this C / N was not conducive to the growth of the composite microbial agent; when C / N was 10-100, the NH4 + -N removal rate was as high as more than 90%, the biomass showed a trend of first increasing, then decreasing, and then increasing, and especially when C / N was 30 and 100, the biomass of the strain was 1.886 and 2.091, respectively, and the NH4 + -N removal rate was as high as 100%, showing that the composite microbial agent had excellent growth and denitrification capacity under these two C / N conditions.

[0122] Effect of nitrogen source concentration on the treatment of tail water by the composite microbial agent

[0123] When C / N=10, different NH4 + Effect of -N concentration on heterotrophic nitrification and denitrification performance of composite bacterial agents Figure 14 As shown in the figure: with the increase of nitrogen source concentration, the biomass also increases accordingly, and the ammonia nitrogen removal rate is as high as more than 99% and there is almost no accumulation of nitrite, but the biomass is relatively low. When the nitrogen source concentration is 120 mg / L, the biomass reaches the highest 1.595, indicating that when the nitrogen source concentration is low (C / N=10), the ammonia nitrogen removal rate of the composite bacterial agent is good, but the growth of the bacteria is general; when C / N=30, different NH4 + Effect of -N concentration on heterotrophic nitrification and denitrification performance of composite bacterial agents Figure 15 As shown in the figure: With the increase of nitrogen source concentration, the biomass and ammonia nitrogen removal rate of the composite bacterial agent showed a trend of first increasing and then decreasing. The growth of the strain was also better than when C / N=10, with the biomass being 1.475, 1.944, 1.950, and 1.875, respectively. The ammonia nitrogen removal rate was as high as 96% or more. In particular, when the nitrogen source concentration was 100 mg / L, the ammonia nitrogen removal rate of the composite bacterial agent was as high as 100%, indicating that the bacteria had excellent growth and denitrification effect when C / N=30 and nitrogen source concentration was 100 mg / L. In summary, the composite bacterial agent has a strong tolerance to nitrogen source concentration. When the nitrogen source concentration is in the range of 50 mg / L to 200 mg / L, the denitrification efficiency of the composite bacterial agent for marine aquaculture tail water is as high as 96% or more.

[0124] Effect of salinity on the treatment of tail water by composite bacterial agent

[0125] like Figure 16 As shown in the figure: the effect of different salinity on the treatment effect of composite bacteria agent on tail water. When the salinity is 1%, 4%, 8% and 12%, the bacterial OD 600 The biomass was relatively stable with a small increase, and the biomass was 1.605, 1.964, 1.926, and 1.644 respectively, and NH4 + -N removal rate is as high as 99%; when the salinity is 16%, the bacterial OD 600 and NH4 + -N removal rates decreased; under extreme salinity conditions of 20%, bacterial growth almost stagnated. This is likely due to the high salinity causing severe dehydration stress on the bacteria, leading to their death and inability to fully utilize their nitrification capacity. Low salinity levels, however, do not provide a suitable growth environment, resulting in slow bacterial growth and low denitrification rates. High salinity generally inhibits bacterial nitrification activity, but the composite inoculant in this design exhibits excellent salt tolerance, maintaining high denitrification efficiency even at a salinity of 12%.

[0126] And previous studies have shown that LJK2 in ammonia nitrogen water body salinity tolerance is 8%, when the salinity is 7%, the cell OD 600 and NH4 + - removal rate is reduced; LJK14 in ammonia nitrogen water body salinity tolerance is 7%, when the salinity is 6%, the cell OD 600 and NH4 + - removal rate is reduced; LJK7 in ammonia nitrogen water body salinity tolerance is 6%, when the salinity is 5%, the cell OD 600 and NH4 + - removal rate is reduced.

[0127] Effect of pH on the effect of tail water treated by composite microbial agent

[0128] pH is one of the main factors affecting biological denitrification process, pH value mainly through the change of cell membrane charge to affect the permeability of cell membrane, and then on the growth characteristics of microorganisms, metabolic enzyme activity and nutrient absorption and conversion have far-reaching influence. The effect of different pH on the effect of tail water treated by composite microbial agent is shown in Figure 17 When pH is 5, due to the pH is too low, the composite microbial agent cannot grow normally, so that the biomass and denitrification rate of the cell is almost 0; when pH is 6, 7 and 8, the biomass of the cell tends to be stable, and the denitrification rate is as high as 100%, which shows that the suitable pH range for the growth of the cell is 6-8; when pH is 9, the biomass of the cell is lower than before, but the denitrification rate is still very high; when pH is 10, due to the pH is too high, part of the enzyme in the composite microbial agent is inactivated, so that the biomass of the composite microbial agent is lower than that when pH is 9, which shows that this pH is not suitable for the growth of the composite microbial agent. Too high or too low pH value will also affect the ionization of organic compounds in the culture medium, thereby indirectly affecting the growth and metabolism of microorganisms.

[0129] In summary, when the carbon source is sodium citrate, C / N is 30, the nitrogen source concentration is 100 mg / L, the salinity is 1%-12%, and the pH is 6.00, the NH4 + - removal rate of the composite microbial agent is optimal, and the growth of the composite microbial agent is best, so the above factors can be selected as the conditions for treating seawater aquaculture tail water in the microbial treatment tank.

[0130] The above-described embodiments are only the preferred specific embodiments of the present application, and the phrase "in an embodiment", "in another embodiment", "in yet another embodiment" or "in other embodiments" in the specification can refer to one or more of the same or different embodiments according to the present disclosure. The usual changes and replacements made by those skilled in the art within the scope of the technical solutions of the present application should be included in the protection scope of the present application.

Claims

1. A salt-tolerant heterotrophic nitrifying composite bacterial agent, characterized in that: It is compounded by the seed liquid of alkaliphilic halomonas LJK2, Marinobacter LJK14 and alkaliphilic halomonas LJK7. The alkaliphilic halomonas LJK2 is deposited in the Guangdong Provincial Microbial Culture Collection Center on December 23, 2021, and the preservation number is GDMCC NO: 62157. The Marinobacter 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 alkaliphilic Halomonas LJK2, Marinobacterium LJK14 and alkaliphilic Halomonas LJK7 seed liquid is 2.0-2.5:1.5-2.0:1.0-1.

5.

2. The salt-tolerant heterotrophic nitrifying composite bacterial agent according to claim 1, characterized in that The volume ratio of the alkaliphilic halomonas LJK2, the Marinobacterium LJK14 and the alkaliphilic halomonas LJK7 seed liquid is 2.1-2.3:1.7-1.9:1.1-1.

3.

3. The salt-tolerant heterotrophic nitrifying composite bacterial agent according to claim 2, characterized in that: The volume ratio of the alkaliphilic Halomonas LJK2, Marinobacterium LJK14 and alkaliphilic Halomonas LJK7 seed liquid is 2.2:1.8:1.

2.

4. The salt-tolerant heterotrophic nitrifying composite bacterial 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 composite bacterial 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 nitrification compound bacterial agent and a carbon source into a high-salt water body with a pH of 6-8, wherein the volume ratio of the salt-tolerant heterotrophic nitrification compound bacterial agent to the water body is 3%-15%, and culturing for 45h-55h.

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 When carbon sources are added to a high-salinity water body, the C / N ratio in the water body is 30-100.

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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