CN118272254A - Salt-tolerant aerobic denitrifying bacteria DWK9 and application thereof - Google Patents
Salt-tolerant aerobic denitrifying bacteria DWK9 and application thereof Download PDFInfo
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- CN118272254A CN118272254A CN202410252922.5A CN202410252922A CN118272254A CN 118272254 A CN118272254 A CN 118272254A CN 202410252922 A CN202410252922 A CN 202410252922A CN 118272254 A CN118272254 A CN 118272254A
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- Prior art keywords
- salt
- dwk9
- denitrifying bacteria
- aerobic denitrifying
- saline
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/007—Contaminated open waterways, rivers, lakes or ponds
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Abstract
The invention discloses a salt-tolerant aerobic denitrifying bacterium DWK9 and application thereof, and belongs to the technical field of biological materials. The strain is named as halomonas Halomonas shizuishanensis DWK and is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC 27980, and the preservation date is 2023, 07 and 24 days. The salt-tolerant aerobic denitrifying bacteria DWK9 has the functions of salt tolerance, aerobic and denitrification, can efficiently reduce the concentration of ammonia nitrogen in saline-alkali soil, and the sewage treatment microbial inoculum containing the strain can be applied to deamination and denitrification treatment of saline-alkali soil culture water bodies, domestic sewage, lakes and other sewage, is particularly suitable for purification and treatment of saline-alkali soil culture water bodies, and solves the problem that the microbial inoculum in the traditional fresh water environment has poor ammonia nitrogen treatment effect in the secondary saline-alkali water culture environment.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a salt-tolerant aerobic denitrifying bacterium DWK9 and application thereof.
Background
In the high-density cultivation process, the continuous bait feeding and the cultivation excrement cause the problem of excessively high concentration of ammonia nitrogen and nitrite nitrogen in the cultivation water body. At present, the common treatment methods at home and abroad mainly comprise: physical, chemical, biological methods. The physical method includes stripping method, adsorption method, etc. for example, NH 4 + in water is added with a large amount of alkali to generate free ammonia, and then other gases are blown into the water to make the free ammonia enter gas phase, so as to achieve the aim of denitrification. Chemical methods include flocculation precipitation, ozone disinfection, electrochemical methods, etc., for example, ozone can directly oxidize ammonia nitrogen into nitrogen, and can also increase dissolved oxygen in water. However, physical and chemical methods have high efficiency, but are liable to cause secondary pollution, and have been limited in aquaculture. Microorganisms are important participants in the processes of ammoxidation, nitrate reduction, nitrous oxide reduction and the like, can also produce organic acid, reduce the alkalinity of water and soil, have nitrogen fixation effect, phosphate dissolution effect and potassium dissolution capability, produce biomass promotion, antagonize pathogenic bacteria, quorum sensing regulation effect (Hayat total, 2010) and the like. The microbial denitrification technology has been widely used in wastewater, source water pretreatment, micro-polluted reservoirs, lakes, wetlands and other environments due to the advantages of low cost, wide adaptability, ecology and regenerability, no secondary pollution to the environment and the like.
Compared with other barrier type soil, the secondary saline-alkali soil has the characteristics of low-lying land topography, shallow groundwater burial depth, unsmooth drainage and the like, the feasibility of the secondary saline-alkali soil as a potential cultivated land resource is determined by rich water resources and the flat topography, and various aquatic products are successfully cultivated in saline-alkali water, so that the combination of saline-alkali soil agriculture and aquaculture greatly promotes grain supply and economic development of saline-alkali areas (Rozema & powers, 2008). Saline-alkali water culture can effectively solve the problem of salt-washing and alkaline-discharging water outlet, however, the saline-alkali water has the characteristics of high pH, high salt, high alkali, high mineralization degree, low biomass and the like, and extreme physicochemical conditions limit the effective colonization of foreign strains (Bonaldo GD et al, 2017). Researchers find that the ammonia nitrogen concentration and the nitrite nitrogen content are high through the detection of the saline-alkali soil cultivation water quality, the effect of adding the conventional biological microbial inoculum or increasing the oxygen content of the water body is not obvious, the secondary saline-alkali water environment has complexity, and the excessive nitrogen greatly limits the fishery utilization of saline-alkali water.
Aerobic denitrification is of much greater interest to water treatment engineers and environmental microbiologists than traditional anaerobic denitrification techniques, as TN and TOC can be removed simultaneously under aerobic conditions (Ji Total, 2015). For aerobic denitrifying bacteria, oxygen is the electron acceptor and the organic carbon acts as the electron donor (Song ZF et al, 2011). Meanwhile, the traditional denitrifying bacteria can only play a denitrification role under anaerobic and low-oxygen conditions, contradict with the high-dissolved-oxygen environment of the culture water body, and the aerobic denitrifying bacteria can play a denitrification role in the high-dissolved-oxygen environment, so that a new way is provided for the biological regulation and control of ammonia nitrogen and nitrite nitrogen in the aquaculture process. In the past few decades, aerobic denitrifying bacteria have not been taxonomically specialized in the presence of Bacillus, rhodococcus, acinetobacter, paracoccus, pseudomonas, enterobacter, and the like. Song ZF et al have found that 10mg/L of nitrite nitrogen is degraded to 0 within 14 hours by aerobic denitrification studies using Bacillus sp. YX-6 strain containing nitrite reductase gene nirS (Song ZF et al, 2011) at a dissolved oxygen concentration of 5.2-5.8 mg/L. Chen Peizhen Rhodococcus CPZ24, which was isolated from pig manure waste water, was able to completely remove 50mg/L of ammoniacal nitrogen within 20 hours (Chen Peizhen et., 2012). Yao Shuo the average removal of ammonia nitrogen and nitrite by the isolated Acinetobacter sp.HA2 at low temperature reached 3.03 and 2.51 mg/(L.times.H) (Yao Shuo et., 2013). Zhang Haihan found that the removal rates of total nitrogen under aerobic and anaerobic conditions were 82% and 85%, respectively, for paracoccus eversum.ks293, and nitrate reductase narG (Zhang Haihan et., 2018). Zhou Maohong Pseudomonas stutzeri.KTB separated from activated sludge was 100% for 4.511mmol/L nitrite and 4.438mmol/L within 18 hours (Zhou Maohong et., 2014). Guo Longjie found that the Enterobactercloacae.HNR had almost all nitrate removed within 30 hours, the average removal rate reached 2.51 mg/(L.times.H), and the optimal C/N ratio 13, pH 8 (Guo Longjie et., 2015). However, the above aerobic denitrifying bacteria studies are mostly isolated from fresh water environments and have limited application in the salinity-containing aquaculture industry.
Although studies on saline-alkali tolerant microbial resources have been conducted, the studies on the evaluation of saline-alkali tolerance of strains and the growth-promoting ability of strains to plants have been focused on many cases. For example, liu Xinpeng et al screen for saline-alkali tolerant bacteria B2222, which can grow in salinity at pH 10 and 8.78% salinity, with higher potassium-dissolving, phosphorus-dissolving, iron-producing carrier capacity (Liu Xinpeng et al, 2022). Jiang Juquan the isolated halomonas in inner mongolian saline-alkali soil can survive at a pH of 6-12 and a salinity of 0-15%, most suitable salinity is 3% (JiangJuquan et., 2013), however, no study has been made on its denitrification performance. Dan Xiaotong isolating the strain in seawater with salinity of 33, the nitrite concentration can be reduced from 32.79mg/L to 0.98mg/L in 40h, and the strain is identified as Halomonas (Dan Xiaotong et al, 2013). Therefore, halomonas is an important component of salt-tolerant aerobic denitrifying bacteria, and is widely existing in inland secondary saline-alkali environments.
In order to effectively promote the efficient and safe application of the ammonia metabolism technology in the saline-alkali water aquaculture industry, the characteristics of high salt and high alkali in the secondary saline-alkali environment are necessary to be taken as the basis, aerobic denitrifying bacteria indigenous strains which are suitable for saline-alkali water and have good denitrification effect are screened, separated and identified from the alkaline water and the characteristics of environmental adaptability, ecological functional efficiency, application safety and the like of the strains are analyzed and evaluated, so that microbial inoculum products and application technologies suitable for practical application of saline-alkali water aquaculture production are developed, and research reports of related strains for purifying the ammonia in the saline-alkali water aquaculture by using halomonas are not seen at present.
Disclosure of Invention
Based on this, the first aspect of the present invention provides a salt-tolerant aerobic denitrifying bacteria DWK9, which is named as halomonas Halomonas shizuishanensis DWK and is preserved in China general microbiological culture collection center (CGMCC), with the preservation number of CGMCC 27980, the preservation date of 2023 and 24 months, and the preservation address of institute of microorganisms, national academy of sciences 3, north Star west road 1, in the korean region of beijing. The species of the strain is determined by carrying out 16SrDNA identification on aerobic denitrifying bacteria and constructing a genus level evolutionary tree and combining with analysis of morphological characteristics and physiological and biochemical characteristics of bacteria, and the strain is shown as SEQ ID NO:1 and the logged-in gene sequence in GenBank, and identifying the strain as halomonas Halomonas shizuishanensis DWK9, can efficiently reduce the ammonia nitrogen concentration in saline-alkali soil, and solves the problem of poor ammonia nitrogen treatment effect of microbial agents in the traditional fresh water environment in the secondary saline-alkali water culture environment.
Preferably, the salt-tolerant aerobic denitrifying bacteria DWK9 have salt-tolerant, aerobic and denitrifying functions.
Preferably, the salt-tolerant aerobic denitrifying bacteria DWK9 is derived from saline-alkali soil.
Preferably, the temperature suitable for growth of the salt-tolerant aerobic denitrifying bacteria DWK9 is 0-45 ℃, the pH value is 7-10, and the salinity is 0-35.
More preferably, the temperature suitable for growth of the salt-tolerant aerobic denitrifying bacteria DWK9 is 28-30 ℃, the pH value is 8-9, and the salinity range is 3-14.
The halomonas Halomonas shizuishanensis DWK is verified by adopting a BTB plate screening method, a salinity tolerance detection method, a most suitable pH range detection method, an ammonia nitrogen removal efficiency detection method, a cultured fish safety test method and the like, and is specifically as follows:
the strain is separated from a low-lying saline-alkali land in a Wukou area of mountain city of Ningxia Hui nationality rocky point in northwest inland secondary saline-alkali lands in China. Firstly, 1g of fresh saline-alkali soil is taken for enrichment and domestication, DM culture medium containing ammonia nitrogen and nitrate nitrogen is adopted for aerobic enrichment, and the culture is carried out for 1 week under the conditions of 28-30 ℃ and the rotating speed of 150-200 rpm/min, so that microorganisms which cannot survive in a high-concentration nitrogen environment are eliminated. Secondly, separating and purifying the enriched matters, and primarily screening according to an aerobic denitrifying bacteria plate separation method, wherein the principle is that bromothymol blue serving as an indicator of a BTB culture medium is blue when the pH value is more than 7.6, and bacterial denitrification is an alcaligenes process, and when denitrifying bacteria grow in a plate, bacterial colonies appear blue or blue halos due to the increase of the pH value, so that pure bacterial cultures are obtained through separation. Culturing the enriched and domesticated bacterial liquid on a BTB culture medium by streaking for 48 hours at 28 ℃, picking out blue single colonies with different forms on a BTB flat plate, and separating and purifying to obtain the bacterial strain with good growth performance. And performing performance detection on the single bacterial strain, inoculating the selected bacterial colony to a DM culture medium containing 100mg/L ammonia nitrogen and 614mg/L nitrate nitrogen, culturing for 3 days at the temperature of 28-30 ℃ and the rotating speed of 150-200 rpm/min, measuring the ammonia nitrogen, nitrite and nitrate nitrogen content in water, and selecting the bacterial strain capable of effectively reducing the ammonia nitrogen concentration of the water body for identification and seed conservation for later use.
The strain can grow at 0-45 ℃ and the optimal temperature range is 28-30 ℃; can grow at pH 7-10, and is most suitable for pH 8-9; the salinity range is 0-35, and the most suitable salinity is 3-14. The strain can reach a peak value after growing for 1-2 days in a culture water nutrition environment under the optimal condition, and the quantity level is maintained at 10 7 CFU/mL during 2-5 days, so that the strain is suitable for most saline-alkali soil.
The second aspect of the invention provides a sewage treatment microbial inoculum, which comprises the salt-tolerant aerobic denitrifying bacteria DWK9.
The third aspect of the invention provides application of the salt-tolerant aerobic denitrifying bacteria DWK9 or the sewage treatment microbial inoculum in sewage deamination and denitrification treatment.
Preferably, the sewage includes saline-alkali soil aquaculture water, domestic sewage and lake sewage.
Preferably, the sewage deamination and denitrification treatment is ammonia nitrogen removal or purification of saline-alkali soil culture water.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a salt-tolerant aerobic denitrifying bacterium DWK9, which is a new bacterium of the genus halomonas, named Halomonas shizuishanensis DWK and deposited with the accession number CMCC27980. The strain is separated from northwest secondary saline-alkali soil, is verified by BTB flat screening, salinity tolerance detection, most suitable pH range detection, ammonia nitrogen removal efficiency detection, cultured fish safety test and other methods, has salt tolerance, aerobic and denitrification functions, is suitable for growth at a temperature of 0-45 ℃, has a pH value of 7-10 and a salinity of 0-35, can be applied to ammonia nitrogen removal of saline-alkali soil culture water, and has great application potential in ammonia nitrogen removal of saline-alkali culture water, domestic sewage and lakes and rivers.
Drawings
FIG. 1 is a phylogenetic tree diagram of the salt tolerant aerobic denitrifying bacteria DWK9 in the examples.
Fig. 2 is a morphological characteristic picture of salt-tolerant aerobic denitrifying bacteria DWK9 under an oil mirror and a transmission electron microscope in the example.
FIG. 3 shows the results of the salt tolerance test of the salt tolerant aerobic denitrifying bacteria DWK9 in the examples.
FIG. 4 shows the results of the salt tolerance test of Na 2SO4 of the salt tolerant aerobic denitrifying bacteria DWK9 in the examples.
FIG. 5 shows the results of the salt tolerance test of Na 2CO3-NaHCO3 of the salt tolerant aerobic denitrifying bacteria DWK9 in the examples.
FIG. 6 shows the pH resistance test results of the salt-tolerant aerobic denitrifying bacteria DWK9 in the example.
FIG. 7 shows the results of temperature resistance test of the salt-tolerant aerobic denitrifying bacteria DWK9 in the examples.
FIG. 8 shows the results of safety detection of the salt tolerant aerobic denitrifying bacteria DWK9 in the examples.
FIG. 9 is a daily monitoring of the ammonia concentration change and zebra fish survival results of salt tolerant aerobic denitrifying bacteria DWK9 in the examples.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.
In the following examples, an aerobic denitrifying bacterium is first isolated and identified, which is identified as halomonas, named halomonas Halomonas shizuishanensis DWK, and is preserved in China general microbiological culture collection center (CGMCC), with the preservation number of CGMCC 27980, the preservation date of 2023 and 24 months, and the preservation address of North Star XU 1 national academy of sciences 3 microbiological institute in the Qingyang area of Beijing. Meanwhile, the salt tolerance, the temperature tolerance, the pH resistance, the ammonia nitrogen and the nitrite removal efficiency of the strain are analyzed, the strain is applied to simulate a secondary saline-alkali water environment, the survival curve of the strain is obtained, and the influence of the strain on the survival curve of the cultured animals and the deamination effect of the strain in an actual culture water body are achieved.
Example 1
Test strain: DWK9 (Halomonas shizuishanensis).
Test fish: the body length of the raw zebra fish is 2-2.5 cm (purchased from the flower and bird market).
Test agent: PBS solution (pH 7.2), bacterial genome kit (full gold), agarose gel (biological organism), bacterial strain physiological and biochemical characteristic identification kit (Qingdao sea Bo), ammonia nitrogen standard solution (100 mg/L), nitrite nitrogen standard solution (100 mg/L), nitrate nitrogen standard solution (100 mg/L) and gas phase molecular absorption spectrometer related solution.
Test instrument: ultra-clean bench, electronic balance, autoclave, biochemical incubator, shaker, oven, centrifuge, enzyme-labeled instrument, PCR instrument, dielectrophoresis instrument, microscope, metal bath, salinity meter, pH meter, gas phase molecular absorption spectrometer.
Test medium:
BTB (bromothymol blue) medium: KNO 3 g, L-asparagine 1g, KH 2PO4 g, ferrous chloride 0.05g, calcium chloride 0.2g, magnesium sulfate 1g, BTB 1mL (1% in ethanol), agar 20g, deionized water 1000mL, pH7.3, 1X 10 5 Pa, and sterilizing for 30min.
DM medium ：Na2HPO4·7H2O 7.9g,NH4Cl 0.3g,MgSO4·7H2O 0.1g,C4H4Na2O4·6H2O 4.7g(0.8351g C), microelement solution 2mL (Scholten, 1999), KNO 3 1.0.0 g, deionized water 1000mL.
LB solid medium: yeast powder 5g, tryptone 10g, naCl 10g, agar powder 15g, deionized water 1000mL, pH 7.2-7.4, salt resistance/pH resistance/temperature resistance detection: 5g of yeast powder, 10g of tryptone, 10g of NaCl and the rest of components are replaced by NaCl according to the required detection salt, and the ratio is calculated according to the detection requirement. Simulated saline-alkali water (7％)：NaHCO3 451.75g,KCl 41.09g,CaCl2 1583.41g,Na2SO4 3364.35g,MgCl2 799.15g,NaCl 763.49g.
Step one: enrichment domestication
And (5) peeling off a white salt shell on the surface of the saline-alkali soil, taking fresh surface soil of 0-30 cm, and filling the fresh surface soil into a sterile centrifuge tube for freezing for later use. Taking 1g of soil sample, filling 100mL of sterile DM liquid culture medium (121 ℃ C., 20 min), adding sterile glass beads, sealing the bottle mouth by using a sealing film, and carrying out enrichment culture for 7d at the speed of 200rpm/min at the temperature of 28 ℃. The cultured bacterial suspension was transferred to fresh DM sterile liquid medium at 1% (V/V) inoculum size and cultured with shaking at 180rpm/min at 28℃for 7d.
Step two: separation and purification
The principle of the primary screening is that bromothymol blue serving as an indicator of a BTB medium is blue when the pH value is more than 7.6, the denitrification of bacteria is an alcaligenes process, and when denitrifying bacteria grow in a plate, a colony appears blue or blue halo due to the rise of the pH value. 10mL of the enriched and domesticated culture solution is taken to 90mL of sterile water, the solution is subjected to gradient dilution, a proper dilution gradient is taken to be coated on a BTB culture medium plate, the culture is carried out for 48 hours at 28 ℃, single colonies of blue halos are selected from the BTB plate, and scribing and separation are continued on the BTB plate, so that pure-cultured single colonies are obtained.
Step three: performance detection
And screening strains capable of efficiently removing ammonia nitrogen by taking the ammonia nitrogen removal rate as a standard. The separated single bacteria are inoculated in a nitrate culture medium for 48 hours at the temperature of 28 ℃ at 180r/min, 10mL of bacteria liquid is taken, the bacteria are centrifuged at 6000X rpm for 10 minutes, the bacteria are washed by PBS, after centrifugation, the bacteria are inoculated in 100mL of DM culture medium taking 78.5mg of NH 4 + -N and 138.62mg/LNO 3 - -N as nitrogen sources (counted by N), the bacteria are subjected to shaking culture at the temperature of 28 ℃ at 180rpm/min for 72 hours, and the culture medium without bacteria is used as a reference, so that the NH 4 + -N, the NO 2 - -N and the NO 3 - -N contents in the supernatant are determined.
Step four: 16SrDNA identification
Taking bacterial liquid of the culture solution for 24 hours, centrifuging at 6700rpm for 1min, and removing the supernatant as much as possible. If the bacterial liquid is too large, the bacterial liquid can be collected by centrifugation for a plurality of times; according to the instructions of the bacterial genome kit (gold of the whole formula), colorless solution RB (containing RNaseA) was added, the bacterial pellet was suspended by shaking, taking care that there should not be small clumps; adding blue solution LB, gently turning up and down, mixing for 4-6 times to enable thalli to be fully cracked to form blue transparent solution, wherein the color of the blue transparent solution is changed from semi-transparent to transparent, and the cracking is complete; adding yellow solution NB, gently mixing for 5-6 times, and standing at room temperature for 2min until the color is changed from blue to yellow; centrifuging at 12000 Xg for 5min, carefully sucking the supernatant, and adding into a centrifugal column; centrifuging at 7300rpm for 1min, discarding effluent, adding into column for multiple times, and centrifuging in the same step to discard effluent; 650 μl of solution WB was added and centrifuged at 7300rpm for 1min, and the effluent was discarded; centrifuging at 7300rpm for 1-2 min to thoroughly remove residual WB; placing the centrifugal column in a clean centrifuge tube, adding 50 mu L of EB (pH > 7.0) into the center of the column, standing for 1min at room temperature, preheating the EB in water bath at 60-70 ℃, centrifuging for 1min at 10000 Xg, eluting DNA, and preserving the eluted DNA at-20 ℃ for later use.
The extracted DNA was subjected to PCR amplification, and the PCR amplification reaction system was as follows: DNA 1uL, primer: upstream and downstream 1uL each, taqMix 210uL containing dNTP, buffer, taq enzyme, ddH 2 O make up 20uL. Primers B27F (AGAGTTTGATCCTGGCTCAG), B1492R (TACGGYTACCTTGTTACGACTT); setting PCR amplification conditions: 95℃for 3min,94℃for 30s,58℃for 30s,72℃for 5min,30 cycles; taking 2uL of PCR products, performing electrophoresis detection imaging by using 2% agarose gel under the conditions of 50V and 30min, and sending the PCR products with the residual amplified success to the biological sequencing after the detection strip is clear and qualified.
The obtained sequence was analyzed by BLAST program with sequence results having a similarity of more than 98% in EZbiocloud gene library, and the analysis result was analyzed using MEGA6.0 analysis evolutionary tree, and the phylogenetic tree of the resulting DWK9 strain was shown in FIG. 1. According to phylogenetic tree analysis, the tree branches of DWK9 were most similar to the strain of Halomonas (Halomonas) lutescens family accession number Q1UT, with a 16sRNA sequence similarity of 99.07%. The self-development value of the branch is as high as 95, so that the strain No. 9 belongs to a new strain different from Q1U (T) of the genus halomonas from the perspective of the evolutionary tree.
The > DWK9 sequence is as follows:
GACCTGGTATACCATGCAGTCGAGCGGTAGCACAGAGAAGCTTGCTTCTTGGGTGACGAGCGGCGGACGGGTGAGTAATGCATAGGAATCTGCCCGATAGTGGGGGATAACCTGGGGAAACCCAGGCTAATACCGCATACGTCCTACGGGAGAAAGGGGGCTCCGGCTCCCGCTATTGGATGAGCCTATGTCGGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACATCGGGACTGAGACACGGCCCGAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAAGAACGCCTAGTGGTTAATACCCATTAGGAAAGACATCACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCCGGTTGTGAAAGCCCCGGGCTCAACCTGGGAACGGCATCCGGAACTGTCAAGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCGGTGTAGCGGTGAAATGCGTAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACCAGCCGTTGGGTGCCTAGCGCACTTTGTGGCGAAGTTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCTGCGAACTTGTGAGAGATCACTTGGTGCCTTCGGGAACGCAGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTATTTGCCAGCACGTAATGGTGGGAACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCCGGTACAAAGGGTTGCCAACTCGCGAGAGTGAGCTAATCCCGAAAAGCCGGTCTCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGACTGCACCAGAAGTGGTTAGCCTAACGCAAGAGTCGATCTGGGTCGG(SEQ ID NO：1).
Adjacent method based on the 16SrRNA gene sequence system development tree, on the relationship node between the DWK9 strain and the Halomonas member, the percentage of Bootstrap based on 1000 resampling (. Gtoreq.70%) was listed.
Step five: morphological and physiological biochemical detection
Colony morphology observation: culturing in LB culture medium at 28deg.C for 72 hr, observing colony shape, size, color, smooth surface, regular edges, raised degree, etc., culturing in LB solid culture medium at 28deg.C for 48 hr, sampling, performing gram staining experiment, observing gram staining result and thallus morphology, size, arrangement, etc. under microscope, and observing lipid particle, cell wall, spore, metachromatic particle staining experiment under optical microscope, and staining method with reference to "common bacterial System identification Manual (Dongxiu bead, etc.). The result shows that the DWK9 colony has smooth edge, round shape, moist surface, light yellow to yellowish white, positive bacterial strain cell wall, negative lipoid grain, spore and metachromatic grain.
Physiological and biochemical characteristics of the strain: the kit is purchased from Qingdao sea blogs, and the operation is shown in related specifications. Oxidase assays and catalase assays, methods are described in the handbook of common bacterial System identification (Dongxiu beads, et al). As shown in FIG. 2, the results show that DWK9 oxidase reaction is negative, catalase reaction is positive, phenylalanine deaminase is negative, ONPG is negative, glucose, maltose and sucrose are not produced by oxidative fermentation, MR-VP reaction is negative, gelatin, starch and esculin hydrolysis are negative, indole can be produced, nitrate reduction can be carried out, and H2S can be produced. Compared with Halomonas lutescensQ1U(＝KP259554T)、Halomonas lionensisRHS90(＝HE661586T)、Halomonas zhaodongensisNEAU-ST10-25T(＝JQ762286T)、Halomonas venustaDSM4743(＝NR042069)、Halomonas meridianaDSM5425(＝AJ306891)、Halomonas hydrothermalisSlthf2(CP023656), with closer relativity, the DWK9 is further proved to be a new bacterium due to the difference of physiological and biochemical characteristics.
Table 1: physiological and biochemical identification kit
| Authentication item | Product name | Shang Pinhao A |
| Glucose oxidative fermentation | Glucose fermentation tube | GB117 |
| Maltose fermentation | Maltose fermentation tube | GB067 |
| Sucrose fermentation | Sucrose fermentation tube | GB169 |
| MR-VP reaction | MR-VP biochemical tube | GS003 |
| ONPG reaction | ONPG biochemical tube | GS009 |
| Starch hydrolysis | Starch hydrolysis culture medium | HB8523 |
| Cellulose hydrolysis | Cellulose congo red culture medium | HB8638 |
| Hydrolysis of esculin | Esculin biochemical tube | GS021 |
| Indole production ability | Indole culture medium | HB4109 |
| Phenylalanine deaminase | Phenylalanine deaminase biochemical tube | GB095 |
| Gelatin liquefaction reaction | Gelatin biochemical tube | SN039 |
| Hydrogen sulfide production capability | Hydrogen sulfide biochemical tube | GB065 |
Step six: salt tolerance test
DWK9 was inoculated into 100mL of LB liquid medium, cultured at 28℃at 180r/min until OD600 = 1.0, the culture broth was inoculated into NaCl-type salt-tolerant medium (NaCl concentrations 0%, 1%, 3%, 7%, 9%, 11%, 14%, 20%, 25%, 30%, 35%) at an inoculum size of 1% (V/V), cultured at 180rpm/min for 7d, at 28℃at 3d and 7d, sampled, and OD 600 of each sample was measured to examine the growth of the strain. The results are shown in figure 3, which shows that the strain can survive under the salinity of 0-20 NaCl concentration, and the most suitable salinity range is 3-14.
Considering that saline alkali water in northwest areas is mainly sulfate type, the growth conditions of DWK9 under the conditions that the concentration of Na 2SO4 is 0%, 1%, 3%, 7%, 9%, 11% and 14% are respectively tested, the culture is carried out at 180rpm/min for 3d and at 28 ℃, and the OD 600 of each sample is measured. The results are shown in figure 4, which shows that the strain can survive under the salinity of 0-14 of Na 2SO4 concentration, and the most suitable salinity range is 5-14.
Considering that saline alkali water in northeast areas is mainly carbonate, the growth conditions of DWK9 under the conditions that the concentration of Na 2CO3-NaHCO3 is 0%, 1%, 3%, 7%, 9%, 11% and 14% are respectively tested, the culture is carried out at 180rpm/min for 3d and at 28 ℃, and the OD 600 of each sample is measured. The results are shown in FIG. 5, which shows that the strain can survive under the salinity of Na 2CO3-NaHCO3 concentration of 0-14, and the most suitable salinity range is 3-11.
Step seven: pH resistance detection
DWK9 was inoculated into 100mL of LB liquid medium, cultured at 28℃at 180r/min to OD 600 =1.0, the culture broth was inoculated into the LB liquid medium in an inoculum size of 1% (V/V), pH was adjusted to 7, 8, 8.5, 9, 9.5, 10, 180rpm/min, and cultured at 28℃for 5d, OD 600 of each sample was measured, and the strain was examined for growth. As a result, as shown in FIG. 6, it was revealed that the strain survived under the conditions of pH 7 to 10, and the most suitable pH was 9.
Step eight: temperature resistance detection
DWK9 was inoculated into 100mL of LB liquid medium, cultured at 28℃at 180r/min to OD 600 =1.0, the culture broth was inoculated into the LB liquid medium in an inoculum size of 1% (V/V), and the temperature was adjusted to 4℃at 10℃at 14℃at 20℃at 28℃at 30℃at 37℃at 41℃at 45℃at 50℃at 180rpm/min, followed by culturing for 7 days, and the OD600 of each sample was measured to detect the growth of the strain. As a result, as shown in FIG. 7, it was revealed that the strain survived at a temperature of 10 to 37℃and a most suitable temperature was 28 to 30 ℃.
Step nine: application security detection
As the strain is selected from northwest regions, the saline-alkali water environment with the salinity of 7% in the northwest regions is simulated, DWK9 is inoculated into sterilized simulated saline-alkali water (with the salinity of 7), the final concentration of water colony is 10 4 CFU/mL, the concentration of the strain is stabilized at 10 6 CFU/mL for 2-4 days, and the growth curve of the strain DWK9 is shown in figure 8.
To further verify the deamination effect of the strain in the aquaculture water, the sterilized zebra fish aquaculture water is used as a basis to test the water, the concentration of ammonia in the water is regulated to 40mg/L by NH 4 Cl, the final concentration of bacterial colonies in the water is 10 4 CFU/mL during inoculation, the water is cultured for one week by adding an aeration pump, the change of the concentration of ammonia in the water and the survival number of the zebra fish are monitored every day, and the result is shown in figure 9, which shows that the ammonia nitrogen removal rate of a treatment group added with DWK9 is 18.22% and 24.26% higher than that of a control group without DWK9, the survival rate of the zebra fish is improved by 3.33% and 6.67% and the survival rate of a bacteria group without adding on day 14 is improved by 10% compared with that of a bacteria group without obvious adverse effect on the cultured organisms.
In conclusion, the halomonas DWK9 has stronger salt tolerance to three types of saline-alkali soil bacteria, wherein the salt content in the environment of sodium chloride (NaCl) is 0-20, the most suitable chloride content is 3-14, the most suitable salt content in the environment of sulfate (Na 2SO4) is 5-14, and the most suitable salt content in the environment of carbonate (Na 2CO3-NaHCO3) is 3-11. Meanwhile, the strain can survive under the condition of pH of 7-10, the most suitable pH is 9, and the strain can survive under the condition of temperature of 10-37 ℃ and the most suitable temperature is 28-30. The brine with the salinity of 7% is simulated, so that the final concentration of bacterial colonies of an inoculated water body is 10 4 CFU/mL, the concentration of DWK9 is stabilized at 10 6 CFU/mL within 2-4 days, and the strain can be applied to ammonia nitrogen removal of saline-alkali soil culture water bodies, and particularly has better potential for removing ammonia nitrogen in saline culture water bodies, domestic sewage and lakes and rivers.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments and that the general principles described herein may be applied to other embodiments without the need for inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (9)
1. A salt-tolerant aerobic denitrifying bacterium DWK9 is named as halomonas Halomonas shizuishanensis DWK and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC 27980, the preservation date of 2023 and 24 months, and the preservation address of North Star Xishu No. 1 and 3 national academy of sciences of China are available in the Chaoyang area of Beijing.
2. The salt tolerant aerobic denitrifying bacteria DWK9 according to claim 1, wherein the salt tolerant aerobic denitrifying bacteria DWK9 has salt tolerant, aerobic and denitrifying functions.
3. The salt tolerant aerobic denitrifying bacteria DWK9 according to claim 1, wherein the salt tolerant aerobic denitrifying bacteria DWK9 is derived from saline-alkali soil.
4. The salt-tolerant aerobic denitrifying bacteria DWK9 according to claim 1, wherein the temperature at which the salt-tolerant aerobic denitrifying bacteria DWK9 is suitable for growth is 0-45 ℃, the pH value is 7-10, and the salinity is 0-35.
5. The salt-tolerant aerobic denitrifying bacteria DWK9 according to claim 4, wherein the temperature at which the salt-tolerant aerobic denitrifying bacteria DWK9 is suitable for growth is 28-30 ℃, the pH value is 8-9, and the salinity range is 3-14.
6. A sewage treatment bacterial agent, characterized in that it comprises the salt-tolerant aerobic denitrifying bacteria DWK9 according to any one of claims 1 to 5.
7. Use of the salt-tolerant aerobic denitrifying bacteria DWK9 according to any one of claims 1 to 5 or the sewage treatment bacterial agent according to claim 6 in deamination and denitrification of sewage.
8. The use according to claim 7, wherein the sewage comprises saline-alkali soil aquaculture water, domestic sewage or lake sewage.
9. The use according to claim 7, wherein the deamination and denitrification treatment of sewage is ammonia nitrogen removal or purification of saline-alkali soil aquaculture water.
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