CN113604379A

CN113604379A - Pseudomonas holothurians with heterotrophic nitrification-aerobic denitrification function and application thereof

Info

Publication number: CN113604379A
Application number: CN202110761877.2A
Authority: CN
Inventors: 舒琥; 孙慧明; 黄文�; 赵洋; 陈琼华; 岳莎
Original assignee: Guangzhou University; Institute of Animal Science of Guangdong Academy of Agricultural Sciences
Current assignee: Guangzhou University; Institute of Animal Science of Guangdong Academy of Agricultural Sciences
Priority date: 2021-07-06
Filing date: 2021-07-06
Publication date: 2021-11-05
Anticipated expiration: 2041-07-06
Also published as: CN113604379B

Abstract

The invention discloses a holothurian strain with heterotrophic nitrification-aerobic denitrification function and application thereof. The Pseudomonas holothurians is named as Pseudomonas perfectoris WM33 (GDMCC No: 61718), has a preservation date of 2021, 06 and 10 days, and is preserved in Guangdong province microbial strain preservation center of Guangdong province microbial research institute of No. 59 building, 5 building, Guangdong province of No. 5 building, of Miehu No. 100 of Miehrland, Guangzhou, City. The strain can be efficiently degraded

And

has the functions of heterotrophic nitrification and aerobic denitrification, has good environmental adaptability and high safety, and has wide application prospect in the field of biological denitrification treatment of aquaculture tail water or other nitrogen-containing sewage.

Description

Pseudomonas holothurians with heterotrophic nitrification-aerobic denitrification function and application thereof

Technical Field

The invention belongs to the field of environmental microorganisms, and particularly relates to pseudomonas oharii with heterotrophic nitrification-aerobic denitrification functions and application thereof.

Background

From the fishery law issued in 1986, the aquaculture industry in China is developing towards intensification and high density. However, in the aquaculture mode, when intensive and high-density development is pursued, excessive residual baits and feces in the water body cannot be decomposed and utilized in time, so that nitrogen elements in the water are seriously accumulated, eutrophication of the aquaculture water body is caused, and sustainable development of aquaculture industry in China is restricted. The aquaculture industry in China is rapidly developed and faces huge environmental challenges.

And

is an important index for evaluating aquaculture water, and the toxicity of the aquaculture water can directly influence the survival of aquaculture objects and the quality of aquatic products. Biological denitrification is a key biochemical process in a biological filter facility. The traditional biological denitrification process is basically established based on anaerobic ammonia oxidation (ANAMMOX), oxygen-limited autotrophic nitrification-denitrification (OLAND), shortcut nitrification and denitrification (SNAD), nitrite complete autotrophic denitrification (CAND) and other mechanisms. The conventional method includes many steps and the cost of removing nutrients from the wastewater is high. Anaerobic-anoxic-aerobicOxygen (A)²O) process is the most commonly used biological nitrogen and phosphorus removal (BNR) process, which requires at least three bioreactors (e.g., anaerobic, anoxic, and aerobic) in series with distinct and complex operating conditions. Conventional biological denitrification techniques have a number of disadvantages: (1) autotrophic nitrifying bacteria grow slowly, and high biomass concentration is difficult to achieve unless investment and operation cost are increased; (2) nitrifying bacteria are very sensitive to pH, DO, T and the like; (3) under the load of high ammonia nitrogen and organic matters, the shock resistance of the autotrophic bacteria is poor; (4) the nitrification and denitrification reactions have different requirements on factors such as organic matters, dissolved oxygen and the like, and the nitrification and denitrification reactions are generally required to be carried out in two independent reactors due to the huge ecological niche difference of functional microorganisms. Heterotrophic nitrification-aerobic denitrification (HN-AD) is a novel biological denitrification technology, not only can successfully overcome the problem of nitrification and denitrification incompatibility caused by different oxygen demands, but also has the advantages of better utilization of organic substrates, higher oxygen resistance, denitrification rate and the like compared with autotrophic organisms, and is widely concerned in recent years. However, research on such HN-AD bacteria has not been completed so far, and further determination of more efficient and stable HN-AD strains obtained by separation and purification is still needed.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a holothurian strain with heterotrophic nitrification-aerobic denitrification functions. The Pseudomonas holothurians with heterotrophic nitrification-aerobic denitrification function can be efficiently degraded

And

has the functions of heterotrophic nitrification and aerobic denitrification. The strain has good environmental adaptability and high safety. Therefore, the method has wide application prospect in the field of biological denitrification treatment of aquaculture tail water or other nitrogen-containing sewage.

The invention also aims to provide the application of the pseudomonas holothurians with the heterotrophic nitrification-aerobic denitrification function.

The purpose of the invention is realized by the following technical scheme: a holothurian with heterotrophic nitrification-aerobic denitrification function is named as holothurian (Pseudomonas perfactomarina) WM33, the preservation number is GDMCC No. 61718, the preservation date is 2021, 06 and 10 days, and the microbial strain preservation center of Guangdong province, which is located in Guangzhou city, Michelia Toxico 100, Dazhou 59, 5 th building, Guangdong province microbial research institute, is preserved.

The application of the pseudomonas holothurians with the heterotrophic nitrification-aerobic denitrification function in the nitrogen-containing sewage denitrification treatment preferably comprises the following steps: inoculating the pseudomonas holothurians with the heterotrophic nitrification-aerobic denitrification function into the nitrogen-containing sewage, and culturing to obtain denitrified wastewater.

The nitrogen-containing sewage is preferably aquaculture tail water.

The carbon source in the culture is at least one of sodium citrate, sodium succinate and sodium acetate; sodium citrate is preferred.

The C/N of the sewage in the culture is 10-40, and the preferable condition is that the C/N is 10.

The pH value of the sewage in the culture is 6-7, and the preferable pH value is 7.

The temperature in the culture is 15-35 ℃, and the preferable condition is that T is 25-35 ℃.

The conditions for the above culture were adjusted to the carbon source and C/N, pH value of the nitrogen-containing wastewater.

Compared with the prior art, the invention has the beneficial effects that:

1. the holothurian WM33 is applied to the field of nitrogen-containing aquaculture tail water treatment, has no adverse effect on aquaculture objects, and has higher aquatic organism safety; and the compound has sensitivity to various common clinical antibiotics such as norfloxacin, streptomycin, tetracycline hydrochloride and the like, and has higher ecological safety. Therefore, the method is suitable for most aquaculture water bodies.

2. The pseudomonas ophiolima WM33 has the functions of heterotrophic nitrification and aerobic denitrification; can utilize various organic carbon sources and has strong tolerance to high-concentration organic carbon, thereby having better water organic carbon removal capability. The strain is particularly suitable for treating nitrogen-containing sewage with high C/N.

3. The Pseudomonas rhodochrous WM33 is applied in the field of nitrogen-containing sewage treatment, and under the condition of complete aerobic condition, the strain can be respectively utilized

And

as the only inorganic nitrogen source, aerobic nitrification and denitrification are carried out; the highest degradation efficiency can respectively reach 76.0 percent, 92.1 percent and 88.0 percent.

4. The Pseudomonas holothurians WM33 can overcome the incompatibility problem of nitrification and denitrification caused by different oxygen demands, and makes it possible to carry out nitrification and denitrification synchronously in the same aerobic reactor. The strain is applied to the microbial denitrification process of the aquaculture water body, is favorable for reducing the occupied area of equipment and the construction cost, improves the treatment efficiency, can also greatly reduce the periodic water change in the aquaculture process, has good economic and environmental benefits and has wide application prospect.

Drawings

FIG. 1 is a colony morphology of Pseudomonas holothurian WM33 of the present invention on nutrient agar plates.

FIG. 2 is a scanning electron microscope image and a thallus size statistical result image of Pseudomonas panhainii WM 33.

FIG. 3 is a graph showing the gram staining results of Pseudomonas holothurian WM33 of the present invention.

FIG. 4 is a graph showing the results of a fish toxicity test of Pseudomonas holothurian WM33 of the present invention.

FIG. 5 is a graph showing the experimental results of the resistance of the general antibiotics of Pseudomonas holothurian WM33 of the present invention.

FIG. 6 is a graph showing the comparison results between the growth and denitrification of Pseudomonas holothurian WM33 under different organic carbon sources and inorganic nitrogen sources.

FIG. 7 is a graph showing the comparison between the growth and denitrification of Pseudomonas holothurian WM33 of the present invention under different pH and inorganic nitrogen source conditions. FIG. 8 is a graph showing the comparison between the growth and denitrification of Pseudomonas holothurian WM33 of the present invention under different C/N and different inorganic nitrogen sources.

FIG. 9 is a graph showing the comparison between the growth and denitrification of Pseudomonas holothurian WM33 of the present invention at different temperatures and under different inorganic nitrogen sources.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

In the experiment

The determination and analysis methods of the three nitrogen elements are all referred to national standards, wherein

The determination and analysis of (A) is carried out according to the Water quality-determination of ammonia nitrogen-Nessler reagent spectrophotometry (GB HJ 535-2009);

the determination and analysis of (A) is carried out according to "determination of Water quality-nitrate Nitrogen-ultraviolet spectrophotometry" (GB HJ/T346-2007);

the determination and analysis of (A) was carried out according to "determination of Water quality-nitrite Nitrogen-spectrophotometry" (GB 7493-87).

The basic culture medium used in the experiment is sterilized by high-pressure steam at 121 ℃ for 20min, and the formula is as follows:

(1) microelement solution (g/L): EDTA 50g, ZnSO₄·7H₂O 5.02g，CuSO₄·5H₂O 1.57g，FeSO₄·7H₂O 5.0g，CoCl₂·6H₂O 1.61g，(NH₄)₆Mo₇O₂·4H₂O 1.1g，CaCl₂·2H₂O 5.5g，MnCl₂·4H₂O 5.06g，pH 6.0；

(2) Enrichment medium (g/L): KH (Perkin Elmer)₂PO₄ 1.5g，MgSO₄·7H₂O 0.01g，Na₂HPO₄7.9g, 6.45g of sodium citrate dihydrate, NaNO₃ 0.8415g，NH₄Cl 0.192g，NaNO₂0.362g, 2mL of trace element solution, and pH 7.2;

(3) BTB solid Medium (g/L): 6.45g of sodium citrate dihydrate, 1mL of 1% BTB (bromothymol blue) ethanol solution, KH₂PO₄ 1.5g，MgSO₄·7H₂O 0.01g，Na₂HPO₄ 7.9g，NaNO₃ 0.8415g，NH₄Cl 0.192g，NaNO₂0.362g, 2mL of trace element solution, 20g of agar and 7.0-7.5 of pH;

(4) nitrogen source only fermentation medium (DM I) (g/L): 6.45g of sodium citrate dihydrate, KH₂PO₄ 1.5g，MgSO₄·7H₂O 0.01g，Na₂HPO₄ 7.9g，NH₄0.6036g of Cl, 2mL of trace element solution and pH of 7.0;

(5) single nitrogen source fermentation medium (DM II) (g/L): 6.45g of sodium citrate dihydrate and MgSO₄·7H₂O 0.01g，KH₂PO₄ 1.5g，Na₂HPO₄ 7.9g，NaNO₃0.9590g, trace element solution 2mL, pH 7.0;

(6) single nitrogen source fermentation medium (DM III) (g/L): 6.45g of sodium citrate dihydrate, KH₂PO₄ 1.5g，MgSO₄·7H₂O 0.01g，Na₂HPO₄ 7.9g，NaNO₂0.375g, 2mL of trace element solution, pH 7.0.

Example 1

(1) Sample collection

The holothurian WM33 is obtained by screening and separating a water sample and a mud sample of a tilapia culture pond in Fushan City (northern latitude N:22 degrees 50 '34' and east longitude E:113 degrees 57 '25') in Guangdong province.

The sample collection is carried out according to a mixed sample collection method in technical Specification for soil environmental monitoring (HJ/T166-2004), a quincuncial point sampling method is adopted for fixed-point sampling, surface layer water, middle layer water, deep layer water and bottom mud are collected from a culture pond and placed in an aseptic sampling bag, and the aseptic sampling bag is refrigerated, transported and stored at 4 ℃ for later use.

(2) Enrichment, separation and screening of heterotrophic nitrification-aerobic denitrification strains

1) Sample pretreatment: 10g of pond bottom mud is taken, a 300mL large-mouth triangular flask filled with 90mL of sterile normal saline with the concentration of 0.9 percent by mass is placed in an ultra-clean workbench, a small amount of glass beads sterilized by high-pressure steam at 121 ℃ for 15min are placed in the flask, and the flask is oscillated for 1h at 180r/min to break up a bottom mud sample, so that microorganisms in the mud sample are fully suspended in the normal saline.

2) Enrichment culture: 22.2mL of the above sediment pretreatment mixture was added to a 500mL conical flask containing 200mL of enrichment medium, and cultured in a shaker at 30 ℃ and 180r/min for 2-3 days. Adding 1mL of NH with the concentration of 5 percent by mass into the enrichment medium every day₄Cl solution to keep in the culture medium

The ion concentration. Taking 10mL of culture water sample, inoculating the culture water sample into a 300mL large-mouth triangular flask containing 90mL of enrichment medium, and oscillating for 1h at 30 ℃ and 180r/min in a shaking table.

3) Sample plate coating: taking 1mL of the pretreated bottom sediment mixed liquid and the surface layer, the middle layer and the deep layer water samples in the step 1) respectively, putting the pretreated bottom sediment mixed liquid and the surface layer, the middle layer and the deep layer water samples into a test tube filled with 9mL of sterile physiological saline in an ultra-clean workbench, and lightly blowing or shaking the test tube by a liquid transfer gun to mix uniformly. Taking out 1mL of the liquid from the test tube, inoculating into a new test tube containing 9mL of sterile physiological saline, repeating the operation, and sequentially diluting the pretreated substrate sludge mixed liquid and the water sample stock solution to 10 degrees by gradient^-2～10^-4And (4) concentration. Respectively take 10^-1～10^-4100-200 mul of stock solution of the substrate sludge and the water sample with concentration gradients are directly coated in a BTB plate culture medium, each gradient is provided with 3 parallel groups and 1 blank control group, and the mixture is inversely cultured in a constant-temperature biochemical incubator for 2-3 days at 30 ℃.

4) Sample (A)Flat coating of the product enrichment liquid: the bacterial suspension after enrichment culture in the step 2) is diluted in a gradient way, and the process is as follows: taking 1mL of the bacterial suspension from the conical flask in the step 2), putting the bacterial suspension into a test tube filled with 9mL of sterile normal saline, and fully mixing the bacterial suspension, namely diluting the bacterial suspension to a concentration of 10^-1. Then sucking 1mL of liquid from the test tube, inoculating the liquid into a new test tube containing 9mL of sterile physiological saline, uniformly mixing, repeating the step, and sequentially diluting to 10^-2～10^-8A concentration gradient. Then 100-200 mul of each mixed solution with each concentration gradient is taken and respectively coated on a prepared BTB solid plate culture medium in advance, the dilution gradient and the date are marked, and the mixed solution is inversely cultured in a biochemical incubator for 2-3 days at 30 ℃.

5) Separation and purification: colonies of different morphologies on the above medium were picked with an inoculating loop. And (3) scribing on the BTB solid plate culture medium by adopting a plate scribing separation method for separation and purification, and after scribing, inversely placing the plate in a constant-temperature biochemical incubator for 2-3 days at 30 ℃ after opening a small opening in an ultraclean workbench and just standing at room temperature for 5 minutes. Repeating the steps, and selecting a single colony to repeatedly streak and purify for 3-4 times. After observation, picking out single bacterial colony without abnormal morphologic bacteria, performing crystal violet single staining, and inspecting the single bacterial colony under a microscope to obtain pure (100 times of oil lens);

6) point-connection primary screening: and (3) picking the purified strain by using an inoculating needle, inoculating the strain into a BTB denitrification identification medium (BTB solid plate medium), and culturing for 2-3 days. Strains with high denitrification capacity are selected according to the growth condition of colonies and the size of blue halos in BTB culture medium around the colonies, and generally, the larger the blue halos, the higher the denitrification capacity. And respectively inoculating the culture solution to nutrient agar slant, culturing at constant temperature of 30 ℃ for 2-3 days, and then preserving the test tube at 4 ℃.

7) Re-screening the nitrification and denitrification performance: inoculating the strain 3-loop activated slant into nutrient broth, culturing at 30 deg.C and 180r/min for 1 day, and measuring OD₆₀₀. Then inoculating with NH at an amount of 1% (v/v)₄Cl、NaNO₃And NaNO₂Performing shake culture at 30 deg.C and 180r/min in DM I, DM II and DM III fermentation culture medium as the only inorganic nitrogen source, and measuring OD of the culture solution at 0h, 24h and 48h₆₀₀5000r/min, 5min lowTaking supernatant after rapid centrifugation, and respectively measuring

Three nitrogen contents.

(3) Identification

1) Morphological and physiological biochemical characterization

The heterotrophic nitrification-aerobic denitrification bacterial strain WM33 is obtained after the screening and separation, bacterial colonies (shown as a figure 1) are white and opaque on nutrient agar, the surface of the bacterial colonies is raised, the bacterial colonies are round, smooth and moist and have flash, and the edges are complete; the strain has a length of 1.43 +/-0.28 mu m and a width of 0.54 +/-0.04 mu m, is straight-rod-shaped and has no flagella (shown in figure 2); is gram-negative (as shown in FIG. 3). The physiological and biochemical characteristics were measured, and the measurement results are shown in Table 1.

TABLE 1 physiological and biochemical characteristics of Strain WM33

Note: "+" is positive; "-" is negative

2) Identification of heterotrophic nitrification-aerobic denitrification strain WM33 by molecular biological method

The DNA extraction of the strain WM33 used Takara Lysis Buffer for Microorganism to Direct PCR lyase. Amplifying the 16S rDNA by taking the extracted DNA as a template, wherein a pair of universal primers (synthesized by Shanghai bioengineering company, Ltd) are adopted for amplification: upstream primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3', respectively; downstream primer (1492 r): 5'-GGCTACCTTGTTACGACTT-3' are provided. PCR reaction (25. mu.L): 2 × Unique^TM12.5. mu.L of Taq Master Mix (With Dye), 1. mu.L of each of the forward primer and the reverse primer at a concentration of 10. mu.M, 1. mu.L (about 50-200ng) of DNA template, ddH₂O9.5. mu.L. The PCR procedure was as follows: 5min at 94 ℃; 1min at 94 ℃, 1min at 55 ℃, 1.5min at 72 ℃ and 30 cycles; 10min at 72 ℃. Results were analyzed by 1% agarose gel electrophoresis. Sequencing of PCR products was performed by Shanghai bioengineering, Inc.

The length of the 16s rDNA sequence of the strain WM33 is 1441bp, and the nucleotide sequence is as follows:

GGCATGGCGGCAGCTACACATGCAAGTCGAGCGGATGACGGGAGCTTGCTCCTTGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTCCGAAAGGGGCGCTAATACCGCATACGTCCTACGGGAGAAAGTGGGGGATCTTCGGACCTCACGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCTCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGACGGTACCACGGTGGATCAGTGC。

the bacterial strain WM33 is judged to be Pseudomonas holothurians (Pseudomonas perfomarina) by integrating the 16s rDNA, bacterial morphology, colony morphology, physiology, biochemistry and other identification items. The related data are consulted to find that no report is available about the purification of inorganic nitrogen in culture tail water or other nitrogen-containing sewage by Pseudomonas marinus (Pseudomonas perfectoriana). The strain is preserved in 10.06.2021 and is located in Guangdong province microbial strain preservation center of No. 59 building 5 building Guangdong province microbial research institute of Mieheli Zhou 100, Guangzhou city, and the preservation number is GDMCC No. 61718.

Example 2

Pseudomonas holothurian WM33 environmental safety evaluation

(1) Fish toxicity test: healthy zebra fish (Danio rerio) (purchased from Guangzhou Hua Bao Hua bird fish and insect wholesale market in Huawan of Argania of Chou bay of Guangzhou, Guangdong province) with the body length within the range of 3 +/-1 cm are selected and temporarily cultured in continuously aerated large water for 30-45 days, during which normal feeding and regular water changing are carried out. After the state is stable, the mixture is randomly distributed into 15L glass jars, an experimental group added with bacterial liquid and a control group added with equal-volume sterile water are arranged, 30 zebra fishes are arranged in each experimental group, and 3 times of the experimental groups are arranged. Taking overnight-cultured bacterial liquid, centrifuging at 3000r/min for 5min, then discarding supernatant, resuspending with sterile PBS buffer solution, repeating for 1-2 times, suspending with sterile water, diluting to obtain bacterial liquids with different concentrations, and determining bacterial concentration and OD₆₀₀And establishing a standard curve between the concentration and the absorbance of the bacteria, and obtaining the OD according to the determined standard curve₆₀₀The relation between the concentration of the bacteria and the bacteria concentration is adjusted to about 1 multiplied by 10 bacteria amount in the experimental water body⁶CFU/mL, blank control added equal amount of sterilized water. During the experiment, the experimental subjects are normally fed, the experimental water body is completely replaced every three days, the method is repeated after water is changed, the bacterial liquid and the sterilized water are respectively added, the survival rate of each group of zebra fishes is recorded, and the experiment lasts for 10 days.

(2) Common antibiotic resistance experiments: the judgment standard of antibiotic resistance test (antibiotic paper drug sensitivity test) is based on the technical requirements of antibiotic drug sensitivity test (WS/T639-. The method comprises the following specific steps:

A) preparing an MHA (Guangdong Huanji Microbiol technology Co., Ltd.) plate, and correcting the pH value to 7.2-7.4;

B) punching a paper sheet with the diameter of about 6mm by using a puncher and qualitative filter paper, and drying for later use after sterilization;

C) preparing paper sheets with corresponding medicine content according to the types of the antibacterial medicines;

D) respectively inoculating the strains into nutrient broth culture media, and culturing at 30 ℃ and 180r/min until logarithmic phase;

E) uniformly coating 100-150 mu L of bacterial liquid on an MHA plate, and drying for 5min at room temperature;

F) attaching paper containing antibiotics to the center of the MHA plate by using sterile forceps, repeating each experiment for 3 times, and additionally arranging 3 paper containing sterile water to be attached to the center of the MHA plate as a blank control;

G) inverting the flat plate within 15min, and culturing at constant temperature of 30 ℃ for 18 h;

H) the zone diameter was measured using an IP54 metal shell number-displaying vernier caliper (jingsida trade ltd, guang city).

TABLE 2 antibiotic resistance evaluation criteria

(3) Results

In a fish toxicity test, the zebra fish is cultured for 10 days under normal conditions, the survival rate of the zebra fish in a control group is 100 percent, and the bacteria content in an experimental group is about 10 percent⁶CFU/mL, higher than the pathogenic dose of common pathogenic bacteria (10)⁴CFU/mL). The survival rate of the experimental group zebra fish is higher than 99 percent, and has no significant difference with the control group (p)>0.05). The Pseudomonas holothurian WM33 is preliminarily judged to have higher aquatic organism safety (see figure 4).

The results of antibiotic resistance experiments with strain WM33 (see Table 3 and FIG. 5) show that Pseudomonas marinus WM33 is sensitive to many common clinical antibiotics used (FIG. 5), indicating its ecological safety. The experiment also provides guidance for taking precautions and emergency measures in the using link of the strain.

TABLE 3 antibiotic resistance experiments

Classes of antibiotics	Size of antibacterial ring (mm)	Species of sensitivity
			Norfloxacin hydrochloride	34.67±1.53	S
Chloromycetin	0	R
			Gentamicin sulfate	20.33±1.53	S
Tetracycline hydrochloride	26±1	S
			Ciprofloxacin	37.33±0.58	S
Ceftazidime	18.67±3.21	I
			Levofloxacin	25±1	S
Streptomycin
		15±1	S

Example 3

Optimal growth and denitrification conditions of pseudomonas marini WM33

(1) Influence of different organic carbon sources on growth and denitrification performance of pseudomonas marini WM33

Four carbon sources of sodium oxalate, sodium succinate, sodium acetate and sodium citrate were selected, and culture conditions of 10C/N, 180r/min, 7.0 pH, and the like were fixed. Based on a DM fermentation culture medium (see the specific embodiment of the invention), the adding amount of four organic carbon sources of sodium oxalate, sodium succinate, sodium acetate and sodium citrate is 0.67g, 0.81g, 0.41g and 1.29g respectively per liter of the culture medium; NH as sole inorganic nitrogen source₄Cl(DMⅠ)、NaNO₃(DMⅡ)、NaNO₂The addition amount of (DM III) is 0.02675g, 0.04g and 0.0345g per liter of culture medium. Inoculating candidate strains into nutrient broth culture medium, culturing at 30 deg.C and 180r/min for 1 day, inoculating with 1% (v/v) of inoculum size, inoculating into the denitrification culture medium with different organic carbon sources, and measuring OD of culture solution at 0h, 8h, 16h, 24h, 32h, 40h, and 48h₆₀₀Centrifuging at low speed of 5000r/min and 5-10 min, taking supernatant, and respectively measuring

Three nitrogen contents. The experiment was set up with 3 technical replicates in the experimental group and a blank control group to which an equal inoculum of saline was added. Analyzing and selecting four different organic carbon sources of sodium oxalate, sodium succinate, sodium acetate and sodium citrate to influence the growth condition and aerobic denitrification of the pseudomonas marini WM 33.

(2) Influence of different C/N on growth and denitrification performance of Pseudomonas holothurian WM33

Sodium citrate is selected as a carbon source of a denitrification culture medium, culture conditions such as 30 ℃, 180r/min, pH 7.0 and the like are fixed, and C/N gradients are set to be 10, 20, 30 and 40. The adding amount of sodium citrate in the culture medium of each gradient is 1.29g/L, 2.58g/L, 3.87g/L and 5.16 g/L; NH as sole inorganic nitrogen source₄Cl(DMⅠ)、NaNO₃(DMⅡ)、NaNO₂(DM III) in an amount of 0.02675g, 0.04g and 0.0345g per liter of the culture medium. Inoculating candidate strains into nutrient broth culture medium, culturing at 30 deg.C and 180r/min for 1 day, and inoculating with 1% (v/v) of inoculumInoculating into the denitrification culture medium, respectively, and measuring OD in culture medium at 0h, 8h, 16h, 24h, 32h, 40h, and 48h₆₀₀Centrifuging at low speed of 5000r/min and 5-10 min, taking supernatant, and respectively measuring

Three nitrogen contents. The experiment was set up with 3 technical replicates in the experimental group and a blank control group to which an equal inoculum of saline was added. The influence of four different C/N types of 10, 20, 30 and 40 on the growth condition and aerobic denitrification of the Pseudomonas holothurian WM33 is analyzed.

(3) Influence of different pH values on growth and denitrification performance of Pseudomonas holothurian WM33

The culture conditions of 10C/N, 30 ℃, 180r/min and sodium citrate as a single organic carbon source are fixed, and pH gradients are set to be 5, 6, 7, 8 and 9. NH as sole inorganic nitrogen source₄Cl(DMⅠ)、NaNO₃(DMⅡ)、NaNO₂(DM III) in an amount of 0.02675g, 0.04g and 0.0345g per liter of the culture medium. Inoculating candidate strains into nutrient broth culture medium, culturing at 30 deg.C and 180r/min for 1 day, inoculating with 1% (v/v) inoculum size, inoculating into denitrification culture medium, and measuring OD of culture solution at 0h, 8h, 16h, 24h, 32h, 40h, and 48h₆₀₀Centrifuging at low speed of 5000r/min and 5-10 min, taking supernatant, and respectively measuring

Three nitrogen contents. The experiment was set up with 3 technical replicates in the experimental group and a blank control group to which an equal inoculum of saline was added. The influence of five different pH values of 5, 6, 7, 8 and 9 on the growth of the Pseudomonas holothurian WM33 and aerobic denitrification is analyzed.

(4) Influence of different temperatures on growth and denitrification performance of pseudomonas holothurian WM33

The culture conditions of 10% C/N, 7.0% pH, 180r/min and sodium citrate as single organic carbon source are fixed, and the temperature gradient is set at 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C and 35 deg.C. NH as sole inorganic nitrogen source₄Cl(DMⅠ)、NaNO₃(DMⅡ)、NaNO₂The addition amount of (DM III) is 0.02675g, 0.04g and 0.0345g per liter of culture medium. Inoculating candidate strains into nutrient broth culture medium, culturing at 30 deg.C and 180r/min for 1 day, inoculating with 1% (v/v) inoculum size, inoculating into denitrification culture medium, and measuring OD of culture solution at 0h, 8h, 16h, 24h, 32h, 40h, and 48h₆₀₀Centrifuging at low speed of 5000r/min and 5-10 min, taking supernatant, and respectively measuring

Three nitrogen contents. The experiment was set up with 3 technical replicates in the experimental group and a blank control group to which an equal inoculum of saline was added. The influence of five different temperatures of 15 ℃, 20 ℃, 25 ℃, 30 ℃ and 35 ℃ on the growth condition and aerobic denitrification of the holothurian WM33 is analyzed.

As can be seen from example 2 (FIG. 6) and example 1 (Table 1), Pseudomonas marinus WM33 was able to grow on a variety of organic carbon sources such as sodium citrate, sodium succinate and sodium acetate. When grown with sodium succinate and sodium acetate, the maximum biomass at plateau was not as high as sodium citrate. Optimum pH for growth and denitrification is 6-7 (FIG. 7); all can grow and degrade three inorganic nitrogen within the range of 10-40C/N, and the Pseudomonas marini WM33 is suitable for the growth of the microorganism with the C/N condition increasing within the range of 10-40

Gradually inhibited degradation ability (fig. 8); temperature gradient experiments show that the growth speed and inorganic nitrogen degradation efficiency of the holothurian WM33 at the temperature of 25-35 ℃ are obviously higher than those of the holothurian WM33 at the temperature of 15-20 ℃ (figure 9) at the temperature of 15-35 ℃, the holothurian WM33 can grow at the temperature of 15-35 ℃ and the C/N is 10-40, the pH is 6-7, and the adaptive range of the C/N and the temperature is larger. The growth rate is reduced at 15 ℃. Within the range of 10-40, the higher the C/N is, the higher the inhibition effect on the utilization of the nitrate is gradually improved. The growth is slow at pH 5 and 15-20 ℃. When the C/N is more than 20, the growth of the nitrate can be prevented from being influenced, and the degradation of the nitrate can be inhibited. In summary, the optimal denitrification conditions for pseudomonas marini WM33 were sodium citrate, C/N10, pH 7, and T35 ℃ as carbon source.

The highest degradation rates can respectively reach 76.0%, 92.1% and 88.0%.

The holothurian WM33 is applied to the field of nitrogen-containing aquaculture tail water treatment, has no adverse effect on aquaculture objects, and has higher aquatic organism safety; and the antibiotic is sensitive to various clinical common antibiotics, and has higher ecological safety. Therefore, the method is suitable for most aquaculture water bodies; it has the functions of heterotrophic nitrification and aerobic denitrification simultaneously; can utilize various organic carbon sources and has strong tolerance to high-concentration organic carbon, thereby having better water organic carbon removal capability. The strain is particularly suitable for treating nitrogen-containing sewage with high C/N; under completely aerobic conditions, the strain can be used separately

And

as the only inorganic nitrogen source, aerobic nitrification and denitrification are carried out; the strain can overcome the incompatibility problem of nitrification and denitrification caused by different oxygen demands, so that the nitrification and denitrification can be synchronously carried out in the same aerobic reactor, and the strain has good economic and environmental benefits and wide application prospect.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

<110> Guangzhou university

Institute of animal science, Guangdong Academy of Agricultural Sciences

<120> a holothurian strain with heterotrophic nitrification-aerobic denitrification function and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1441

<212> DNA

<213> Pseudomonas pansuis WM33(Pseudomonas perfectorina WM33)

<400> 1

ggcatggcgg cagctacaca tgcaagtcga gcggatgacg ggagcttgct ccttgattca 60

gcggcggacg ggtgagtaat gcctaggaat ctgcctggta gtgggggaca acgttccgaa 120

aggggcgcta ataccgcata cgtcctacgg gagaaagtgg gggatcttcg gacctcacgc 180

tatcagatga gcctaggtcg gattagctag ttggtgaggt aaaggctcac caaggcgacg 240

atccgtaact ggtctgagag gatgatcagt cacactggaa ctgagacacg gtccagactc 300

ctacgggagg cagcagtggg gaatattgga caatgggcga aagcctgatc cagccatgcc 360

gcgtgtgtga agaaggtctt cggattgtaa agcactttaa gttgggagga agggcagtaa 420

gttaatacct tgctgttttg acgttaccga cagaataagc accggctaac tctgtgccag 480

cagccgcggt aatacagagg gtgcaagcgt taatcggaat tactgggcgt aaagcgcgcg 540

taggtggttc gttaagttgg atgtgaaagc cccgggctca acctgggaac tgcatccaaa 600

actggcgagc tagagtatgg tagagggtgg tggaatttcc tgtgtagcgg tgaaatgcgt 660

agatatagga aggaacacca gtggcgaagg cgaccacctg gactgatact gacactgagg 720

tgcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg 780

tcaactagcc gttggaatcc ttgagatttt agtggcgcag ctaacgcatt aagttgaccg 840

cctggggagt acggccgcaa ggttaaaact caaatgaatt gacgggggcc cgcacaagcg 900

gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggcct tgacatgcag 960

agaactttcc agagatggat tggtgccttc gggaactctg acacaggtgc tgcatggctg 1020

tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgta acgagcgcaa cccttgtcct 1080

tagttaccag cacgttatgg tgggcactct aaggagactg ccggtgacaa accggaggaa 1140

ggtggggatg acgtcaagtc atcatggccc ttacggcctg ggctacacac gtgctacaat 1200

ggtcggtaca gagggttgcc aagccgcgag gtggagctaa tctcacaaaa ccgatcgtag 1260

tccggatcgc agtctgcaac tcgactgcgt gaagtcggaa tcgctagtaa tcgcgaatca 1320

gaatgtcgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt 1380

gggttgcacc agaagtagct agtctaacct tcgggaggac ggtaccacgg tggatcagtg 1440

c 1441

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer (27F)

<400> 2

agagtttgat cctggctcag 20

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer (1492r)

<400> 3

ggctaccttg ttacgactt 19

Claims

1. A holothurian with heterotrophic nitrification-aerobic denitrification function is characterized in that: the holothurian pseudomonad with heterotrophic nitrification-aerobic denitrification function is named as holothurian pseudomonad (Pseudomonas perfactomarina) WM33, the preservation number is GDMCC No. 61718, the preservation date is 2021, 06 and 10 days, and the microbial strain preservation center of Guangdong province, which is located in Guangzhou city, Michelia Tokoro No. 100, Dazhou No. 59, building 5, Guangdong province microbial research institute, is preserved.

2. The use of Pseudomonas holothurian with heterotrophic nitrification-aerobic denitrification capability as claimed in claim 1 in the denitrification of nitrogen-containing wastewater.

3. The use of Pseudomonas holothurian with heterotrophic nitrification-aerobic denitrification function in nitrogen-containing wastewater denitrification treatment according to claim 2, characterized by comprising the steps of: inoculating the pseudomonas holothurians with the heterotrophic nitrification-aerobic denitrification function into the nitrogen-containing sewage, and culturing to obtain denitrified wastewater.

4. The use of Pseudomonas holothurian with heterotrophic nitrification-aerobic denitrification function according to claim 1 or 2 in nitrogen-containing wastewater denitrification treatment, wherein: the nitrogen-containing sewage is aquaculture tail water.

5. The use of Pseudomonas holothurian with heterotrophic nitrification-aerobic denitrification function in nitrogen-containing wastewater denitrification treatment according to claim 2, wherein: the carbon source in the culture is at least one of sodium citrate, sodium succinate and sodium acetate.

6. The use of Pseudomonas holothurian with heterotrophic nitrification-aerobic denitrification function in nitrogen-containing wastewater denitrification treatment according to claim 2, wherein: the C/N ratio of the sewage in the culture is 10-40.

7. The use of Pseudomonas holothurian with heterotrophic nitrification-aerobic denitrification function in nitrogen-containing wastewater denitrification treatment according to claim 2, wherein: the pH value of the sewage in the culture is 6-7.

8. The use of Pseudomonas holothurian with heterotrophic nitrification-aerobic denitrification function in nitrogen-containing wastewater denitrification treatment according to claim 2, wherein: the temperature in the culture is 15-35 ℃.