CN114231431B

CN114231431B - Application of rhodococcus toonapus WM36 in denitrification of nitrogen-containing sewage in treatment of salt fresh water

Info

Publication number: CN114231431B
Application number: CN202110798557.4A
Authority: CN
Inventors: 黄文�; 孙慧明; 舒琥; 陈国建; 陈冰
Original assignee: Zhuhai Nongfubao Agricultural Technology Co ltd; Institute of Animal Science of Guangdong Academy of Agricultural Sciences
Current assignee: Zhuhai Nongfubao Agricultural Technology Co ltd; Institute of Animal Science of Guangdong Academy of Agricultural Sciences
Priority date: 2021-07-15
Filing date: 2021-07-15
Publication date: 2023-03-14
Anticipated expiration: 2041-07-15
Also published as: CN114231431A

Abstract

The invention discloses application of rhodococcus toonapus WM36 in denitrification of nitrogen-containing sewage in saline water. The Rhodococcus rhodochrous WM36 has salinity adaptability of 0-12% and is suitable for saline water culture environment with alternately changed salinity. The rhodococcus toonae WM36 is applied to the field of nitrogenous aquaculture tail water treatment, has no adverse effect on aquaculture objects, and has higher aquatic organism safety; and the antibiotic composition is sensitive to various common clinical antibiotics such as norfloxacin, streptomycin, gentamicin sulfate, tetracycline hydrochloride, ciprofloxacin, levofloxacin and the like, has high ecological safety, has good economic and environmental benefits, and has wide application prospect.

Description

Application of rhodococcus toonapus WM36 in denitrification of nitrogen-containing sewage in treatment of salt fresh water

The technical field is as follows:

the invention belongs to the field of culture tail water treatment, and particularly relates to application of rhodococcus toonaportus WM36 in denitrification of nitrogen-containing sewage in saline and fresh water.

Background art:

the saline-fresh water culture is a mode for carrying out aquaculture in water areas with varying salinity, such as estuaries.

The sea bass, also known as Lateolabrax maculitus, is the main species of sea bass cultured in China. Lateolabrax japonicus usually lives in offshore sea areas, and particularly likes to live in estuary salt-water lakes. Lateolabrax japonicus belongs to carnivorous fishes and is extremely violent in sex. Under natural conditions, young shrimps and podosomes are mainly prey on, and adults take shrimps and fishes as staple food. Under the condition of artificial breeding, the feed is eaten by artificial compound pellet feed. The lateolabrax japonicus muscle has high nutritive value, rich amino acid composition, rich high-quality amino acid essential for human body and balanced proportion. The fatty acid is mainly unsaturated fatty acid, and the highly unsaturated fatty acid has high content of EPA and DHA, which is called as DHA king. However, the production mode of sea bass breeding is extensive, the supporting facilities are simple and crude, and the typical characteristics of the existing breeding such as high density and strong feeding cause the deterioration of the breeding environment, the increase of the disease occurrence frequency, the instability of the breeding yield and quality, the aggravation of the breeding risk and the influence on the market competitiveness and the breeding benefit. In recent years, intensive culture of sea bass, including seedling source and quality, feed nutrition, disease control and culture management, has received attention. Pollution to culture water areas and culture quality become keys influencing the quality improvement and the efficiency improvement of the sea bass culture.

And

when the water body concentration is too high, the main overproof item in a sea bass culture area is also an important index for evaluating the culture water body, and the toxicity of the main overproof item and the important index can directly influence the survival of an aquaculture object and the quality of aquatic products. Biological denitrification is a key biochemical process in a biological filter facility. Heterotrophic nitrification-aerobic denitrification (HN-AD) as a novel biological denitrification technology can successfully overcome the problem of incompatibility in nitrification and denitrification caused by different oxygen demands, compared with autotrophic organisms, the heterotrophic organisms also have the advantages of better utilization of organic substrates, higher oxygen resistance, higher denitrification rate and the like, and in recent yearsHave received a lot of attention. 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.

The method for degrading inorganic nitrogen in water by using microorganisms is an important method for treating the culture tail water. At present, nitrobacteria, denitrifying bacteria, bacillus subtilis and other microbial agents are developed. However, salinity of the aquiculture water area varies alternately within 0.5-12.0%, and strain screening and application for inorganic nitrogen removal of wide-salinity aquiculture water body still need to be enhanced at present.

The invention content is as follows:

the invention aims to provide application of Rhodococcus toona (Rhodococcus rhododendri) WM36 in denitrification for treating nitrogen-containing sewage in salt fresh water. The rhodococcus toonae WM36 has wide salinity adaptability, can be applied to removal of inorganic nitrogen elements in the saltwater and freshwater, has high-efficiency heterotrophic nitrification and aerobic denitrification functions, has the characteristics of high safety of aquatic organisms, high environmental safety and the like, and has great application value in the aspect of denitrification treatment of the saltwater and freshwater aquaculture tail water.

Preferably, the brackish water is tail water of the brackish water aquaculture.

Preferably, the denitrification is to remove nitrogen-containing sewage in the salt-water

Three inorganic nitrogen elements.

Preferably, the Rhodococcus toosendan WM36 is inoculated to the nitrogen-containing sewage of the salt-water, and the sewage is denitrified by culturing.

Preferably, the pH value of the nitrogen-containing sewage of the brackish water is 7.

Preferably, the temperature of the culture is 15-35 ℃.

Preferably, the C/N of the nitrogen-containing sewage of the brackish water is 10 to 40.

Preferably, the salinity growth range of the nitrogen-containing sewage of the saline and fresh water is 0-12 per mill.

Preferably, sodium citrate is used as the carbon source for the culture, and the culture has the characteristics of C/N =10, pH =7, T =35 ℃ and SAL = 0-12 per mill.

The saline water refers to a water area with salinity between fresh water and seawater, and is mostly found at river and sea junctions. Salinity fluctuation caused by rising tide and falling tide.

The Rhodococcus rhodochrous WM36 has salinity adaptability of 0-12% and is suitable for saline water culture environment with alternately changed salinity. The rhodococcus toonae WM36 is applied to the field of nitrogenous aquaculture tail water treatment, has no adverse effect on aquaculture objects, and has higher aquatic organism safety; and the antibiotic composition is sensitive to various common clinical antibiotics such as norfloxacin, streptomycin, gentamicin sulfate, tetracycline hydrochloride, ciprofloxacin, levofloxacin and the like, has high ecological safety, and has good economic and environmental benefits and wide application prospect.

Rhodococcus rhododenii WM36 was deposited at 10.06.2021 with the culture Collection of microorganisms of Guangdong province (GDMCC) with the following deposition addresses: building 5 of first furnance, large yard, 100, building 59, guangdong province, guangzhou, china, zip code: 510070, accession number GDMCC No.61717.

Description of the drawings:

FIG. 1 is a graph of colony morphology results of Rhodococcus toosendan WM36 on nutrient agar plates;

FIG. 2 is a scanning electron microscope image and a thallus size statistical result image of Rhodococcus toosendan WM 36;

FIG. 3 is a graph showing the gram stain results of Rhodococcus toosendan WM 36;

FIG. 4 is a graph showing the results of a fish toxicity test of Rhodococcus toosendan WM 36;

FIG. 5 is a graph showing the results of a conventional antibiotic resistance test of Rhodococcus toosendan WM 36;

FIG. 6 is a graph showing the comparative results of growth and denitrification of Rhodococcus toosendan WM36 under different organic carbon sources and different inorganic nitrogen sources;

FIG. 7 is a graph showing the comparison results of growth and denitrification of Rhodococcus toosendan WM36 under different pH and inorganic nitrogen source conditions;

FIG. 8 is a graph showing the comparison of growth and denitrification of Rhodococcus toosendan WM36 under different C/N and different inorganic nitrogen sources;

FIG. 9 is a graph showing the comparison of growth and denitrification of Rhodococcus toosendan WM36 at different temperatures and under different inorganic nitrogen sources;

FIG. 10 is a graph showing the comparison between the growth and denitrification of Rhodococcus toosendan WM36 under different salinity gradients and different inorganic nitrogen sources;

FIG. 11 is a field view of a sample taken from Showa Xincun (east longitude E:113 ° 36 '41', northern latitude N:22 ° 19 '84') in the Douchai district of Zhuhai, guangdong province;

FIG. 12 is a graph showing the denitrification treatment application results of Rhodococcus toosendan WM36 in tail water from the Bingcun saltwater and freshwater aquaculture in the Bingmen area of Zhuhai City, guangdong province;

the specific implementation mode is as follows:

the present invention will be described in further detail with reference to the following examples, but the scope of 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:

microelement solution (g/L): EDTA 50g, znSO ₄ ·7H ₂ O 5.02g，CuSO ₄ ·5H ₂ O 1.57g，FeSO ₄ ·7H ₂ O 5.0g， CoCl ₂ ·6H ₂ O 1.61g，(NH ₄ ) ₆ Mo ₇ O ₂ ·4H ₂ O 1.1g，CaCl ₂ ·2H ₂ O 5.5g，MnCl ₂ ·4H ₂ O 5.06g，pH 6.0；

Enrichment medium (g/L): KH (Perkin Elmer) ₂ PO ₄ 1.5g，MgSO ₄ ·7H ₂ O 0.01g，Na ₂ HPO ₄ 7.9g, 6.45g sodium citrate dihydrate, naNO ₃ 0.8415g，NH ₄ Cl 0.192g，NaNO ₂ 0.362g, 2mL of trace element solution, and pH 7.2;

BTB solid Medium (g/L): 6.45g of sodium citrate dihydrate 1% of BTB (bromothymol blue) in ethanol 1mL, KH ₂ PO ₄ 1.5g，MgSO ₄ ·7H ₂ O 0.01g，Na ₂ HPO ₄ 7.9g，NaNO ₃ 0.8415g，NH ₄ Cl 0.192g，NaNO ₂ 0.362g, 2mL of trace element solution, 20g of agar and 7.0-7.5 of pH;

nitrogen source only fermentation medium (DM I) (g/L): 6.45g of sodium citrate dihydrate ₂ PO ₄ 1.5g，MgSO ₄ ·7H ₂ O 0.01g，Na ₂ HPO ₄ 7.9g，NH ₄ Cl 0.6036g, trace element solution 2mL, pH 7.0;

single nitrogen source fermentation medium (DM II) (g/L): 6.45g of sodium citrate dihydrate ₄ ·7H ₂ O 0.01g，KH ₂ PO ₄ 1.5g，Na ₂ HPO ₄ 7.9g，NaNO ₃ 0.9590g, 2mL of microelement solution, pH 7.0;

single nitrogen source fermentation Medium (DM III) (g/L): 6.45g of sodium citrate dihydrate ₂ PO ₄ 1.5g，MgSO ₄ ·7H ₂ O 0.01g，Na ₂ HPO ₄ 7.9g，NaNO ₂ 0.375g, 2mL of trace element solution, pH 7.0.

Example 1

1. Sample collection

The water quality and sediment sample is obtained by screening and separating a water sample and a sediment mixture of a freshwater aquaculture pond; sample collection according to a mixed sample collection method in technical Specification for soil environmental monitoring (HJ/T166-2004), a quincunx 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 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 strains

Sample pretreatment: taking pond sediment and a water sample respectively, putting the pond sediment and the water sample into a large-mouth triangular flask filled with sterile physiological saline water in a super clean workbench, putting a small amount of sterilized glass beads, and oscillating to break up the sediment sample so as to enable microorganisms in the sediment sample to be fully suspended in the physiological saline water.

Primary enrichment culture and plate coating: 22.2mL of the mixed liquid is added into a 500mL conical flask filled with 200mL of enrichment medium, and cultured for 2-3 days at 180r/min in a shaking table at 30 ℃. Adding 1mL of NH with the concentration of 5 percent by mass into the enrichment medium every day ₄ Cl solution to keep in the culture medium

The ion concentration. 10mL of water sample is taken and inoculated into a 300mL large-mouth triangular flask containing 90mL of enrichment medium, and the mixture is shaken for 1h at 180r/min in a shaking table at 30 ℃. And (3) respectively taking 1mL of the pretreated mud sample stock solution in the step (1), putting the mud sample stock solution into a test tube filled with 9mL of sterile physiological saline in an ultra-clean workbench, and lightly blowing or vibrating and uniformly mixing by using a liquid transfer gun. Taking out 1mL of liquid from the test tube, inoculating into a new test tube containing 9mL of sterile physiological saline, repeating the operation, and sequentially diluting the substrate sludge and the water sample stock solution to 10 in a gradient manner ^-2 ～10 ^-8 And (4) concentration. Respectively take 10 ^-2 ～10 ^-8 100-200 mul of the substrate sludge with concentration gradient and the stock solution with water sample concentration are coated in a BTB solid plate culture medium, each gradient is provided with 3 parallel groups, 1 blank control group, the control group is coated with the same amount of sterile physiological saline, and the inversion culture is carried out for 2-3 days in a constant temperature biochemical incubator at 30 ℃.

Separation, purification and preliminary screening: the colonies with different morphologies were picked with an inoculating loop. And (3) streaking on a BTB solid plate medium by adopting a plate streaking separation method for separation and purification, and selecting a single colony for repeated streaking and purification for 3-4 times. Point-connection primary screening: the purified strain is picked by using an inoculating needle and inoculated in a BTB denitrification identification culture medium for 2 to 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. Inoculating the strain on a slant, culturing at 30 ℃ for 2-3 days, and preserving the test tube at 4 ℃.

Re-screening the nitrification and denitrification performance: inoculating the activated slant of the strain obtained above into a nutrient broth, inoculating with NH at an inoculum size of 1% ₄ Cl、NaNO ₃ And NaNO ₂ Performing 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 48h ₆₀₀ . Centrifuging at low speed of 5000r/min for 5min, collecting supernatant, and measuring respectively

Three nitrogen contents. Thus strain WM36 was obtained.

3. Identification of strain species

Extracting DNA of the strain WM36, carrying out 16S rDNA amplification by taking the strain DNA as a template, and amplifying an upstream primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3', downstream primer (1492R): 5'-GGCTACCTTGTTACGACTT-3'. PCR was performed using a conventional amplification system. 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 is 1423bp, and the nucleotide sequencing result is as follows (the nucleotide sequence is shown as SEQ ID NO. 1):

GGAAGGCGGGTGCTTACCATGCAGTCGAGCGGTAAGGCCCTTTCGGGGGTACACG AGCGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCAGAGGCTGCATGGCTTTTGGTGGAAAGGTT TACTGGTGCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACG GCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGG ACGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTA GGCGGTTTGTCGCGTCGTCTGTGAAAACCAGCAGCTCAACTGCTGGCTTGCAGGCGATA CGGGCAGACTTGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTG AGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTTCCTTCCACGGGATCCGTGCCGTAGCTAACGCATTAA GCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGT TTGACATATACCGGAAAGCCGTAGAGATACGGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAA CCCTTGTCCTGTGTTGCCAGCGCGTAATGGCGGGGACTCGCAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCAC ACATGCTACAATGGCCGGTACAGAGGGCTGCGATACCGTGAGGTGGAGCGAATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTA GTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGGAGGGAG CCGTCGAAGGTGATCGTCAGTT。

the strain is gram-reaction negative (figure 3), the length of the strain is 1.42 +/-0.31 mu m, the width is 0.56 +/-0.06 mu m, and the strain is straight rod-shaped and has no flagellum (figure 2); colonies were white and opaque on nutrient agar, dry on the surface, round, flat, with intact edges (fig. 1).

TABLE 1 physiological and biochemical characteristics of Strain WM36

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

And (3) judging the strain to be Rhodococcus toosendan (Rhodococcus rhodochrous) by integrating the 16s rDNA sequencing result, the bacterial morphology, the colony morphology, the physiology and biochemistry and other identification items of the strain. The related data is consulted to find that no report is available about the purification of inorganic nitrogen in the tail water of cultivation or other nitrogen-containing sewage by using Rhodococcus toona (Rhodococcus rhododendri). The strain is named as Rhodococcus ailanthi (Rhodococcus rhododendri) WM36, and the strain is preserved in Guangdong province microorganism culture collection center (GDMCC) at 11 days 06 and 2021, and the preservation number is GDMCC No:61717, the preservation address is No. 59 building No. 5 building of No. 100 Dazhong Jie of Guangzhou city, the postcode: 510070.

4. WM36 environmental safety evaluation of erythrococcus toonae

1. Fish toxicity test: healthy zebra fish (Danio rerio) (purchased from Guangzhou Hua Bao Hua bird fish and insect wholesale market in Huawan of Arganian district of Chou bay of Guangzhou, guangdong province) with the body length within the range of 3 +/-1 cm are selected to be temporarily cultured and stabilized in a continuously aerated large water body for 30 to 45 days, and during the period, the zebra fish are normally fed and the water is periodically changed. After the state is stable, the materials are randomly distributed into 15L glass jars, experimental groups added with bacterial liquid and control groups added with isometric 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, discarding supernatant, resuspending with sterile PBS buffer solution, repeating for 1-2 times, suspending with sterile water, and obtaining OD according to determined standard curve ₆₀₀ The 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 body ⁶ CFU/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: antibiotic resistance experiments (antibiotic paper drug sensitivity tests) were carried out according to the technical requirements of antibiotic drug sensitivity tests (WS/T639-2018) (see Table 2) and the experimental method was carried out according to the requirements of the "paper diffusion method" in the above standards. The method comprises the following specific steps:

a) Preparing MHA (Guangdong Huanji microbial science and technology Limited) 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) Inoculating the strains into nutrient broth culture medium respectively, and culturing at 30 deg.C and 180r/min to 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 plate within 15min, and culturing at constant temperature of 30 ℃ for 18h;

h) Measuring the diameter of the inhibition zone by using an IP54 metal shell digital vernier caliper (Yongkang Jingsida trade Co., ltd.);

TABLE 2 antibiotic resistance evaluation criteria

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 percent ⁶ CFU/mL, higher than the pathogenic dose of common pathogenic bacteria (10) ⁴ CFU/mL). The survival rate of the experimental group zebra fish is higher than 96.7 percent, and the survival rate is not obviously different from that of the control group (p)>0.05). The rhodococcus toonapus WM36 is preliminarily judged to have higher biological safety of aquatic organisms (see figure 4). The results of antibiotic resistance experiments with the strain WM36 (Table 3 and FIG. 5) show that the Rhodococcus toosendan WM36 is sensitive to a variety of common clinical antibiotics used (FIG. 5), indicating that it is ecologically safe. The experiment also provides guidance for taking precautions and emergency measures in the using link of the strains.

TABLE 3 antibiotic resistance experiments

5. Optimum growth and denitrification conditions of rhodococcus toonae WM36

Influence of different organic carbon sources on growth and denitrification performance of rhodococcus toosendan WM36

SelectingFour carbon sources, sodium oxalate, sodium succinate, sodium acetate and sodium citrate, fixed C/N =10, 30 ℃,180r/min, pH =7.0. On the basis of a DM fermentation culture medium, 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 source ₄ Cl(DMⅠ)、 NaNO ₃ (DMⅡ)、NaNO ₂ The addition amount of (DM III) per liter of culture medium is 0.02675g, 0.04g and 0.0345g. 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, taking culture solution at 0h, 8h, 16h, 24h, 32h, 40h and 48h, measuring OD600 and 5000r/min, centrifuging at low speed for 5-10 min, and measuring supernatant

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 rhodococcus toosendan WM36.

(2) Influence of different C/N on growth and denitrification performance of rhodococcus toonapus WM36

Selecting sodium citrate as a denitrification culture medium carbon source, fixing conditions of 30 ℃,180r/min, pH =7.0 and the like, and setting C/N gradients 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 5.16g/L; NH as sole inorganic nitrogen source ₄ Cl(DMⅠ)、NaNO ₃ (DMⅡ)、NaNO ₂ (DM III) is added into 0.02675g, 0.04g and 0.0345g of culture medium per liter respectively. 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, measuring OD600 in culture medium at 0h, 8h, 16h, 24h, 32h, 40h, and 48h, and measuring with 5000rCentrifuging at low speed for 5-10 min, taking supernatant, and 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 the influence of four different C/N of 10, 20, 30 and 40 on the growth condition and aerobic denitrification of rhodococcus toosendan WM36.

(3) Influence of different pH values on growth and denitrification performance of rhodococcus toonae WM36

Setting the conditions of C/N =10, 30 ℃,180r/min, sodium citrate as a single organic carbon source and the like, and setting pH gradients of 5, 6, 7, 8 and 9. NH as sole inorganic nitrogen source ₄ Cl(DMⅠ)、NaNO ₃ (DMⅡ)、NaNO ₂ The addition amount of (DM III) per liter of culture medium is 0.02675g, 0.04g and 0.0345g. Inoculating candidate strains in nutrient broth culture medium, culturing at 30 deg.C and 180r/min for 1 day, inoculating with 1% (v/v) inoculum size, measuring OD600 of culture medium at 0h, 8h, 16h, 24h, 32h, 40h, and 48h, centrifuging at low speed of 5-10 min, and measuring supernatant

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 pH values of 5, 6, 7, 8 and 9 on the growth condition and aerobic denitrification of rhodococcus toosendan WM36 is analyzed.

(4) Influence of different temperatures on growth and denitrification performance of rhodococcus toonae WM36

Fixing the conditions of C/N =10, pH =7.0, 180r/min, sodium citrate as a single organic carbon source and the like, and setting the temperature gradient at 15 ℃,20 ℃, 25 ℃,30 ℃ and 35 ℃. NH as sole inorganic nitrogen source ₄ Cl(DMⅠ)、NaNO ₃ (DMⅡ)、NaNO ₂ The addition amount of (DM III) per liter of culture medium is 0.02675g, 0.04g and 0.0345g. Inoculating candidate strains into nutrient broth culture medium, culturing at 30 deg.C and 180r/min for 1 day, and inoculating at 1% (v/v)The amounts of the components are respectively inoculated into the denitrification culture medium, culture solution is taken for 0h, 8h, 16h, 24h, 32h, 40h and 48h to measure the OD600 of the culture solution, 5000r/min is carried out, supernatant is taken for respective measurement after 5-10 min low-speed centrifugation

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 the influence of five different temperatures of 15 ℃,20 ℃, 25 ℃,30 ℃ and 35 ℃ on the growth condition and aerobic denitrification of the rhodococcus toosendan WM36.

(5) Influence of different salinity on growth and denitrification performance of rhodococcus toonapus WM36

The method comprises the steps of fixing conditions of C/N =10, pH =7.0, 180r/min, T =30 ℃ sodium citrate as a single organic carbon source and the like, and setting salinity gradients of the culture medium to be 0 per thousand, 4 per thousand, 8 per thousand and 12 per thousand by changing and adjusting the concentration of NaCl in the culture medium. NH as sole inorganic nitrogen source ₄ Cl(DMⅠ)、NaNO ₃ (DMⅡ)、NaNO ₂ (DM III) is added into 0.02675g, 0.04g and 0.0345g of culture medium per liter respectively. Inoculating candidate strains in nutrient broth culture medium, culturing at 30 deg.C and 180r/min for 1 day, inoculating with 1% (v/v) inoculum size, measuring OD600 of culture medium at 0h, 8h, 16h, 24h, 32h, 40h, and 48h, centrifuging at low speed of 5-10 min, and measuring supernatant

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 the influence of four different salinity of 0 per thousand, 4 per thousand, 8 per thousand and 12 per thousand on the growth condition and the denitrification of the rhodococcus toosendan WM36.

As can be seen from comparative analysis of FIG. 6, rhodococcus Torae WM36 was able to grow on organic carbon sources such as sodium citrate and sodium acetate. The growth speed is reduced when sodium acetate is used for growth; most suitable for raw meatThe pH for growth and denitrification was 7 (FIG. 7); can grow and degrade within the range of 10-40C/N

And the C/N condition is in the range of 20-40,

and

is inhibited (fig. 8); temperature gradient experiments show that the growth speed and inorganic nitrogen degradation efficiency of rhodococcus toosendan WM36 at T =35 ℃ are obviously more than 15-30 ℃ (FIG. 9); in the salinity gradient experiment, the growth speed and the inorganic nitrogen degradation efficiency of the strain WM36 are not affected in the salinity range of 0-12 per mill (figure 10), which shows that the strain WM36 has wide application range to salinity. In summary, the optimal growth and denitrification conditions of the bacteria are that sodium citrate is used as a carbon source, C/N =10, pH =7, T =35 ℃, and the salinity growth range is 0-12 per thousand.

The highest can reach 82.6 percent, 94.6 percent and 86.1 percent respectively.

6. Application of rhodococcus toonae WM36 in tail water of saline-fresh water culture

Water samples (figure 11) of a culture pond and an ecological ditch in a Zhao Xincun saline-fresh water culture area in a sluice gate area of the Zhuhai city are collected, and an optical salinity meter is used for measuring the salinity value of water at a sampling point on site. And (4) refrigerating and transporting the water sample back to a laboratory for later use, and completing the inorganic nitrogen index determination of the water sample on the same day. Taking 60-100mL water samples of the aquaculture pond and the ecological ditch respectively, filtering to remove solid particles and suspended matters, filtering and sterilizing by using a 0.22 mu m/25mm needle head type filter, and transferring into a conical flask sterilized by high-pressure steam for later use. Inoculating the strain WM36 into a nutrient broth culture medium, activating for 18h at 30 ℃ at 180r/min, respectively inoculating the bacterial liquid in the logarithmic phase into two water samples according to the inoculation amount of 1% (v/v), and performing shake culture at 30 ℃ at 180 r/min. Taking water samples at 0h, 12h, 24h, 48h and 72h to determine the OD ₆₀₀ Centrifuging at low speed of 5000r/min and 5-10 min, taking supernatant and determining respectively

Three nitrogen contents.

Through analysis, the mean value of the basic water quality data of the aquaculture pond and the ecological ditch in the saline-fresh water aquaculture area is shown in table 4.

TABLE 4 Water quality data of saltwater and freshwater aquaculture area

Since the ecological ditches in the culture area are connected with the river of Zhaoxing village, the salinity of the water body is changed alternately (figure 11), which causes the salinity of the culture pond and the ecological ditches in the table 4 to be inconsistent. According to the data of the inorganic nitrogen treatment result, the strain WM36 can be simultaneously suitable for the water body of an aquaculture pond with the salinity of 2 per thousand and the tail water of ecological ditch aquaculture with the salinity of 8 per thousand, and can grow and degrade the inorganic nitrogen of the purified water body. After being treated by the strain WM36 for 72 hours, the water in the water sample of the aquaculture pond

The highest degradation rates can respectively reach 68.2%, 95.2% and 85.4%, and none of the degradation rates

Accumulating; in tail water sample of ecological ditch culture

The highest degradation rates can respectively reach 44.8%, 87.5% and 69.5%, and none of the degradation rates

Accumulation (fig. 12). The strain has important application value in the aspect of tail water treatment capacity of saline-fresh water culture.

The Rhodococcus toosendan WM36 is applied to the field of tail water treatment of nitrogen-containing salt fresh water aquaculture, and the strain has wide salinity application range, 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 being applied to the aquaculture water body of fresh water and saline water (the salinity is less than or equal to 12 per thousand); it has the functions of heterotrophic nitrification and aerobic denitrification simultaneously; can utilize various organic carbon sources, has better water organic carbon removal capacity, has good economic and environmental benefits and wide application prospect.

The above-mentioned embodiments are not intended to limit the embodiments of the present invention, but to exemplify the present invention. Other variations and modifications of the above-described embodiments will be apparent to those skilled in the art. It is intended that all such alterations and modifications are included in the scope of the embodiments and claims of the invention.

Sequence listing

<110> animal science institute of academy of agricultural sciences of Guangdong province

Zhuhai augmentation science and technology Co Ltd

Application of <120> rhodococcus toona WM36 in denitrification of nitrogen-containing sewage in saline water

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1423

<212> DNA

<213> Rhodococcus toonae WM36 (Rhodococcus rhododenii)

<400> 1

ggaaggcggg tgcttaccat gcagtcgagc ggtaaggccc tttcgggggt acacgagcgg 60

cgaacgggtg agtaacacgt gggtgatctg ccctgcactt cgggataagc ctgggaaact 120

gggtctaata ccggatatga ccagaggctg catggctttt ggtggaaagg tttactggtg 180

caggatgggc ccgcggccta tcagcttgtt ggtggggtaa tggcctacca aggcgacgac 240

gggtagccga cctgagaggg tgaccggcca cactgggact gagacacggc ccagactcct 300

acgggaggca gcagtgggga atattgcaca atgggcgaaa gcctgatgca gcgacgccgc 360

gtgagggatg acggccttcg ggttgtaaac ctctttcagc agggacgaag cgaaagtgac 420

ggtacctgca gaagaagcac cggccaacta cgtgccagca gccgcggtaa tacgtagggt 480

gcgagcgttg tccggaatta ctgggcgtaa agagctcgta ggcggtttgt cgcgtcgtct 540

gtgaaaacca gcagctcaac tgctggcttg caggcgatac gggcagactt gagtactgca 600

ggggagactg gaattcctgg tgtagcggtg aaatgcgcag atatcaggag gaacaccggt 660

ggcgaaggcg ggtctctggg cagtaactga cgctgaggag cgaaagcgtg ggtagcgaac 720

aggattagat accctggtag tccacgccgt aaacggtggg cgctaggtgt gggtttcctt 780

ccacgggatc cgtgccgtag ctaacgcatt aagcgccccg cctggggagt acggccgcaa 840

ggctaaaact caaaggaatt gacgggggcc cgcacaagcg gcggagcatg tggattaatt 900

cgatgcaacg cgaagaacct tacctgggtt tgacatatac cggaaagccg tagagatacg 960

gccccccttg tggtcggtat acaggtggtg catggctgtc gtcagctcgt gtcgtgagat 1020

gttgggttaa gtcccgcaac gagcgcaacc cttgtcctgt gttgccagcg cgtaatggcg 1080

gggactcgca ggagactgcc ggggtcaact cggaggaagg tggggacgac gtcaagtcat 1140

catgcccctt atgtccaggg cttcacacat gctacaatgg ccggtacaga gggctgcgat 1200

accgtgaggt ggagcgaatc ccttaaagcc ggtctcagtt cggatcgggg tctgcaactc 1260

gaccccgtga agtcggagtc gctagtaatc gcagatcagc aacgctgcgg tgaatacgtt 1320

cccgggcctt gtacacaccg cccgtcacgt catgaaagtc ggtaacaccc gaagccggtg 1380

gcctaaccct tgtggaggga gccgtcgaag gtgatcgtca gtt 1423

Claims

1. Rhodococcus toonae (C.sinensis)Rhodococcus rhodnii) The application of WM36 in the denitrification of the nitrogen-containing sewage in the salty fresh water is realized, and the number of the Rhodococcus ailanthoides WM36 is as follows: GDMCC No:61717The salinity range of the nitrogen-containing sewage of the saline and fresh water is 4-12 per mill; the denitrification is to remove nitrogen-containing sewage in the salt-water

Three inorganic nitrogen elements.

2. The use according to claim 1, wherein the brackish water is a saltwater aquaculture tail water.

3. The use of claim 1, wherein the wastewater is denitrified by inoculating rhodococcus toosendan WM36 in the nitrogen-containing wastewater of the brackish water and culturing.

4. The use according to claim 1, wherein the saltwater nitrogen-containing wastewater has a pH of 7.

5. The use according to claim 3, wherein the incubation is carried out at a temperature of 35 ℃.

6. The use according to claim 1, wherein the C/N of the nitrogen-containing sewage of the brackish water is 10 to 40.

7. The use of claim 3, wherein the culture is performed with sodium citrate as a carbon source, C/N =10, pH =7, T =35 ℃, and salinity ranging from 4% o to 12% o.