CN111471611B - Rhodococcus ruber HDRR1 for purifying inorganic nitrogen and phosphorus in tail water of seawater pond culture and application thereof - Google Patents
Rhodococcus ruber HDRR1 for purifying inorganic nitrogen and phosphorus in tail water of seawater pond culture and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/105—Phosphorus compounds
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
Abstract
The invention discloses Rhodococcus ruber HDRR1 and Rhodococcus ruber HDRR1(Rhodococcus ruber HDRR1) for purifying inorganic nitrogen and phosphorus in tail water of seawater pond culture, wherein the preservation name is Rhodococcus ruber HDRR1, the preservation number is CCTCC M20191008, the preservation date is 12 months and 4 days in 2019, the preservation unit is the China center for type culture preservation, and the preservation address is Wuhan in China. The strain HDRR1 has strong purification capacity on inorganic nitrogen and phosphorus in culture tail water, has good environmental adaptability and has no adverse effect on fish and shrimp culture. Also discloses application of the rhodococcus ruber HDRR1 in purifying inorganic nitrogen and phosphorus in tail water of seawater pond culture.
Description
Technical Field
The invention belongs to the technical field of microbial purification of seawater pond culture water bodies, and particularly relates to rhodococcus ruber HDRR1 for purifying inorganic nitrogen and phosphorus in seawater pond culture tail water and application thereof.
Background
In 2018, the total yield of aquaculture in China exceeds 5000 ten thousand tons, and accounts for more than 78% of the total supply of the aquaculture. If the management is not good in the aquaculture process, the residual feed, the culture metabolites and the aquatic organism residues are gathered in the water environment to form self-derived culture tail water or pollutants. The tail water mainly contains ammonia nitrogen, Total Inorganic Nitrogen (TIN), phosphorus, organic matters, fouling organisms and the like in terms of the form of chemical substances contained in the tail water (slow rising 2011; great fly and the like 2002). For example, harmful nitrogen such as ammonia nitrogen and nitrite nitrogen is very easily accumulated in a large amount in high-density intensive culture water (Zhongping 2013), which is a main stress factor (Tovar, et al, 2000) affecting aquaculture organisms, has serious toxic action, causes disease resistance of cultured prawns to be reduced and even induces diseases or death (Jiang reamou et al, 2004), and is one of the key technical links of aquaculture (Liu Xing, 2011). Moreover, if the aquaculture tail water is directly discharged into nearby water areas without treatment, eutrophication of the nearby water areas can be caused, environmental disasters such as water bloom, red tide and the like are caused, and sustainable development of aquaculture industry is severely restricted (Liujian and the like, 2017).
Good effect can be obtained by utilizing beneficial microorganisms to treat the culture water body or tail water. At present, the traditional treatment method of the aquaculture tail water mainly comprises a physical method, a chemical method, a biomembrane method, a plant treatment method, an artificial wetland comprehensive treatment method and the like. Wherein, the biological method utilizes the physiological and ecological effect of specific microorganisms to remove potential pollution sources in water and has the advantages of environmental protection, difficult generation of secondary pollution, strong sustainability and the like (Wang Ying, etc., 2013). Most of the current research focuses on nitrifying bacteria, denitrifying bacteria, lactobacillus plantarum, bacillus subtilis, light bacteria and the like. Although the application of microorganism to purification and treatment of aquaculture water or tail water has wide application prospect, the method also has a lot of problems in the practical application process, and the technical requirement on the targeted control of the flora function is relatively high. The reason is closely related to various factors such as the physiological and ecological characteristics of different types of microorganisms, the environmental adaptability of the dominant population, the interaction relationship between the dominant population and the ecological function of the indigenous bacteria, and the like. For example, heterotrophic microorganisms which are easy to culture are easy to degrade soluble or suspended organic matters in a water body, but the purification efficiency of total inorganic nitrogen and phosphorus such as ammonia nitrogen, nitrite nitrogen, phosphate and the like is relatively low; some strains may have good purification effect in a pure culture state of a specific nutrient solution, and once the strains enter a complex natural water environment state, the strains lose functions or even cannot survive due to the influence of external factors. Therefore, the method aims at excavating excellent strain resources with wide environmental adaptation spectrum and gives consideration to the function synergistic effect of the excellent strain resources and other dominant populations in the target environment so as to realize the directional maintenance of the ecological function of the microbial community of the water body, and the method has important significance for realizing and improving the application effect of purifying the water body environment by the microbes.
As nitrobacteria and denitrifying bacteria are relatively difficult to separate and purify, most researchers at home and abroad adopt activated sludge for enrichment culture, less pure bacteria expansion culture mode is adopted, and the formation of a pure bacteria preparation product which can be applied to the aquaculture industry in a large scale is more rarely reported (Yangning, 2003; Wang Juan, 2006). Although the microbial immobilization technology is concerned and beneficial exploration and attempt are carried out, most of the technologies still stay in the small-scale and small-range application experiment stage, and the large-scale industrial application effect of directed culture of single microorganisms is not achieved.
Disclosure of Invention
The invention aims to provide the Rhodococcus ruber HDRR1 for purifying inorganic nitrogen and phosphorus in tail water of seawater pond culture, and the bacterial strain HDRR1 has strong purification capability on inorganic nitrogen and phosphorus in the tail water of the culture, has good environmental adaptability and has no adverse effect on fish and shrimp culture.
The invention also aims to provide application of the rhodococcus ruber HDRR1 in purifying inorganic nitrogen and phosphorus in tail water of seawater pond culture.
The first object of the present invention can be achieved by the following technical solutions: the rhodococcus ruber HDRR1 for purifying inorganic nitrogen and phosphorus in tail water of seawater pond culture is Rhodococcus ruber HDRR1(Rhodococcus ruber HDRR1), the preservation name is rhodococcus ruber HDRR1, the preservation number is CCTCC M20191008, the preservation date is 12 months and 4 days in 2019, the preservation unit is China center for type culture preservation, and the preservation address is Wuhan, China.
The second object of the present invention can be achieved by the following technical solutions: the Rhodococcus ruber HDRR1 is applied to purifying inorganic nitrogen and phosphorus in tail water of seawater pond culture.
The environment of the seawater culture pond water body and tail water is greatly different from that of the water body in the background technology, and the indigenous strains with inorganic nitrogen and phosphorus purification function are preferably obtained from the seawater culture pond water body based on the seawater culture water body environment. And evaluating the adaptability of the strain to different aquaculture water body environments, the nitrogen and phosphorus purification efficiency, the application safety and other characteristics, and further researching and developing a microbial inoculum product and an application technology suitable for practical application of aquaculture production. If the specific actual requirements of the cultured organisms and the production on safety, high efficiency and sustainable development are neglected by simply referring to or according to the relevant details in the technical field of sewage treatment engineering, the formed microorganism products or the relevant technologies cannot be effectively applied and popularized in the mariculture industry. At present, no relevant research report about purifying inorganic nitrogen and phosphorus in mariculture water body or tail water by rhodococcus ruber exists in the industry.
Therefore, the Rhodococcus ruber HDRR1 is obtained by separating and screening in the environment of the seawater intensified culture pond, and the Rhodococcus ruber HDRR1 has obvious removal effect on inorganic nitrogen and phosphorus in culture tail water and has no obvious adverse effect on litopenaeus vannamei during the high-density zero-water-change culture process. Can be used as an alternative strain resource for researching and developing aquaculture microbial inoculum products.
Compared with the prior art, the invention has the following advantages:
(1) the Rhodococcus ruber HDRR1 is selected from tail water in the middle and later periods of prawn intensive zero-water-change aquaculture, has good environmental adaptability, has no adverse effect on aquaculture organisms, and is suitable for most aquaculture pond water bodies.
(2) The Rhodococcus ruber HDRR1 has a remarkable effect of removing inorganic nitrogen and phosphorus in shrimp intensive zero-water-change aquaculture tail water, and the purification time is determined according to the specific condition of nitrogen and phosphorus concentration to be removed in water in the application process.
(3) The Rhodococcus ruber HDRR1 can achieve good application effect when applied to water quality purification regulation and control of intensive culture and removal of inorganic nitrogen and phosphorus in tail water, is beneficial to greatly reducing water body replacement in the culture production process, can avoid the need of configuring expensive water quality purification equipment, and can provide technical support for researching and developing or implementing culture water environment directional regulation and control technology in future and promoting development of green, efficient and healthy culture industry for realizing ecological and environmental protection.
Drawings
FIG. 1 is a graph of the growth of Rhodococcus ruber HDRR1 in sterilized pond tail water in example 3;
FIG. 2 is the variation of the tail water phosphate concentration of Rhodococcus ruber HDRR1 at different salinity in example 3;
FIG. 3 is the variation of the ammonia nitrogen concentration of the tail water of the Rhodococcus ruber HDRR1 in example 3 at different salinity;
FIG. 4 is the variation of nitrate nitrogen concentration of tail water at different salinity for Rhodococcus ruber HDRR1 in example 3;
FIG. 5 is the change of the TIN concentration of tail water at different salinity of the HDRR1 of Rhodococcus ruber in example 3;
FIG. 6 is the tail water phosphate concentration change at different temperatures for the Rhodococcus ruber HDRR1 in example 3;
FIG. 7 shows the variation of ammonia nitrogen concentration in tail water at different temperatures in example 3 with Rhodococcus ruber HDRR 1;
FIG. 8 is the variation of the nitrate nitrogen concentration of tail water at different temperatures for Rhodococcus ruber HDRR1 in example 3;
FIG. 9 shows the TIN concentration of tail water at different temperatures for Rhodococcus ruber HDRR1 in example 3;
FIG. 10 is the tail water phosphate concentration change at different pH's for the Rhodococcus ruber HDRR1 in example 3;
FIG. 11 is the change of the ammonia nitrogen concentration of the tail water at different pH values of the Rhodococcus ruber HDRR1 in example 3;
FIG. 12 is the change in nitrate nitrogen concentration of tail water at different pH for Rhodococcus ruber HDRR1 in example 3;
FIG. 13 is the variation of the TIN concentration of tail water at different pH values for Rhodococcus ruber HDRR1 in example 3.
Detailed Description
The invention is further illustrated, but is not intended to be in any way limited, by the following examples and figures.
Example 1 screening and cultivation of Rhodococcus ruber HDRR1 for purifying inorganic nitrogen and phosphorus in seawater Pond culture Tail Water
1. Material preparation
1.1, sources of bacteria
Collecting tail water samples of 60-90 days of culture in an intensive prawn culture pond in the town of the plain sea of the county of Heidong, Guangdong, and performing isolated culture by using a selective culture medium plate.
1.2 culture Medium
(1) Selective liquid medium: CH (CH) 3 COONa: 1g, yeast extract: 1g, MgSO 4 ·7H 2 O： 0.4g、NaCl：0.1g、CaCl 2 ·2H 2 O：0.05g、NaHCO 3 ：0.3g、KH 2 PO 4 : 1g and 1mL of trace element solution, and dissolving the above medicines in distilled water respectively to 1000mL, and adjusting the pH to 7.0.
Solution of trace elements: EDTA: 2.5g, ZnSO 4 ·7H 2 O：10.95g、MnSO 4 ·H 2 O：1.54g、 CuS0 4 ·5H 2 O：0.39g、CoCl 2 ·6H 2 O：0.2g、FeSO 4 ·7H 2 O: 7g, glutamic acid: 0.02g, the above drugs are dissolved in distilled water respectively, and dissolved to 1000mL, pH7.0.
(2) Selective solid plate medium: on the basis of the selective liquid culture medium, 20g/L agar powder is added to prepare a solid plate culture medium.
2. Screening culture of strains
Selecting the tail water (cultured for 60-90 days) of the prawn intensive zero-water-change culture pond in the open sea town of Guangdong Heidong county, filtering the collected water sample by using a mixed cellulose ester filter membrane (with the aperture of 0.22 mu m), and placing the filter membrane in a selective liquid culture medium for shake culture for 2-6 days at the temperature of 25-35 ℃ and the illumination intensity of 2000-6000 lx; and (3) carrying out streak separation on the cultured bacterial liquid on a selective solid plate culture medium to obtain single colonies, culturing for 3-5 days, selecting the single colonies with different forms, and selecting strains with good growth performance. And then, re-inoculating the strain to a selective liquid culture medium, and performing shake cultivation for 3-5 days at the temperature of 30-35 ℃, the illumination intensity of 2000-6000 lx and the rotation speed of 100-200 rpm.
Adding different bacteria solutions into sterilized culture water (NH) with adjusted ammonia nitrogen and active phosphate concentration 4 Adjusting the concentration of ammonia in the water body to 10-20 mg/L with Cl, and using KH 2 PO 4 Adjusting the concentration of phosphate in the water body to 15-20 mg/L), and illuminating at 30-35 ℃ and with the illumination intensity of 2000-6000 lx and 10%Carrying out shaking table amplification culture at 0-200 rpm for 3-5 days.
And selecting a strain capable of effectively reducing the concentration of ammonia nitrogen, phosphate and Total Inorganic Nitrogen (TIN) in the water body to perform strain identification and preservation for later use. The Rhodococcus ruber HDRR1 shows good growth performance in the primary screening process, and has good removal effect on ammonia nitrogen, phosphate and Total Inorganic Nitrogen (TIN).
Example 2 identification of the screened Rhodococcus ruber HDRR1
The invention carries out 16S rDNA molecular identification on the screened rhodococcus ruber HDRR1, and determines the species of the strain from the molecular level by combining the morphological characteristics and physiological and biochemical characteristics of bacteria. The analysis of the 16S rDNA sequence mainly comprises the following steps:
1. extraction of bacterial genomic DNA:
(1) picking a single colony by using a sterile toothpick and inoculating the colony in an enlarged culture medium for culture;
(2) centrifuging 1.5mL of bacteria culture solution at 10000rpm (11,500 Xg) for 1 min, and sucking the supernatant as far as possible;
(3) adding 200 mu L of buffer solution GA into the thallus sediment, oscillating until the thallus is completely suspended, adding 180 mu L of lysozyme with the final concentration of 20mg/mL, and treating for more than 30 minutes at 37 ℃;
(4) adding 20 mu L of proteinase K solution into the tube, and uniformly mixing;
(5) adding 220 mu L buffer solution GB, oscillating for 15 seconds, standing at 70 ℃ for 10 minutes, enabling the solution to become clear, and centrifuging briefly to remove water drops on the inner wall of the tube cover;
(6) adding 220 mu L of absolute ethyl alcohol, fully oscillating and uniformly mixing for 15 seconds, and centrifuging briefly to remove water drops on the inner wall of the tube cover;
(7) adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (placing the adsorption column into a collecting pipe), centrifuging at 12000rpm (13,400 Xg) for 30 s, pouring out waste liquid, and placing an adsorption column CB3 into the collecting pipe;
(8) adding 500 μ L buffer GD into adsorption column CB3, centrifuging at 12000rpm (13,400 × g) for 30 s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;
(9) adding 700 μ L of rinsing liquid PW into adsorption column CB3, centrifuging at 12000rpm (13,400 × g) for 30 s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;
(10) adding 500 μ L of rinsing solution PW into adsorption column CB3, centrifuging at 12000rpm (13,400 Xg) for 30 s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;
(11) the adsorption column CB3 was returned to the collection tube, centrifuged at 12000rpm (13,400 Xg) for 2 minutes, and the waste liquid was discarded. Placing the adsorption column CB3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material;
(12) transferring the adsorption column CB3 into a clean centrifugal tube, dripping 50-200 mu L of elution buffer TE into the middle part of an adsorption film in a hanging manner, standing at room temperature for 2-5 minutes, centrifuging at 12000rpm (13,400 Xg) for 2 minutes, and collecting the solution into the centrifugal tube;
(13) the recovered DNA fragment was subjected to agarose gel electrophoresis and ultraviolet spectrophotometer to determine the concentration and purity.
2. PCR amplification of 16S rDNA Gene
The universal bacterial primers used for the amplification of 16S rDNA were synthesized by Biotechnology (Shanghai) limited, and the forward primers (8f) were: 5'-AGAGTTTGATCCTGGCTCAG-3', respectively; the reverse primer (1492r) is: 5'-GGTTACCTTGTTACGACTT-3' is added. The 50 μ L PCR reaction included: mu.L of sterilized double distilled water, 1. mu.L of each primer, 4. mu.L of dNTPs (2.5mmol/L), 1. mu.L of Tapase, 5. mu.L of 10 XPCR buffer, and 1. mu.L of DNA template (DNA recovered from the extraction of the above-mentioned bacterial genomic DNA). PCR conditions: 3 minutes at 95 ℃, 1 minute at 48 ℃,2 minutes at 72 ℃ for 30 cycles; 10 minutes at 72 ℃.
3. 16S rDNA sequencing
After the amplification, the PCR product was detected by 1.0% agarose gel electrophoresis and sequenced by Biotechnology engineering (Shanghai) Co., Ltd. The sequence is determined (specifically shown as the sequence table SEQ ID NO: 1):
TAGGAGGGGGCGGCTTGCTTACCATGCAGTCGAACGATGAAGCCCAGCTTGCTGGGTGGATTAGTGGCG AACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACTGGGTCTAATAC CGGATAGGACCTCGGGATGCATGTTCCGGGGTGGAAAGGTTTTCCGGTGCAGGATGGGCCCGCGGCCTA TCAGCTTGTTGGTGGGGTAACGGCCCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCC ACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGC AAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGTACCGACG AAGCGCAAGTGACGGTAGGTACAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTA GGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCTGTGAA AACCCGCAGCTCAACTGCGGGCTTGCAGGCGATACGGGCAGACTTGAGTACTGCAGGGGAGACTGGAAT TCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAG TAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAA ACGGTGGGCGCTAGGTGTGGGTTTCCTTCCACGGGATCCGTGCCGTAGCTAACGCATTAAGCGCCCCGC CTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATG TGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATACACCGGACCGCCCCAGAGATGG GGTTTCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT AAGTCCCGCAACGAGCGCAACCCTTGTCCTGTGTTGCCAGCACGTAATGGTGGGGACTCGCAGGAGACT GCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCAC ACATGCTACAATGGCCGGTACAGAGGGCTGCGATACCGCGAGGTGGAGCGAATCCCTTAAAGCCGGTCT CAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACG CTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAA GCCGGTGGCCTAACCCCTCGTGGGAGGGAGCCGTCGAAGGTGATCCGCGCT。
4. colony morphology and physiological characteristics of Rhodococcus ruber HDRR1
The colony morphology and physiological characteristics of the Rhodococcus ruber HDRR1 are shown in Table 1 below.
TABLE 1 colony morphology, physiological characteristics of Rhodococcus ruber HDRR1
5. Identification of Rhodococcus ruber HDRR1
The 16S rDNA gene sequence of the strain is compared with the registered gene sequence in GenBank, and the result shows that the strain is Rhodococcus ruber HDRR1(Rhodococcus ruber). The results of 16S rDNA gene sequence analysis, biochemical identification, morphological characteristics and the like are integrated. The strain HDRR1 was identified as Rhodococcus ruber (Rhodococcus ruber). By referring to the relevant data, no research report on the purification of inorganic nitrogen and phosphorus in the prawn intensive culture water body or tail water of the prawn intensive culture water body by rhodococcus ruber 1(Rhodococcus ruber) is available. The strain is preserved in China center for type culture collection (CCTCC M20191008) in 2019, 12 months and 4 days, and the preservation address is Wuhan, in particular to the China center for type culture collection of Wuhan university at Lojia mountain of Wuchang, Wuhan city, Hubei province.
Example 3 Effect of Rhodococcus ruber HDRR1 on removal of inorganic Nitrogen and phosphorus from seawater Pond culture Tail Water
1. Growth of the Strain
The strain Rhodococcus ruber HDRR1 obtained in example 1 was adjusted to an initial concentration of 10 4 ～10 6 Inoculating CFU/mL into sterilized intensive culture pond tail water, and increasing the bacterial load to 10 within about 2 days 8 CFU/mL, the bacterial load is always stable at 10 within 3-10 days 8 The growth curve of the CFU/mL number level, Rhodococcus ruber HDRR1 is shown in FIG. 1.
2. Removal effect of inorganic nitrogen and phosphorus in culture tail water with different salinity by using strain
The tail water (water salinity 25) of the sterilized prawn intensive culture pond is used as a basic test water body control, and no Rhodococcus ruber HDRR1 is added in the test process. Adding bacteria group to adjust salinity of water body to 5, 10, 25, 40 with distilled water and sea salt, and mixing the Rhodococcus ruber HDRR1 obtained in example 1 according to the ratio of 10 4 ～10 6 And (3) inoculating the CFU/mL into test water bodies with different salinity, performing shake culture for 10 days at the temperature of 35 ℃, the illumination intensity of 2000-6000 Lx, the pH value of the water body of 7.0-8.5 and the rpm of 100-200, and setting 4 parallel test samples in each group. The change of the phosphate, ammonia nitrogen, nitrate nitrogen and Total Inorganic Nitrogen (TIN) concentration in the water body is monitored every 2 days.
The results show that:
as shown in figure 2, when the salinity of the water body is 5-40, the phosphate concentration of the water body with the bacteria is reduced from 16.669-17.619 mg/L to 6.904-9.289 mg/L on the 8 th day, the average removal rate is 44.3-60.0%, and the removal rate on the 2 nd day is 34.6-50.3%.
As shown in figure 3, when the salinity of the water body is 5-40, the ammonia nitrogen concentration is reduced from 16.611-18.335 mg/L to 0.055-1.692 mg/L on day 4, the average removal rate is 89.8-99.7%, and the removal rate on day 2 is 90.7-96.6%.
As shown in FIG. 4, when the salinity of the water body is 5-25, the nitrate nitrogen concentration is reduced from 3.872-4.126 mg/L to 1.848-2.600 mg/L on the day 2, and the average removal rate is 32.9-55.2%; when the salinity is 40, the nitrate nitrogen concentration has no obvious change.
As shown in figure 5, when the salinity of the water body is 5-40, the concentration of the TIN is reduced from 31.281-32.466 mg/L to 9.572-13.947 mg/L on the 2 nd day, and the average removal rate is 57.0-70.4%.
As shown in FIGS. 2 to 5, the concentration ranges of phosphate, ammonia nitrogen and Total Inorganic Nitrogen (TIN) in the control group are 17.280 to 19.113mg/L, 18.419 to 19.038mg/L and 33.156 to 33.207mg/L respectively, and basically have no obvious change compared with the initial concentration value.
When the salinity is 5-40, the range of the monitored bacterial load of the HDRR1 during the test is 2.4 multiplied by 10 8 CFU/mL～ 7.0×10 8 CFU/mL。
Therefore, the Rhodococcus ruber HDRR1 has good adaptability to the salinity of a water body, can be used for purifying inorganic nitrogen and phosphorus in the seawater culture tail water with the salinity of 5-40, and has a better effect of removing the inorganic nitrogen and phosphorus in the seawater culture tail water when the salinity is 5-25.
3. Effect of removing inorganic nitrogen and phosphorus in tail water of culture of bacterial strains at different temperatures
The tail water (water salinity 25) of the sterilized prawn intensive culture pond is used as a basic test water body control and is cultured at constant temperature of 30 ℃, wherein the Rhodococcus ruber HDRR1 is not added. Addition of bacterial groups the Rhodococcus ruber HDRR1 obtained in example 1 was adjusted to 10 4 ～10 6 The CFU/mL is inoculated into test water bodies with different temperatures, the culture temperatures are respectively set to be 10 ℃,20 ℃, 30 ℃,40 ℃, the illumination intensity is 2000-6000 Lx, the pH value of the water body is 7.0-8.5, shaking culture is carried out at 100-200 rpm for 10 days, and 4 parallel test samples in each group are set. The change of the phosphate, ammonia nitrogen, nitrate nitrogen and Total Inorganic Nitrogen (TIN) concentration in the water body is monitored every 2 days.
The results show that:
as shown in figure 6, when the water temperature is 20-30 ℃, the phosphate concentration of the water body with the bacteria is reduced from 14.987-15.782 mg/L to 8.494-10.217 mg/L on the 4 th day, and the average removal rate is 31.8-46.2%.
As shown in figure 7, when the water temperature is 20-40 ℃, the ammonia nitrogen concentration is reduced from 19.038-20.333 mg/L to 0.191-0.844 mg/L on day 4, the average removal rate is 95.7-99.1%, and the removal rate is 81.6-97.9% on day 2.
As shown in figure 8, when the water temperature is 20-40 ℃, the nitrate nitrogen concentration is reduced from 2.567-3.518 mg/L to 1.759-2.235 mg/L on day 8, and the average removal rate is 31.5-36.5%.
As shown in figure 9, when the water temperature is 20-40 ℃, the TIN concentration is reduced from 32.135-34.266 mg/L to 12.399-13.765 mg/L on the 4 th day, the average removal rate is 57.2-63.8%, and the removal rate on the 2 nd day is 44.8-58.7%.
As shown in FIGS. 6 to 9, the concentration of phosphate, ammonia nitrogen and Total Inorganic Nitrogen (TIN) in the control group varied in the ranges of 14.656 to 19.046mg/L, 14.634 to 20.901mg/L and 30.187 to 37.539mg/L, respectively, and there was almost no significant change in the initial concentration value.
The range of the monitored bacterial load of HDRR1 during the test period is 1.9 multiplied by 10 when the temperature is 20-40 DEG C 8 CFU/mL～6.2×10 8 CFU/mL, bacterial load range at 10 ℃ of 1.5X 10 7 CFU/mL～9.0×10 7 CFU/mL。
In general, the rhodococcus erythropolis HDRR1 can adapt to the temperature condition of a culture water body during culture production, has good temperature adaptability, and can effectively reduce the inorganic nitrogen and phosphorus concentration of seawater culture tail water when being applied to a water body regulation and control technical link.
4. Removal effect of inorganic nitrogen and phosphorus in culture tail water with different pH values of bacterial strain
The tail water (water body salinity of 25 and pH value of 8.0) of the sterilized prawn intensive culture pond is used as a basic test water body control and is cultured at constant temperature of 35 ℃, wherein the Rhodococcus ruber HDRR1 is not added. Addition of bacterial groups the Rhodococcus ruber HDRR1 obtained in example 1 was adjusted to 10 4 ～10 6 CFU/mL is inoculated into test water bodies with different pH values, the pH values are respectively set to be 4, 6, 8 and 10, and the illumination intensity is highCulturing for 10 days at the constant temperature of 35 ℃ by using a shaking table at the temperature of 2000-6000 Lx and the rpm of 100-200, wherein 4 parallel test samples are arranged in each group. The change conditions of the phosphate, ammonia nitrogen, nitrate nitrogen and Total Inorganic Nitrogen (TIN) concentration in the water body are monitored every 2 days.
The results show that:
as shown in FIG. 10, when the pH of the water body is 6-8, the effect of removing nitrogen and phosphorus in the water body is good, the concentration of phosphate in the water body with the bacterium group on day 6 is reduced from 15.289-16.513 mg/L to 9.900-10.963 mg/L, and the average removal rate is 28.3-40.0%.
As shown in FIG. 11, when the pH of the water body is 6-8, the ammonia nitrogen concentration is reduced from 19.271-19.915 mg/L to 0.540-1.456 mg/L at the 4 th day, the average removal rate is 92.7-97.2%, and the removal rate is 93.4-94.8% at the 2 nd day.
As shown in figure 12, when the pH of the water body is 6-8, the nitrate nitrogen concentration is reduced from 4.220mg/L to 2.159-3.017 mg/L at the 8 th day, and the average removal rate is 28.5-48.8%.
As shown in FIG. 13, when the pH of the water body is 6-8, the TIN concentration is reduced from 33.105-34.069 mg/L to 11.203-12.501 mg/L on day 4, the average removal rate is 63.3-66.2%, and the removal rate on day 2 is 61.0-63.1%.
As shown in FIGS. 11 to 13, the concentration of phosphate, ammonia nitrogen and Total Inorganic Nitrogen (TIN) in the control group varied in the ranges of 15.160 to 17.738mg/L, 16.557 to 19.099mg/L, 4.220 to 4.450mg/L and 32.087 to 33.420mg/L, respectively, and there was almost no significant change in the initial concentration value.
When the pH value is 6-8, the range of the monitored bacterial load of the HDRR1 during the test period is 2.4 multiplied by 10 8 CFU/mL～ 7.5×10 8 The bacterial count ranges between CFU/mL, pH4 and pH10 of 4.5X 10 5 CFU/mL～1.8×10 7 CFU/mL。
In general, the Rhodococcus erythropolis HDRR1 can adapt to the pH condition of a culture water body (the pH value is 6-10, and preferably 6-8) during the culture production period, and can effectively reduce the concentration of inorganic nitrogen and phosphorus in seawater culture tail water when being applied to a water body regulation and control technical link.
5. Application effect of strain HDRR1 in high-density zero-water-change aquaculture production of litopenaeus vannamei
The bacterial strain HDRR1 is prepared into a microbial inoculum by amplification culture in a laboratory and is sent to a Litopenaeus vannamei farmer in Yangmuic village of Lofeng city, Guangdong province for application. The cultivation adopts an intensive cultivation mode of high density zero water change, and the concentration of the used bacteria is 10 4 ～10 6 CFU/mL. Proper amount of brown sugar and the microbial preparation are added into water in 15 days of seedling release, and the mixture is repeatedly used for 3-4 times. The bacteria preparation is added periodically every 10-15 days during the later culture period, and is matched with other water environment regulating agents, for example, quicklime water and the like are utilized to stabilize the total alkalinity of the water body to 150-260 CaCO 3 The pH value is stabilized within 7.0-8.2 within the range of mg/L. In the whole cultivation process, the ejector is utilized to keep the water body flowing and enhance oxygenation.
The results show that: the culture application effect of the bacterial preparation is good, and the survival rate of the litopenaeus vannamei cultured in 60 days can reach more than 80.8 percent, which shows that the bacterial preparation has no obvious adverse effect on cultured organisms. Multiple on-site monitoring shows that the water temperature of the aquaculture water body is 28-31 ℃, the salinity is 25-30, the DO is more than 4.5mg/L, and the pH is 7.0-7.35, so that the concentration of ammonia nitrogen in the water body is 0.155mg/L and the concentration of nitrite nitrogen is 0.149mg/L, which are measured by the portable water quality monitoring kit, and the water quality conditions, particularly the conditions of the concentration of ammonia nitrogen and the concentration of nitrite can meet the requirement of healthy growth of the cultured prawns.
The invention is not limited to the specific embodiments described above, which are intended to illustrate the use of the invention in detail, and functionally equivalent production methods and technical details are part of the disclosure. Indeed, those skilled in the art can, based on the foregoing description, find various modifications as may be required and which are within the scope of the claims appended hereto.
Sequence listing
<110> Shenzhen test base of south China institute for aquatic science and research in the south China sea
South China aquatic product research institute
<120> Rhodococcus ruber HDRR1 for purifying inorganic nitrogen and phosphorus in tail water of seawater pond culture and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1431
<212> DNA
<213> Rhodococcus ruber (Rhodococcus ruber)
<400> 1
taggaggggg cggcttgctt accatgcagt cgaacgatga agcccagctt gctgggtgga 60
ttagtggcga acgggtgagt aacacgtggg tgatctgccc tgcacttcgg gataagcctg 120
ggaaactggg tctaataccg gataggacct cgggatgcat gttccggggt ggaaaggttt 180
tccggtgcag gatgggcccg cggcctatca gcttgttggt ggggtaacgg cccaccaagg 240
cgacgacggg tagccggcct gagagggcga ccggccacac tgggactgag acacggccca 300
gactcctacg ggaggcagca gtggggaata ttgcacaatg ggcgcaagcc tgatgcagcg 360
acgccgcgtg agggatgacg gccttcgggt tgtaaacctc tttcagtacc gacgaagcgc 420
aagtgacggt aggtacagaa gaagcaccgg ccaactacgt gccagcagcc gcggtaatac 480
gtagggtgcg agcgttgtcc ggaattactg ggcgtaaaga gctcgtaggc ggtttgtcgc 540
gtcgtctgtg aaaacccgca gctcaactgc gggcttgcag gcgatacggg cagacttgag 600
tactgcaggg gagactggaa ttcctggtgt agcggtgaaa tgcgcagata tcaggaggaa 660
caccggtggc gaaggcgggt ctctgggcag taactgacgc tgaggagcga aagcgtgggt 720
agcgaacagg attagatacc ctggtagtcc acgccgtaaa cggtgggcgc taggtgtggg 780
tttccttcca cgggatccgt gccgtagcta acgcattaag cgccccgcct ggggagtacg 840
gccgcaaggc taaaactcaa aggaattgac gggggcccgc acaagcggcg gagcatgtgg 900
attaattcga tgcaacgcga agaaccttac ctgggtttga catacaccgg accgccccag 960
agatggggtt tcccttgtgg tcggtgtaca ggtggtgcat ggctgtcgtc agctcgtgtc 1020
gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gtcctgtgtt gccagcacgt 1080
aatggtgggg actcgcagga gactgccggg gtcaactcgg aggaaggtgg ggacgacgtc 1140
aagtcatcat gccccttatg tccagggctt cacacatgct acaatggccg gtacagaggg 1200
ctgcgatacc gcgaggtgga gcgaatccct taaagccggt ctcagttcgg atcggggtct 1260
gcaactcgac cccgtgaagt cggagtcgct agtaatcgca gatcagcaac gctgcggtga 1320
atacgttccc gggccttgta cacaccgccc gtcacgtcat gaaagtcggt aacacccgaa 1380
gccggtggcc taacccctcg tgggagggag ccgtcgaagg tgatccgcgc t 1431
Claims (2)
1. The Rhodococcus ruber HDRR1 for purifying inorganic nitrogen and phosphorus in tail water of seawater pond culture is characterized in that: rhodococcus ruber (C.)Rhodococcus ruber) HDRR1 with preservation number of CCTCC M20191008.
2. The Rhodococcus ruber HDRR1 of claim 1 for purifying inorganic nitrogen and phosphorus in seawater pond culture tail water.
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