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

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 stinkbug WM36 in Nitrogen Removal of Nitrogenous Wastewater from Salty Fresh Water

Technical field:

The invention belongs to the field of aquaculture tail water treatment, and in particular relates to the application of Rhodococcus stinkbug WM36 in the denitrification of saline and fresh water nitrogen-containing sewage.

Background technique:

Brackishwater aquaculture is a form of aquaculture in waters of varying salinity such as estuaries.

Sea bass, also known as flower perch, scientific name Lateolabrax maculatus, is the main species of sea bass cultured in my country. Perch usually live in offshore waters, but especially like to live in estuary brackish water lakes. Perch is a carnivorous fish with a ferocious nature. Under natural conditions, larvae mainly prey on mysis and amphipods, while adults mainly eat shrimp and fish. Under artificial breeding conditions, they ingest artificial compound pellet feed. Sea bass muscle has high nutritional value, and its amino acid composition is rich in high-quality amino acids necessary for the human body, and the proportion is balanced. Fatty acids are mainly unsaturated fatty acids, and the content of high unsaturated fatty acids EPA and DHA is high, known as the king of DHA. However, the extensive production methods of seabass farming, poor supporting facilities, and the typical characteristics of "high-density, intensive feeding" have resulted in the deterioration of the breeding environment, the increase in the frequency of diseases, the instability of the production and quality of the culture, the increased risk of breeding, and the impact on Market competitiveness and farming benefits. In recent years, the technical issues of seabass intensive farming, including the source and quality of seedlings, feed nutrition, disease prevention and breeding management, etc. have attracted people's attention. Pollution of aquaculture waters and aquaculture quality have become the key factors affecting the quality and efficiency of seabass aquaculture.

以及

等水体浓度过高是海鲈养殖区主要的超标项，亦是评价养殖水体很重要的指标，它们的毒性也会直接影响水产养殖对象的生存和水产品质量。生物脱氮是生物滤池设施中的关键生化过程。异养硝化-好氧反硝化(HN-AD)作为一种新型的生物脱氮技术，不但能够成功克服不同需氧量引起的硝化反硝化不相容问题，与自养生物相比，异养生物还拥有对有机底物更好的利用、更高的耐氧性和脱氮率等优势，近年来受到广泛关注。但至今为止对此类HN-AD细菌的研究尚未完善，仍需要分离纯化得到更多的高效、稳定的 HN-AD菌株作进一步确定。

as well as

Excessive concentration in water bodies such as sea bass is the main over-standard item in seabass farming areas, and it is also an important indicator for evaluating aquaculture water bodies. Their toxicity will also directly affect the survival of aquaculture objects and the quality of aquatic products. Biological nitrogen removal is a key biochemical process in biofilter facilities. Heterotrophic nitrification-aerobic denitrification (HN-AD), as a new type of biological denitrification technology, can not only successfully overcome the incompatibility of nitrification and denitrification caused by different oxygen demands, but compared with autotrophic organisms, heterotrophic Biology also has the advantages of better utilization of organic substrates, higher oxygen tolerance and nitrogen removal rate, and has received extensive attention in recent years. However, the research on this type of HN-AD bacteria has not been perfected so far, and more efficient and stable HN-AD strains still need to be isolated and purified for further confirmation.

The use of microorganisms to degrade inorganic nitrogen in water is an important method for the treatment of aquaculture tail water. At present, bacteria agents such as nitrifying bacteria, denitrifying bacteria, and Bacillus subtilis have been developed. However, the salinity of brackish and fresh water aquaculture waters changes alternately, ranging from 0.5‰ to 12.0‰. At present, the screening and application of bacteria for the removal of inorganic nitrogen in euryotic brackish and fresh water aquaculture waters still needs to be strengthened.

Invention content:

The object of the present invention is to provide the application of Rhodococcus rhodnii WM36 in the denitrification of saline and fresh water nitrogenous sewage. Rhodococcus stinkbug WM36 has a wide range of salinity adaptability, can be used to remove inorganic nitrogen elements in brackish water and has high-efficiency heterotrophic nitrification and aerobic denitrification functions, and also has the characteristics of high aquatic biological safety and environmental safety. , has great application value in the denitrification treatment of brackish water aquaculture tail water.

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

三种无机氮元素。Preferably, the denitrification is to remove nitrogenous sewage in brackish water

Three inorganic nitrogen elements.

Preferably, Rhodococcus stinkbug WM36 is inoculated in brackish water nitrogen-containing sewage, and cultured to denitrify sewage.

Preferably, the pH value of the brackish water nitrogenous sewage is 7.

Preferably, the temperature of the cultivation is 15°C to 35°C.

Preferably, the C/N in the salty fresh water and nitrogen-containing sewage is 10-40.

Preferably, the salinity growth range of the salty fresh water and nitrogen-containing sewage is 0-12‰.

Preferably, the culture uses sodium citrate as carbon source, C/N=10, pH=7, T=35°C, SAL=0-12‰.

The brackish water mentioned in the present invention refers to the water area whose salinity is between fresh water and sea water, and is mostly found at the confluence of rivers and seas. Fluctuations in salinity due to high tide and low tide.

The rhodococcus rhodnii (Rhodococcus rhodnii) WM36 of the present invention has a salinity adaptability of 0-12‰, and can adapt to the brackish and fresh water aquaculture environment where the salinity changes alternately. The Rhodococcus stinkii WM36 of the present invention is applied to the field of nitrogen-containing aquaculture tail water treatment, has no adverse effects on aquaculture objects, and has high biological safety of aquatic organisms; Mycin, tetracycline hydrochloride, ciprofloxacin, levofloxacin and other common clinical antibiotics are sensitive, have high ecological safety, have good economic and environmental benefits, and have broad application prospects.

Rhodococcus rhodnii WM36 was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on June 10, 2021. The preservation address is: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, postcode: 510070, and the preservation number is GDMCC No. 61717.

Description of drawings:

Fig. 1 is the bacterium colony morphology result figure of Rhodococcus stinkbug WM36 on nutrient agar plate;

Fig. 2 is the scanning electron micrograph of Rhodococcus stinkbug WM36 and thalline size statistical result figure;

Fig. 3 is the Gram stain result figure of Rhodococcus stinkbug WM36;

Fig. 4 is the fish toxicity test result figure of Rhodococcus stinkbug WM36;

Fig. 5 is the result figure of the commonly used antibiotic resistance experiment of Rhodococcus stinkbug WM36;

Figure 6 is a graph showing the growth and denitrification effect comparison results of Rhodococcus stinkbug WM36 under different organic carbon sources and different inorganic nitrogen sources;

Figure 7 is a graph showing the growth and denitrification effect comparison results of Rhodococcus stinkbug WM36 under different pH and different inorganic nitrogen source conditions;

Figure 8 is a graph showing the growth and denitrification effects of Rhodococcus stinkbug WM36 under different C/N and different inorganic nitrogen source conditions;

Fig. 9 is a graph showing the growth and denitrification effect comparison results of Rhodococcus stinkbug WM36 at different temperatures and different inorganic nitrogen sources;

Figure 10 is a graph showing the growth and denitrification effect comparison results of Rhodococcus stinkbug WM36 under different salinity gradients and different inorganic nitrogen source conditions;

Figure 11 is a sampling site map in Zhaoxin Village, Doumen District, Zhuhai City, Guangdong Province (east longitude E: 113°36′41″, north latitude N: 22°19′84″);

Figure 12 is a graph showing the application results of denitrification treatment of Rhodococcus stinkbug WM36 in brackish water aquaculture tail water in Zhaoxin Village, Doumen District, Zhuhai City, Guangdong Province;

Detailed ways:

Below in conjunction with specific embodiment the present invention is described in further detail, but protection scope of the present invention is not limited thereto:

三种氮元素的测定与分析方法均参考于国标，其中

的测定与分析根据《水质-氨氮的测定-纳氏试剂分光光度法》(GB HJ535-2009)；

的测定与分析根据《水质-硝酸盐氮的测定-紫外分光光度法》(GB HJ/T346-2007)；

的测定与分析根据《水质-亚硝酸盐氮的测定-分光光度法》(GB 7493-87)。In experiment

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

Determination and analysis according to "Water Quality - Determination of Ammonia Nitrogen - Nessler's Reagent Spectrophotometry" (GB HJ535-2009);

Determination and analysis according to "Water Quality - Determination of Nitrate Nitrogen - Ultraviolet Spectrophotometry" (GB HJ/T346-2007);

The determination and analysis are based on "Water Quality - Determination of Nitrite Nitrogen - Spectrophotometric Method" (GB 7493-87).

The basal medium used in the experiment was sterilized by high-pressure steam at 121°C for 20 minutes, and its formula is as follows:

Trace element 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;

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

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

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

Single nitrogen source fermentation medium (DMⅡ) (g/L): sodium citrate dihydrate 6.45g, MgSO ₄ ·7H ₂ O 0.01g, KH ₂ PO ₄ 1.5g, Na ₂ HPO ₄ 7.9g, NaNO ₃ 0.9590g , trace element solution 2mL, pH 7.0;

Single nitrogen source fermentation medium (DMⅢ) (g/L): sodium citrate dihydrate 6.45g, KH ₂ PO ₄ 1.5g, MgSO ₄ 7H ₂ O 0.01g, Na ₂ HPO ₄ 7.9g, NaNO ₂ 0.375g , trace element solution 2mL, pH 7.0.

Example 1

1. Sample collection

The water quality and sediment samples of the present invention are obtained by screening and separating from freshwater aquaculture pond water samples and sediment mixtures; sample collection is according to the "mixed sample collection method" in "Technical Specifications for Soil Environment Monitoring" (HJ/T 166-2004), The plum blossom point sampling method was adopted to collect samples from the surface, middle and deep layers of water and sediment in sterile sampling bags, and refrigerated, transported and stored at 4°C for later use.

2. Strain enrichment, isolation and screening

Sample pretreatment: Take pond sediment and water samples respectively, put them into a large-mouthed Erlenmeyer flask filled with sterile physiological saline in the ultra-clean workbench, and put a little sterilized glass beads, oscillate to break up the sediment samples, Suspend the microorganisms in the mud sample fully in saline.

离子浓度。水样则取10ml接种于含90ml富集培养基的300mL大口三角烧瓶中，摇床内30℃，180r/min振荡1h。分别取(1) 中经过预处理的泥样原液1mL，于超净工作台内接入装有9mL无菌生理盐水的试管中，移液枪轻轻吹打或震荡混匀。从该试管取出1mL液体接入到新的装有9mL无菌生理盐水的试管中，重复该操作，依次把底泥和水样原液梯度稀释为10^-2～10^-8浓度。分别取10^-2～10^-8浓度梯度的底泥和水样浓度的原液100μL～200μL，涂布于BTB固体平板培养基中，每个梯度设置3 个平行组，1个空白对照组，对照组以等量的无菌生理盐水涂布，在恒温生化培养箱中30℃倒置培养2～3天。Preliminary enrichment culture and plate coating: Take 22.2mL of the above mixed liquid and add it to a 500mL Erlenmeyer flask filled with 200mL enrichment medium, and culture in a shaker at 30°C and 180r/min for 2-3 days. Add 1 mL of NH ₄ Cl solution with a concentration of 5% by mass to the enriched medium every day to keep the concentration in the medium

ion concentration. Take 10ml of the water sample and inoculate it into a 300mL Erlenmeyer flask containing 90ml enriched medium, shake in a shaker at 30°C and 180r/min for 1h. Take 1mL of the pretreated mud sample stock solution in (1), put it into a test tube containing 9mL sterile normal saline in the ultra-clean workbench, and gently blow or oscillate with a pipette to mix. Take 1mL of the liquid from the test tube and put it into a new test tube containing 9mL of sterile physiological saline, repeat the operation, and sequentially dilute the bottom sludge and water sample stock solution to a concentration of 10 ^-2 ~ 10 ^-8 . Take 100 μL to 200 μL of stock solutions of sediment and water samples with a concentration gradient of 10 ^-2 to 10 ^-8 respectively, and spread them on the BTB solid plate medium. Set up 3 parallel groups for each gradient, 1 blank control group, and control group. The group was coated with an equal amount of sterile saline, and cultured upside down at 30°C for 2 to 3 days in a constant temperature biochemical incubator.

Separation, purification and primary screening: Use an inoculation loop to pick the above-mentioned colonies of different forms. Use plate streak separation method to streak on BTB solid plate medium for isolation and purification, pick a single colony and repeatedly streak and purify for 3 to 4 times. Spot screening: Use an inoculation needle to pick and purify the strain and spot it in the BTB denitrification identification medium for 2 to 3 days. According to the growth of the colony and the size of the blue halo in the BTB medium around the colony, the strains with high denitrification ability were selected. Generally speaking, the larger the blue halo, the higher the denitrification ability. Inoculate on a slant and incubate at a constant temperature of 30°C for 2 to 3 days, then store the test tube at 4°C.

三种氮元素含量。由此获得菌株WM36。Re-screening of nitrification and denitrification performance: Use an inoculation loop to inoculate the activated slant of the above-mentioned strains in the nutrient broth, and then inoculate them with NH ₄ Cl, NaNO ₃ and NaNO ₂ as the only inorganic nitrogen source with an inoculation amount of 1%. In the DMⅠ, DMⅡ and DMⅢ fermentation medium, 30 ℃, 180r/min shaking culture, 0h, 24h, 48h to take the culture solution to measure its OD ₆₀₀ . 5000r/min, after 5min low-speed centrifugation, take the supernatant and measure

three types of nitrogen content. Thus, strain WM36 was obtained.

3. Identification of strain species

The DNA of strain WM36 was extracted, and 16S rDNA was amplified using the strain DNA as a template. The upstream primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3' and the downstream primer (1492R): 5'-GGCTACCTTGTTACGACTT-3' were amplified. PCR was performed using a conventional amplification system. 1% agarose gel electrophoresis analysis results. PCR product sequencing was completed by Shanghai Bioengineering Co., Ltd. The 16s rDNA sequence length of the strain is 1423bp, and the nucleotide sequencing results are as follows (the nucleotide sequence is shown in SEQ ID NO.1):

GGAAGGCGGGTGCTTACCATGCAGTCGAGCGGTAAGGCCCTTTCGGGGGTACACG AGCGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCAGAGGCTGCATGGCTTTTGGTGGAAAGGTT TACTGGTGCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACG GCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGG ACGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCTGTGAAAACCAGCAGCTCAACTGCTGGCTTGCAGGCGATA CGGGCAGACTTGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTG AGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTTCCTTCCACGGGATCCGTGCCGTAGCTAACGCATTAA GCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGT TTGACATATACCGGAAAGCCGTAGAGATACGGCCCCCCTTGTGGTCGGTATACAGGTGGTGCA TGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTGTTGCCAGCGCGTAATGGCGGGGACTCGCAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCAC ACATGCTACAATGGCCGGTACAGAGGGCTGCGATACCGTGAGGTGGAGCGAATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTA GTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGGAGGGAG CCGTCGAAGGTGATCGTCAGTT。

Gram-negative bacterial strain (Figure 3), the bacterium is 1.42 ± 0.31 μm long, 0.56 ± 0.06 μm wide, straight rod-shaped, without flagella (Figure 2); the colony is white and opaque on the nutrient agar, and the surface is dry. It is round and flat with complete edges (Figure 1).

Table 1 Physiological and biochemical characteristics of strain WM36

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

Based on the 16s rDNA sequencing results of the strain, bacterial morphology, colony morphology, and physiological and biochemical identification items, it was determined that the strain was Rhodococcus rhodnii. After reviewing relevant information, it was found that there is no relevant report on the purification of inorganic nitrogen in aquaculture tail water or other nitrogen-containing sewage with Rhodococcus rhodnii. The strain is named Rhodococcus rhodnii WM36. The strain has been preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on June 11, 2021. The preservation number is GDMCC No: 61717, and the preservation address is Guangzhou City. 5th Floor, Building 59, Compound, No. 100 Middle Road, Zip Code: 510070.

4. Environmental safety assessment of Rhodococcus stinkbug WM36

1. Fish toxicity test: choose a healthy zebrafish (Danio rerio) within the scope of 3 ± 1cm in body length (purchased in Huadiwan Flower, Bird, Fish and Insect Wholesale Market, Huadiwan, Fangcun, Liwan District, Guangzhou City, Guangdong Province), They are temporarily raised and stabilized in a large water body with continuous aeration for 30 to 45 days, during which time they are fed normally and water is changed regularly. After the state was stable, they were randomly assigned to 15L glass tanks, and the experimental group added with bacterial solution and the control group added with equal volume of sterile water were set up. There were 30 zebrafish in each experimental group, and 3 replicates were set up in each group. Take the overnight cultured bacterial solution, centrifuge at 3000r/min for 5min, discard the supernatant, resuspend with sterile PBS buffer, repeat 1-2 times, then suspend with sterilized water, and obtain OD ₆₀₀ and bacterial concentration according to the measured standard curve According to the relationship between them, the amount of bacteria in the experimental water was adjusted to about 1×10 ⁶ CFU/mL, and the same amount of sterilized water was added to the blank control group. During the experiment, the experimental subjects were fed normally and the experimental water body was completely replaced every three days. After the water was changed, the above method was repeated to add the bacterial solution and sterilized water respectively, and the survival rate of each group of zebrafish was recorded. The experiment lasted for 10 days.

2. Commonly used antibiotic resistance test: the evaluation criteria of antibiotic resistance test (antibiotic disc susceptibility test) is based on "Technical Requirements for Antimicrobial Susceptibility Test" (WS/T 639-2018) (see Table 2), the experimental The method is carried out according to the requirements of the "disc diffusion method" in the above-mentioned standard. Specific steps are as follows:

a) Prepare MHA (Guangdong Huankai Microbial Technology Co., Ltd.) plates, and correct the pH to 7.2-7.4;

b) Use a puncher and qualitative filter paper to punch out a paper disc with a diameter of about 6 mm, dry it after sterilization for later use;

c) According to the types of antibacterial drugs, make paper sheets corresponding to the drug content;

d) Take the strains and inoculate them in the nutrient broth medium respectively, and cultivate them at 30°C and 180r/min until the logarithmic phase;

e) Take 100-150 μL of bacterial solution and evenly spread it on the MHA plate, and dry it at room temperature for 5 minutes;

f) Use sterile tweezers to paste the paper sheet containing antibiotics on the center of the MHA plate, repeat each experiment 3 times, and set another 3 paper sheets containing sterile water on the center of the MHA plate as a blank control;

g) Invert the plate within 15 minutes, and incubate at a constant temperature of 30°C for 18 hours;

h) Use IP54 metal shell digital display vernier caliper (Yongkang Jingsida Trading Co., Ltd.) to measure the diameter of the inhibition zone;

Table 2 Criteria for judging antibiotic tolerance

In the fish toxicity test, under normal conditions for 10 days, the survival rate of the zebrafish in the control group was 100%, and the bacterial content in the experimental group was about 10 ⁶ CFU/mL, which was higher than the pathogenic dose of common pathogenic bacteria (10 ⁴ CFU/mL mL). The survival rate of zebrafish in the experimental group was higher than 96.7%, which was not significantly different from that in the control group (p>0.05). It is preliminarily judged that Rhodococcus stinkbug WM36 has high aquatic biological safety (see Figure 4). The results of the antibiotic resistance test of strain WM36 showed (Table 3 and Figure 5) that Rhodococcus stinkbug WM36 was sensitive to a variety of common clinical antibiotics used (Figure 5), indicating that its ecological security is relatively high. The experiment also provides guidance for the prevention and emergency measures in the use of strains.

Table 3 Antibiotic resistance experiments

5. Optimal growth and denitrification conditions of Rhodococcus stinkbug WM36

Effects of different organic carbon sources on the growth and denitrification performance of Rhodococcus stinkbug WM36

三种氮元素含量。实验设置3个技术重复的实验组以及一个空白对照组，对照组加入等接种量的生理盐水。分析选择草酸钠、琥珀酸钠、乙酸钠以及柠檬酸钠四种不同的有机碳源对椿象红球菌WM36生长情况及好氧反硝化作用的影响。Select four carbon sources of sodium oxalate, sodium succinate, sodium acetate and sodium citrate, fix C/N=10, 30°C, 180r/min, pH=7.0. Based on DM fermentation medium, sodium oxalate, sodium succinate, sodium acetate, and sodium citrate are added in amounts of 0.67g, 0.81g, 0.41g, and 1.29g per liter of medium; as a single inorganic nitrogen The amount of NH ₄ Cl (DMⅠ), NaNO ₃ (DMⅡ) and NaNO ₂ (DMⅢ) per liter of culture medium was 0.02675g, 0.04g and 0.0345g respectively. Take the candidate strains and inoculate them in the nutrient broth medium respectively, cultivate them at 30°C and 180r/min for 1 day, and then insert the above-mentioned denitrification medium with different organic carbon sources at an inoculum amount of 1% (v/v). In 0h, 8h, 16h, 24h, 32h, 40h, 48h, take the culture fluid to measure its OD600, 5000r/min, centrifuge at low speed for 5-10min, take the supernatant to measure respectively

three types of nitrogen content. The experiment set up 3 experimental groups with technical repetitions and a blank control group, and the control group was added with the same inoculum volume of normal saline. The effects of choosing four different organic carbon sources of sodium oxalate, sodium succinate, sodium acetate and sodium citrate on the growth and aerobic denitrification of Rhodococcus stinkbus WM36 were analyzed.

(2) Effects of different C/N on the growth and denitrification performance of Rhodococcus stinkbug WM36

三种氮元素含量。实验设置3个技术重复的实验组以及一个空白对照组，对照组加入等接种量的生理盐水。分析10、20、30、40四种不同的C/N 对椿象红球菌WM36生长情况及好氧反硝化作用的影响。Sodium citrate was selected as the carbon source of the denitrification medium, the conditions were fixed at 30°C, 180r/min, pH=7.0, and the C/N gradient was set to 10, 20, 30, 40. The amount of sodium citrate in each gradient medium was 1.29g/L, 2.58g/L, 3.87g/L, 5.16g/L; NH ₄ Cl (DMⅠ), NaNO ₃ (DMⅡ) as a single inorganic nitrogen source , NaNO ₂ (DMⅢ) added per liter of culture medium were 0.02675g, 0.04g, 0.0345g. The candidate strains were inoculated in the nutrient broth medium respectively, cultivated at 30°C and 180r/min for 1 day, and then inserted into the above-mentioned denitrification medium with an inoculum amount of 1% (v/v), and in 0h, 8h, 16h, 24h, 32h, 40h, 48h, take the culture fluid to measure its OD600, 5000r/min, after 5-10min low-speed centrifugation, take the supernatant to measure respectively

three types of nitrogen content. The experiment set up 3 experimental groups with technical repetitions and a blank control group, and the control group was added with the same inoculum volume of normal saline. Analyze the effects of 10, 20, 30, 40 four different C/N on the growth of Rhodococcus stinkbug WM36 and aerobic denitrification.

(3) Effects of different pH on the growth and denitrification performance of Rhodococcus stinkbug WM36

三种氮元素含量。实验设置3个技术重复的实验组以及一个空白对照组，对照组加入等接种量的生理盐水。分析5、6、7、8、9四种不同的pH 对椿象红球菌WM36生长情况及好氧反硝化作用的影响。Fix the conditions of C/N=10, 30°C, 180r/min, and sodium citrate as the single organic carbon source, and set the pH gradient to 5, 6, 7, 8, and 9. NH ₄ Cl (DMⅠ), NaNO ₃ (DMⅡ) and NaNO ₂ (DMⅢ) as a single inorganic nitrogen source were 0.02675g, 0.04g and 0.0345g per liter of medium, respectively. The candidate strains were inoculated in the nutrient broth medium respectively, cultivated at 30°C and 180r/min for 1 day, and then inserted into the above-mentioned denitrification medium with an inoculum amount of 1% (v/v), and in 0h, 8h, 16h, 24h, 32h, 40h, 48h, take the culture fluid to measure its OD600, 5000r/min, after 5-10min low-speed centrifugation, take the supernatant to measure respectively

three types of nitrogen content. The experiment set up 3 experimental groups with technical repetitions and a blank control group, and the control group was added with the same inoculum volume of normal saline. The effects of four different pH values of 5, 6, 7, 8, and 9 on the growth of Rhodococcus stinkbug WM36 and aerobic denitrification were analyzed.

(4) Effects of different temperatures on the growth and denitrification performance of Rhodococcus stinkbug WM36

三种氮元素含量。实验设置3个技术重复的实验组以及一个空白对照组，对照组加入等接种量的生理盐水。分析15℃、20℃、25℃、30℃、 35℃五种不同的温度对椿象红球菌WM36生长情况及好氧反硝化作用的影响。Fix the conditions of C/N=10, pH=7.0, 180r/min, and sodium citrate as the single organic carbon source, and set the temperature gradient at 15°C, 20°C, 25°C, 30°C, and 35°C. NH ₄ Cl (DMⅠ), NaNO ₃ (DMⅡ) and NaNO ₂ (DMⅢ) as a single inorganic nitrogen source were 0.02675g, 0.04g and 0.0345g per liter of medium, respectively. The candidate strains were inoculated in the nutrient broth medium respectively, cultivated at 30°C and 180r/min for 1 day, and then inserted into the above-mentioned denitrification medium with an inoculum amount of 1% (v/v), and in 0h, 8h, 16h, 24h, 32h, 40h, 48h, take the culture fluid to measure its OD600, 5000r/min, after 5-10min low-speed centrifugation, take the supernatant to measure respectively

three types of nitrogen content. The experiment set up 3 experimental groups with technical repetitions and a blank control group, and the control group was added with the same inoculum volume of normal saline. The effects of five different temperatures of 15°C, 20°C, 25°C, 30°C and 35°C on the growth of Rhodococcus stinkbug WM36 and aerobic denitrification were analyzed.

(5) Effects of different salinity on the growth and denitrification performance of Rhodococcus stinkbug WM36

三种氮元素含量。实验设置3个技术重复的实验组以及一个空白对照组，对照组加入等接种量的生理盐水。分析0‰、4‰、8‰、12‰四种不同的盐度对椿象红球菌WM36生长情况及脱氮作用的影响。Fix the conditions of C/N=10, pH=7.0, 180r/min, T=30°C sodium citrate as the single organic carbon source, and set the medium salinity gradient to 0‰ by changing and adjusting the concentration of NaCl in the medium , 4‰, 8‰, 12‰. NH ₄ Cl (DMⅠ), NaNO ₃ (DMⅡ) and NaNO ₂ (DMⅢ) as a single inorganic nitrogen source were 0.02675g, 0.04g and 0.0345g per liter of medium, respectively. The candidate strains were inoculated in the nutrient broth medium respectively, cultivated at 30°C and 180r/min for 1 day, and then inserted into the above-mentioned denitrification medium with an inoculum amount of 1% (v/v), and in 0h, 8h, 16h, 24h, 32h, 40h, 48h, take the culture fluid to measure its OD600, 5000r/min, after 5-10min low-speed centrifugation, take the supernatant to measure respectively

three types of nitrogen content. The experiment set up 3 experimental groups with technical repetitions and a blank control group, and the control group was added with the same inoculum volume of normal saline. The effects of four different salinities of 0‰, 4‰, 8‰ and 12‰ on the growth and denitrification of Rhodococcus stinkbus WM36 were analyzed.

而C/N条件在20-40范围内时，

和

的降解能力受到抑制(图8)；温度梯度实验表明，椿象红球菌WM36在T＝35℃时的生长速度和无机氮降解效率明显要大于15-30℃(图9)；盐度梯度实验中，菌株WM36在0～12‰的盐度范围内，生长速度和无机氮降解效率皆不受影响(图10)，说明菌株WM36对盐度的适应范围广。综上所述，该菌最佳生长和脱氮条件为碳源使用柠檬酸钠、C/N＝10、pH＝7、T＝35℃，盐度生长范围为0～12‰。

最高可分别达到82.6％、94.6％、86.1％。It can be seen from the comparative analysis of Figure 6 that Rhodococcus stinkbug WM36 can grow with organic carbon sources such as sodium citrate and sodium acetate. The growth rate slows down when using sodium acetate to grow; the most suitable pH for growth and denitrification is 7 (Figure 7); it can grow and degrade in the range of C/N 10-40

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

and

The degradation ability of is inhibited (Fig. 8); Temperature gradient experiment shows, the growth rate and the inorganic nitrogen degradation efficiency of Rhodococcus stinkbug WM36 when T=35 ℃ are obviously greater than 15-30 ℃ (Fig. 9); In salinity gradient experiment , the growth rate and inorganic nitrogen degradation efficiency of the strain WM36 were not affected in the salinity range of 0-12‰ (Fig. 10), indicating that the strain WM36 has a wide adaptability to salinity. In summary, the optimal growth and denitrification conditions for the bacteria were sodium citrate as carbon source, C/N=10, pH=7, T=35℃, and the growth range of salinity was 0-12‰.

The highest can respectively reach 82.6%, 94.6%, 86.1%.

6. Application of Rhodococcus stinkbug WM36 in brackish water aquaculture tail water

三种氮元素含量。Collect water samples from aquaculture ponds and ecological ditches in Zhaoxin Village, Doumen District, Zhuhai City (Figure 11), and use an optical salinometer to measure the salinity value of the water at the sampling point. Refrigerated at 4°C and transported back to the laboratory for standby, and the determination of the inorganic nitrogen index of the water sample was completed on the same day. Take 60-100mL water samples from aquaculture ponds and ecological ditches respectively, filter to remove solid particles and suspended solids, use a 0.22μm/25mm syringe filter to filter and sterilize, and transfer to a conical flask that has been sterilized by high pressure steam for later use. Take the strain WM36 and inoculate it in the nutrient broth medium, activate it at 30°C, 180r/min for 18h, and then inoculate the bacterial solution in the logarithmic phase into the two water samples with an inoculation amount of 1% (v/v). , 30°C, 180r/min shaking culture. Take water samples at 0h, 12h, 24h, 48h, and 72h to measure their OD ₆₀₀ , centrifuge at 5000r/min, 5-10min at a low speed, and take supernatants to measure respectively

three types of nitrogen content.

After analysis, the average value of the basic water quality data of aquaculture ponds and ecological ditches in brackish water aquaculture areas is shown in Table 4.

Table 4 Water quality data of brackish water aquaculture area

降解率最高可分别达到68.2％、95.2％、85.4％，期间无

积累；生态沟渠养殖尾水水样中的

降解率最高可分别达到44.8％、87.5％、69.5％，期间无

积累(图12)。说明本发明菌种在咸淡水养殖尾水处理能力方面具有重要的应用价值。Since the ecological ditch in the breeding area is connected to the river in Zhaoxin Village, the salinity of the water body changes alternately (Figure 11), resulting in inconsistent salinity in the water body of the aquaculture pond and the ecological ditch in Table 4. From the data of inorganic nitrogen treatment results, it can be known that the strain WM36 can adapt to the water body of aquaculture pond with a salinity of 2‰ and the tail water of an ecological ditch with a salinity of 8‰, and can grow in it and degrade the inorganic nitrogen in the purified water. After using the strain WM36 to treat for 72h, the water samples in the culture pond

The highest degradation rate can reach 68.2%, 95.2%, 85.4%, respectively, during which no

accumulation; in the tail water samples of ecological ditch aquaculture

The highest degradation rate can reach 44.8%, 87.5%, 69.5%, respectively, during which no

accumulation (Figure 12). It shows that the bacterial strain of the present invention has important application value in the treatment capacity of brackish and fresh water aquaculture tail water.

The Rhodococcus stinkii WM36 of the present invention is applied to the field of nitrogen-containing salty and fresh water aquaculture tail water treatment. The strain has a wide range of salinity adaptability, has no adverse effects on aquaculture objects, and has high biological safety of aquatic organisms; and is suitable for various clinical Commonly used antibiotics are sensitive and have high ecological security. Therefore, it is suitable for aquaculture water applications in fresh water and brackish water (salinity ≤ 12‰); it has the functions of heterotrophic nitrification and aerobic denitrification at the same time; it can use a variety of organic carbon sources, and has a good ability to remove organic carbon in water bodies. It has good economic and environmental benefits and broad application prospects.

The foregoing embodiments are not intended to limit the embodiments of the present invention, but are examples for illustrating the present invention. For researchers familiar with this field, other changes and modifications can still be made on the basis of the above-mentioned embodiments. All extensions or modifications made within the scope of the embodiments or technical solutions of the present invention are still within the scope of the patent coverage of the present invention.

sequence listing

<110> Institute of Animal Science, Guangdong Academy of Agricultural Sciences

Zhuhai Jiali Technology Co., Ltd.

<120> Application of Rhodococcus stinkbug WM36 in Nitrogen Removal of Brackish Water Nitrogenous Wastewater

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1423

<212> DNA

<213> Rhodococcus stinkbug WM36 (Rhodococcus rhodnii)

<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 gacggggcc 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 atgtccagggg 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 (7)

1. The application of Rhodococcus rhodnii ( Rhodococcus rhodnii ) WM36 in the denitrification of saline and fresh water nitrogen-containing sewage, the preservation number of the Rhodococcus stink bug WM36: GDMCC No: 61717, the salinity range of the saline and fresh water nitrogen-containing sewage 4‰~12‰; the denitrification is to remove nitrogenous sewage in brackish water

Three inorganic nitrogen elements. 2. The application according to claim 1, characterized in that, the brackish water is brackish water aquaculture tail water. 3. The application according to claim 1, characterized in that, inoculate Rhodococcus stinkrubia WM36 in saline and fresh water nitrogenous sewage, and cultivate to denitrify sewage. 4. The application according to claim 1, characterized in that the pH value of the brackish water nitrogenous sewage is 7. 5. The application according to claim 3, characterized in that the temperature of the cultivation is 35°C. 6. The application according to claim 1, characterized in that the C/N in the brackish water and nitrogen-containing sewage is 10-40. 7. The application according to claim 3, characterized in that, the cultivation uses sodium citrate as carbon source, C/N=10, pH=7, T=35°C, salinity range=4‰~12 ‰.

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