CN107828679B - Rhodococcus roseus strain XHRR1 for purifying ammonia in aquaculture water and application thereof - Google Patents
Rhodococcus roseus strain XHRR1 for purifying ammonia in aquaculture water and application thereof Download PDFInfo
- Publication number
- CN107828679B CN107828679B CN201711026615.1A CN201711026615A CN107828679B CN 107828679 B CN107828679 B CN 107828679B CN 201711026615 A CN201711026615 A CN 201711026615A CN 107828679 B CN107828679 B CN 107828679B
- Authority
- CN
- China
- Prior art keywords
- xhrr1
- strain
- ammonia
- rhodococcus
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- 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
-
- 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
- C02F2101/16—Nitrogen compounds, e.g. ammonia
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a Rhodococcus roseus strain XHRR1 for purifying ammonia in aquaculture water, wherein the preservation name of the strain XHRR1 is as follows: rhodococcus roseus XHRR1, class name: rhodococcus rhodochrous XHRR1, deposited in units of: china center for type culture Collection, the collection addresses are: wuhan, China, the preservation date is: 8, 3 months in 2017, the preservation number is: CCTCC NO: m2017437. The bacterial strain XHRR1 has strong purification capacity on ammonia in the aquaculture water body, good environmental adaptability and no adverse effect on prawn aquaculture. Also discloses application of the Rhodococcus roseus strain XHRR1 in purifying ammonia in aquaculture water.
Description
Technical Field
The invention belongs to the technical field of rhodococcus roseus, and particularly relates to a rhodococcus roseus strain XHRR1 for purifying ammonia in aquaculture water and application thereof.
Background
In a high-density intensive aquaculture mode, the efficient control of the water environment is one of the key technical links of the whole production process. Ammonia nitrogen has serious toxic action on aquatic animals, and is easy to accumulate in large quantity in high-density aquaculture water (Zhongping 2013), so that the effective regulation of the content of harmful nitrogen in the water is extremely important (Liuxing country 2011). At present, the common treatment methods at home and abroad mainly comprise three methods, namely physical, chemical, biological and the like. The physical method utilizes the adsorption capacity of solid to purify ammonia nitrogen, common materials mainly comprise zeolite powder, dolomite powder, coral sand and the like, but because the adsorption and purification capacity is limited (rich capacity 2016), the stress of harmful nitrogen such as ammonia nitrogen and the like on cultivated organisms is relieved in a water discharge mode in the cultivation production process. The chemical method purifies harmful substances in the water body by applying the strong oxidation effect of the chemical oxidant, but the problem of accumulation of potential pollution sources cannot be thoroughly eradicated during production and application, the chemical method usually needs to be used for many times to achieve a good effect, the use cost is relatively high, and potential safety risks of the water body ecological environment are easily caused if the chemical method is improperly operated (the forest rainbow 2006). The biological method utilizes specific microorganisms to absorb and convert harmful substances such as ammonia nitrogen, nitrite and the like in the water body, and has the advantages of environmental friendliness, difficulty in generating secondary pollution, strong sustainability and the like (2013 such as the royal mai). Plum blossom, lingling and the like (2008) introduce nitrobacteria with different concentrations into a water body for culturing the tilapia fries, and the results show that when the concentration of the initial nitrobacteria is 100CFU/L, the ammonia nitrogen content is reduced by 25.05 percent compared with a control group, the fry survival rate is improved by 7.58 percent compared with the control group, and the weight is increased by 46.15 percent; the results of the research on the Australian silverbass by the Beam champion and the like (2005) show that the accumulation of ammonia nitrogen and nitrite can be relieved and the culture environment can be improved by putting nitrobacteria in advance and supplementing the nitrobacteria to the water body at regular intervals of 7-10 days. Therefore, the nitrifying bacteria are scientifically applied in the aquaculture process, so that the problem of harmful nitrogen accumulation is solved, and good effects of purifying water quality and promoting healthy growth of cultured organisms are achieved. Autotrophic nitrifying bacteria have been reported mainly including nitrifying bacillus (Nitrobacter) (zhengjinlai et al 2003), nitrating spinifera (nitrospinona) (li jun wen et al 2004), nitrating coccus (Nitrococcus) (Bartosch, et al 1999), nitrating spira (Nitrospira) (Koops, et al 1990), Nitrosomonas (nitrosolomas) (Grunditz 2001), Nitrosospira (Nitrospira) (Shaw, et al 2006), nitrosophyllum (Nitrosolobus) (Webster 1996), Nitrosococcus (Nitrosococcus) (kloz, et al 2006), Nitrosovibrio (nitrosoucobio) (Ida, et al 2004). Wherein the autotrophic ammonia-oxidizing bacteria belong to Proteobacteria (Proteobacteria) subclass beta and subclass gamma; heterotrophic nitrifiers include bacteria, fungi, actinomycetes, etc. (Spiller, et al 1976), such as: arthrobacter globiformis (Arthrobacter globiformis) (2009, Elazan et al), Pseudomonas aeruginosa (Prunus aeruginosa) (2015, Liqian et al), Aspergillus parasiticus (Aspergillus parasiticus) (2006, Hawai et al). However, most researchers at home and abroad use activated sludge to enrich and culture nitrifying bacteria and rarely use pure bacteria expansion culture methods (Yangning 2003; Juan 2006) because nitrifying bacteria are difficult to separate and purify. The enrichment of nitrifying bacteria in Jinshigang et al (1998) by activated sludge shows that the concentration of nitrifying bacteria in sludge which is not enriched is 12.5-20 times higher than that in the nitrifying bacteria in the sludge which is not enriched after 1-13 weeks of enrichment culture when the temperature is 30 ℃, the pH value is 6.5-8.0 and the dissolved oxygen content is higher than 2.0 mg/L. Shan and Obbard (2001) achieved the desired effect of treating lobster culture wastewater with immobilized nitrifying bacteria. Liuling (2012) studies the freshwater nitrifying bacteria and the seawater nitrifying bacteria in an immobilized manner, and as a result, the ammonia nitrogen removal rate of the freshwater nitrifying bacteria is 0.12 mg/g.h, and the ammonia nitrogen removal rate of the seawater nitrifying bacteria is 0.13 mg/g.h.
The Rhodococcus can survive in polluted environment under natural conditions and can be used as inoculation medium for biological decontamination, and the bacteria of the Rhodococcus have transformation and degradation effects on many compounds and can be used as biosurfactant and bioflocculant. The cell surface active substances of the rhodococcus, such as mycolic acid, can reduce the surface tension of an interface, so that hydrophilic compounds can enter thallus cells more easily, and the invasion surface of thallus is increased; in addition, the Rhodococcus produces glycolipid flocculation substances formed by polypeptide and lipid aggregation, which can flocculate many suspended matters and help to remove the suspended matters in waste water or waste treatment (Hua Gou Gen 2003). The rhodococcus has large genome and linear plasmid, which is beneficial to contain a large amount of oxidase and other enzyme system substances, so that the rhodococcus can fully utilize energy and carbon sources from organic compounds and can also produce carotenoid (cumin 2016).
Although much interest is paid to microbial immobilization technology and many beneficial researches and attempts are made, most of the microbial immobilization technology still only stays in a small-scale and small-range application experiment stage at present, and the high-efficiency application effect of large-scale industrialization is not achieved. In order to effectively promote the safe and efficient application of related technologies in the aquaculture industry, it is necessary to screen and obtain indigenous strains with harmful nitrogen purification function from the aquaculture water environment, analyze the characteristics of the strains such as environmental adaptability, ecological functional efficiency, application safety and the like, and further develop microbial inoculum products and application technologies suitable for practical application in aquaculture production. But not simply apply relevant details in the sewage treatment engineering technology, neglecting the specific actual requirements of the cultured organisms and the production on safety, efficiency and sustainable development. No research report about related strains for purifying ammonia in aquaculture water by Rhodococcus rhodochrous (Rhodococcus rhodochrous) is found at present.
Disclosure of Invention
The invention aims to provide a Rhodococcus roseus strain XHRR1 for purifying ammonia in aquaculture water, wherein the strain XHRR1 has strong purification capability on ammonia in aquaculture water, has good environmental adaptability and has no adverse effect on aquaculture of prawns.
The invention also aims to provide the application of the Rhodococcus roseus strain XHRR1 in purifying ammonia in aquaculture water.
The first purpose of the invention is realized by the following technical scheme: a Rhodococcus roseus strain XHRR1 for purifying ammonia in aquaculture water, wherein the preservation name of the strain XHRR1 is Rhodococcus roseus XHRR1, and the classification name is as follows: rhodococcus rhodochrous XHRR1, deposited in China center for type culture Collection with the deposit addresses: wuhan, China, the preservation date is: 8, 3 months in 2017, the preservation number is: CCTCC NO: m2017437.
The screening, separating and identifying process of the rhodococcus roseus XHRR1 comprises the following steps: selecting a later-stage culture water body (for 50-75 days of culture) of the prawn intensive high-density culture pond, filtering a water sample by using a 0.22-micron mixed cellulose ester filter membrane, and placing the filter membrane with the gathered microorganism sample in a photosynthetic bacteria liquid culture medium for closed shake culture for 3-5 days at the temperature of 25-30 ℃ and the illumination intensity of 2500-4000 lx; and (3) performing streak culture on the cultured bacterial liquid on a photosynthetic bacteria solid plate culture medium for 3-5 days, selecting single colonies with different forms, and separating and purifying to obtain the strain with good growth performance. Then, the strain is inoculated to a photosynthetic bacteria liquid culture medium, the temperature is 28-30 ℃, the illumination intensity is 2500-4000 lx, and the shaking table is used for carrying out enlarged culture for 3-5 days at 150-200 rpm. Adding different bacteria solutions into the adjusted NH4Sterilized culture water of Cl concentration (as NH)4Cl is used for adjusting the concentration of ammonia in the water body to 10-30 mg/L), the temperature is 28-30 ℃, and light is emittedAnd performing shaking table enlarged culture for 3-5 days at the intensity of 2500-4000 lx and the rotation speed of 150-200 rpm. And selecting strains capable of effectively reducing the ammonia concentration of the water body for strain identification and seed preservation for later use.
Separating to obtain bacterial strain from water body in middle and later culture periods of intensive culture pond, pure culturing, and adding NH into bacterial liquid4High concentration ammonia (as NH) in Cl4Cl adjusting the concentration of ammonia in the water body to 10-30 mg/L), analyzing the influence of the strain on the purification effect of the ammonia in the water body, obtaining the strain capable of efficiently purifying the ammonia in the aquaculture water body, and determining the culture condition of the strain.
The Rhodococcus rhodochrous XHRR1 can normally grow at the temperature of 15-35 ℃, the salinity of 5-45 and the pH of 6-9, and the optimal conditions are that the temperature is 15-35 ℃, the salinity of 25-45 and the pH is 7.0-8.5. The growth can reach the peak value about 2 days under the optimal condition and in the nutrient environment of the culture water body, and the growth is maintained at 10 days in 2-5 days8Number level of CFU/mL. The strain is suitable for most ponds.
The second purpose of the invention is realized by the following technical scheme: the Rhodococcus roseus strain XHRR1 is applied to purifying ammonia in aquaculture water.
The Rhodococcus roseus XHRR1 can reach a peak value after growing for about 2 days in the nutrient environment of aquaculture water, and is maintained at 10 within 2-5 days8Number level of CFU/mL. The strain is suitable for most ponds. The strain is placed in a culture water body containing high ammonia nitrogen, when the salinity of the water body is 5-45, the temperature is 30 ℃, and the removal rate of ammonia in the water body of a bacterium adding group can reach 79-99 percent in 3 days; when the salinity reaches 25-45, the removal rate of ammonia in the water body is more than 90%. When the water temperature is 15-35 ℃ and the salinity is 25, the removal rate of ammonia in the water body of the bacterium adding group can reach more than 90 percent in 3 days; when the water temperature is lower than 15 ℃ or higher than 40 ℃, the ammonia removal effect of the rhodococcus roseus XHRR1 is obviously influenced.
Moreover, when the strain is applied to an experimental aquaculture water body at a concentration of 107CFU/mL, the 7-day survival rate of the litopenaeus vannamei can reach over 90 percent by testing, which shows that the strain has no obvious adverse effect on aquaculture organisms.
The environment of the aquaculture pond is greatly different from the water environment in the background technology, so that the indigenous strains with the nitrification function are screened and obtained on the basis of the aquaculture water environment, and the characteristics of environmental adaptability, ecological function efficiency, application safety and the like of the strains are analyzed and evaluated, so that microbial inoculum products and application technology suitable for practical application of aquaculture production are developed. But not simply apply relevant details in the sewage treatment engineering technology, neglecting the specific actual requirements of the cultured organisms and the production on safety, efficiency and sustainable development. Therefore, the separated and screened rhodococcus roseus XHRR1 has an obvious effect of removing ammonia in the aquaculture water body, and litopenaeus vannamei and weever have no obvious adverse effect. Provides theoretical and technical support for further research and development of nitrobacteria microbial inoculum suitable for practical application requirements of aquaculture and matched technology.
Compared with the prior art, the invention has the following advantages:
(1) the Rhodococcus roseus XHRR1 has an obvious effect of removing ammonia in an intensive aquaculture water body, and has no adverse effect on aquaculture organisms;
(2) the Rhodococcus rhodochrous XHRR1 in the invention is used for screening water bodies in the middle stage of intensive culture, has good environmental adaptability, and is suitable for application in most culture pond water bodies;
(3) the Rhodococcus rhodochrous XHRR1 can achieve good application effect when being applied to water quality purification regulation and control of intensive culture, is beneficial to greatly reducing water body replacement in the culture production process, can be used without configuring expensive water quality purification equipment, and can provide technical reserve and support for further research and development or implementation of culture water environment directional regulation and control technology and promotion of development of aquatic high-efficiency healthy culture industry for realizing ecological environmental protection.
Drawings
FIG. 1 is a growth curve of the strain Rhodococcus roseus XHRR1 in example 3;
FIG. 2 is a graph showing the variation of the strain Rhodococcus roseus XHRR1 in example 3 in purifying ammonia in water at different salinity;
FIG. 3 is a graph showing the variation of ammonia in purified water at different temperatures according to the strain Rhodococcus roseus XHRR1 in example 3;
FIG. 4 is the effect of the strain Rhodococcus roseus XHRR1 on Litopenaeus vannamei in example 4.
Detailed Description
The invention is further illustrated, but not limited in any way, by the following examples in connection with the accompanying drawings.
Example 1 screening and culture of Rhodococcus roseus XHRR1 for purifying Ammonia in aquaculture Water
1. Material preparation
1.1, sources of bacteria
Collecting water samples for 50-75 days of culture in an intensive prawn culture pond, and performing isolated culture by using a photosynthetic bacteria culture medium plate.
1.2 culture Medium
(1) Photosynthetic bacteria liquid culture medium: CH (CH)3COONa: 1g, yeast extract: 1g, MgSO4·7H2O：0.4g、NaCl：0.1g、CaCl2·2H2O：0.05g、NaHCO3：0.3g、KH2PO4: 1g of trace element solution and 1mL of trace element solution, and the above medicines are respectively dissolved in distilled water, and the volume is determined to be 1000mL, and the pH value is 7.0.
Solution of trace elements: EDTA: 2.5g, ZnSO4·7H2O：10.95g、MnSO4·H2O：1.54g、CuS04·5H2O：0.39g、CoCl2·6H2O：0.2g、FeSO4·7H2O: 7g of the above medicines are respectively dissolved in distilled water, and the volume is up to 1000m1, pH 7.0.
(2) Photosynthetic bacteria solid plate culture medium: on the basis of a photosynthetic bacteria liquid culture medium, 20g/L agar powder is added to prepare a solid plate culture medium.
2. Screening culture of strains
Selecting a later-stage culture water body (for 50-75 days of culture) of the prawn intensive high-density culture pond, filtering a water sample by using a 0.22-micron mixed cellulose ester filter membrane, and placing the filter membrane with the gathered microorganism sample in a photosynthetic bacteria liquid culture medium for closed shake culture for 3-5 days at the temperature of 25-30 ℃ and the illumination intensity of 2500-4000 lx; putting the cultured bacterial liquid on a photosynthetic bacteria solid plateAnd (3) carrying out streak culture on the culture medium for 3-5 days, selecting single colonies with different forms, and separating and purifying to obtain the strain with good growth performance. Then, the strain is inoculated to a photosynthetic bacteria liquid culture medium, the temperature is 28-30 ℃, the illumination intensity is 2500-4000 lx, and the shaking table is used for carrying out enlarged culture for 3-5 days at 150-200 rpm. Adding different bacteria solutions into the adjusted NH4Sterilized culture water of Cl concentration (as NH)4And adjusting the concentration of ammonia in the water body to 10-30 mg/L by Cl), performing shaking table enlarged culture for 3-5 days at the illumination intensity of 2500-4000 lx and the rotation speed of 150-200 rpm at the temperature of 28-30 ℃. And selecting a strain capable of effectively reducing the ammonia concentration of the water body for strain identification.
Separating to obtain bacterial strain from water body in middle and later culture periods of intensive culture pond, pure culturing, and adding NH into bacterial liquid4Analyzing the influence of the strain on the purification effect of the ammonia in the water body in the culture water body containing high-concentration ammonia prepared by Cl to obtain the strain capable of efficiently purifying the ammonia in the culture water body, and determining the culture condition of the strain.
Example 2 identification of Rhodococcus roseus XHRR1 for ammonia purification in aquaculture waters
The invention identifies 16S rDNA molecules of rhodococcus roseus XHRR1 for purifying ammonia in the culture water body, and determines the species of the strain from the molecular level and by combining the analysis of the morphological characteristics and physiological and biochemical characteristics of bacteria. The 16S rDNA sequence analysis 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) taking 1.5mL of bacteria culture solution, centrifuging at 10000rpm (11,500g) for 1 minute, 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 μ L buffer solution GB, shaking for 15 s, standing at 70 deg.C for 10 min, cleaning the solution, and centrifuging briefly to remove water droplets 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 (the adsorption column is placed into a collecting pipe), centrifuging at 12000rpm (13,400 Xg) for 30 s, pouring off 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 (13,400g) 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 (13,400g) for 30 s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;
(10) adding 500 μ L of rinsing liquid PW into adsorption column CB3, centrifuging at 12000rpm (13,400g) 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 (13,400g) 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, suspending and dripping 50-200 mu L of elution buffer TE into the middle part of the adsorption film, standing at room temperature for 2-5 minutes, centrifuging at 12000rpm (13,400g) for 2 minutes, and collecting the solution into the centrifugal tube;
(13) DNA concentration and purity measurement
The recovered DNA fragment is subjected to agarose gel electrophoresis and ultraviolet spectrophotometer to detect the concentration and purity.
2. PCR amplification of 16S rDNA Gene
The universal bacterial primers used for the 16S rDNA amplification were synthesized by England Weiji (Shanghai) trade company Limited, and the forward primers (8f) were: 5'-AGAGTTTGATCCTGGCTCAG-3', respectively; the reverse primer (1492r) is: 5'-GGTTACCTTGTTACGACTT-3' are provided. The 50 μ L PCR reaction included: sterilized double distilled water (37. mu.L), primers (1. mu.L each), dNTPs (2.5mmol/L), Tapase (1. mu.L), 10 XPCR buffer (5. mu.L), and DNA template (1. mu.L). And (3) PCR reaction 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 the Weijie Weiji (Shanghai) trade Co., Ltd. The sequence was determined as follows:
1 acggctccct cccacaaggg gttaggccac cggcttcggg tgttaccgac tttcatgacg
61 tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg cagcgttgct gatctgcgat
121 tactagcgac tccgacttca cggggtcgag ttgcagaccc cgatccgaac tgagaccggc
181 tttaagggat tcgctccacc tcacggtatc gcagccctct gtaccgacca ttgtagcatg
241 tgtgaagccc tggacataag gggcatgatg acttgacgtc gtccccacct tcctccgagt
301 tgaccccggc agtctcctgc gagtccccac catcacgtgc tggcaacaca ggacaagggt
361 tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac agccatgcac
421 cacctgtcta ccggccacaa gggaaaccac atctctgcag tcgtccggta catgtcaaac
481 ccaggtaagg ttcttcgcgt tgcatcgaat taatccacat gctccgccgc ttgtgcgggc
541 ccccgtcaat tcctttgagt tttagccttg cggccgtact ccccaggcgg ggcgcttaat
601 gcgttggcta cggcacggat cccgtggaag gaaacccaca cctagcgccc accgtttacg
661 gcgtggacta ccagggtatc taatcctgtt cgctacccac gctttcgctc ctcagcgtca
721 gttactgccc agagacccgc cttcgccacc ggtgttcctc ctgatatctg cgcatttcac
781 cgctacacca ggaattccag tctcccctgc agtactcgag tctgcccgta tcgcctgcaa
841 gcccgcagtt gagctgcggg atttcacaga cgacgcgaca aaccgcctac gagctcttta
901 cgcccagtaa ttccggacaa cgctcgcacc ctacgtatta ccgcggctgc tggcacgtag
961 ttggccggtg cttcttctcc cactaccgtc acttgcgctt cgtcatgggt gaaagaggtt
1021 tacaacccga aggccgtcat ccctcacgcg gcgtcgctgc atcaggcttg cgcccattgt
1081 gcaatattcc ccactgctgc ctcccgtagg agtctgggcc gtgtctcagt cccagtgtgg
1141 ccggtcgccc tctcaggccg gctacccgtc gtcgccttgg taggccatta ccccaccaac
1201 aagctgatag gccgcgggct catcctgcac cgaaaaactt tccaccccag aacatgcatc
1261 ccgaggtcat atccggtatt agacccagtt tcccaggctt atcccagagt gcagggcaga
1321 tcacccacgt gttactcacc cgttcgccac taatccaccc agcaagctgg gcttcatcgt
1381 tcgactgcat
4. the colony morphology of the rhodococcus roseus XHRR1 and the physiological characteristics of the rhodococcus roseus XHRR1 for purifying the ammonia in the aquaculture water body are shown in the following table 1.
TABLE 1 Rhodococcus roseus XHRR1 colony morphology, physiological characteristics
5. Identification of Rhodococcus roseus XHRR1
The 16S rDNA gene sequence of the strain is compared with the registered gene sequence in GenBank for analysis, and the Biolog MicroState TM System bacteria identification System is used for identifying the biochemical characteristics of the strain respectively, and the result shows that the strain is Rhodococcus rhodochrous (Rhodococcus rhodochrous). The results of 16S rDNA gene sequence analysis, biochemical identification, morphological characteristics and the like are integrated. The strain XHRR1 was identified as Rhodococcus rhodochrous (Rhodococcus rhodochrous). No research report on purification of ammonia in shrimp pond water by Rhodococcus rhodochrous (Rhodococcus rhodochrous) is available after referring to relevant data.
The collection name of the strain XHRR1 is Rhodococcus roseus XHRR1, and the classification name is: rhodococcus rhodochrous XHRR1, deposited in China center for type culture Collection with the deposit addresses: wuhan, China, the preservation date is: 8, 3 months in 2017, the preservation number is: CCTCC NO: m2017437.
Example 3 Small-Scale application of Rhodococcus roseus XHRR1 for purifying Ammonia in aquaculture Water
1. Growth of the Strain
The strain Rhodococcus roseus XHRR1 obtained in example 1 was pressed at 106Inoculating CFU/mL into water in a sterilized aquaculture pond, wherein the bacteria concentration is stabilized at 10 within 2-5 days8The growth curve of strain XHRR1 at CFU/mL number level is shown in FIG. 1.
2. Removing effect of bacterial strain on ammonia in aquaculture water with different salinity
Testing water body with sterilized culture pond water (water body salinity of 25) as basis, and taking NH as reference4And regulating the concentration of ammonia in the water body to be about 24mg/L by Cl, and regulating the salinity of the water body to be 5, 15, 25, 35 and 45 by using distilled water and sea salt. The strain Rhodococcus roseus XHRR1 obtained in example 1 was pressed at 106And (3) inoculating the CFU/mL into an experimental water body, and performing shake culture at 30 ℃, the illumination intensity of 2500-4000 lx and the rotation speed of 150-200 rpm for 3 days. The ammonia concentration in the water was monitored daily. The result is shown in figure 2, when the salinity of the water body is 5-45, the removal rate of ammonia in the water body with the bacterium group can reach 79-99% in 3 days; when the salinity reaches 25-45, the removal rate of ammonia in the water body is more than 90%, and the ammonia concentration of a control group (taking a test culture system with the salinity of 25 as a reference and not adding the Rhodococcus XHRR1) is not greatly changed in the whole monitoring process. Therefore, the XHRR1 bacteria have good salinity adaptability, can be suitable for purifying ammonia in aquaculture water bodies with different salinity, and has better effect compared with the water body with the salinity of more than 25.
3. Removing effect of ammonia in culture water body of bacterial strain at different temperatures
Testing water body with sterilized culture pond water (water body salinity of 25) as basis, and taking NH as reference4And regulating the concentration of ammonia in the water body to be about 24mg/L by using Cl. The strain Rhodococcus roseus XHRR1 obtained in example 1 was pressed at 106And inoculating the CFU/mL into an experimental water body, setting the culture temperature to be 5 ℃, 15 ℃, 25 ℃, 30 ℃, 35 ℃ and 45 ℃ respectively, and performing shake culture for 3 days under the conditions of illumination intensity of 2500-4000 lx and 150-200 rpm. The ammonia concentration in the water was monitored daily. As shown in FIG. 3, the results were obtained when the water temperature was 15 to 35 ℃The removal rate of ammonia in the water body of the bacterium adding group can reach more than 90 percent in 3 days; the ammonia removal effect of Rhodococcus roseus XHRR1 was significantly affected when the water temperature was below 15 ℃ or above 40 ℃ and the ammonia concentration of the control group (reference test culture at 30 ℃ without Rhodococcus added XHRR1) did not change much throughout the monitoring. In general, the XHRR1 strain can adapt to the temperature condition of a culture water body during culture production, has good temperature adaptability, and can effectively reduce the concentration of ammonia in the water body when being scientifically applied to a water body regulation and control technical link, thereby achieving the effect of optimizing culture water quality.
Example 4 Effect of the Strain XHRR1 on Litopenaeus vannamei
The influence of the strains on litopenaeus vannamei is tested in Shanshen and Shenzhen breeding bases. The salinity of the aquaculture water body is 25-28, the pH value is 7.5-8.1, the dissolved oxygen is 6.0-6.7 mg/L, the temperature is 28-32 ℃, and the volume of the water body is 200L. Experimental group is 107The bacteria were applied at CFU/mL concentration, and no bacteria were added to the control group, 3 replicates per group. The initial body length of the Litopenaeus vannamei is tested to be 6.7 +/-0.5 cm, and the body weight is tested to be 2.8 +/-0.2 g. The test period was 7 days. The results are shown in fig. 4, and the survival rates of the prawns in the bacterium-adding group and the control group at the end of the test are 92.7% and 93.3%, respectively. It can be seen that 10 is added in the aquaculture water body7When the strain is applied at the concentration of CFU/mL, the strain has no obvious adverse effect on cultured organisms.
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. In fact, a person skilled in the art, on the basis of the preceding description, will be able to find different modifications according to his own needs, which modifications are intended to be within the scope of the claims appended hereto.
Sequence listing
<110> research institute for aquatic products in south China sea
Chaozhou city honest agriculture science and technology Limited
<120> Rhodococcus roseus strain XHRR1 for purifying ammonia in aquaculture water and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1390
<212> DNA
<213> Rhodococcus roseus strain XHRR1(Rhodococcus rhodochrous XHRR1)
<400> 1
acggctccct cccacaaggg gttaggccac cggcttcggg tgttaccgac tttcatgacg 60
tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg cagcgttgct gatctgcgat 120
tactagcgac tccgacttca cggggtcgag ttgcagaccc cgatccgaac tgagaccggc 180
tttaagggat tcgctccacc tcacggtatc gcagccctct gtaccgacca ttgtagcatg 240
tgtgaagccc tggacataag gggcatgatg acttgacgtc gtccccacct tcctccgagt 300
tgaccccggc agtctcctgc gagtccccac catcacgtgc tggcaacaca ggacaagggt 360
tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac agccatgcac 420
cacctgtcta ccggccacaa gggaaaccac atctctgcag tcgtccggta catgtcaaac 480
ccaggtaagg ttcttcgcgt tgcatcgaat taatccacat gctccgccgc ttgtgcgggc 540
ccccgtcaat tcctttgagt tttagccttg cggccgtact ccccaggcgg ggcgcttaat 600
gcgttggcta cggcacggat cccgtggaag gaaacccaca cctagcgccc accgtttacg 660
gcgtggacta ccagggtatc taatcctgtt cgctacccac gctttcgctc ctcagcgtca 720
gttactgccc agagacccgc cttcgccacc ggtgttcctc ctgatatctg cgcatttcac 780
cgctacacca ggaattccag tctcccctgc agtactcgag tctgcccgta tcgcctgcaa 840
gcccgcagtt gagctgcggg atttcacaga cgacgcgaca aaccgcctac gagctcttta 900
cgcccagtaa ttccggacaa cgctcgcacc ctacgtatta ccgcggctgc tggcacgtag 960
ttggccggtg cttcttctcc cactaccgtc acttgcgctt cgtcatgggt gaaagaggtt 1020
tacaacccga aggccgtcat ccctcacgcg gcgtcgctgc atcaggcttg cgcccattgt 1080
gcaatattcc ccactgctgc ctcccgtagg agtctgggcc gtgtctcagt cccagtgtgg 1140
ccggtcgccc tctcaggccg gctacccgtc gtcgccttgg taggccatta ccccaccaac 1200
aagctgatag gccgcgggct catcctgcac cgaaaaactt tccaccccag aacatgcatc 1260
ccgaggtcat atccggtatt agacccagtt tcccaggctt atcccagagt gcagggcaga 1320
tcacccacgt gttactcacc cgttcgccac taatccaccc agcaagctgg gcttcatcgt 1380
tcgactgcat 1390
Claims (2)
1. Rhodococcus roseus for purifying ammonia in aquaculture waterRhodococcusrhodochrous) Strain XHRR1, characterized by: the collection name of the strain XHRR1 is: rhodococcus roseus XHRR1, deposited in units: china center for type culture Collection, the collection addresses are: wuhan, China, the preservation date is: 8, 3 months in 2017, the preservation number is: CCTCC NO: m2017437.
2. Rhodococcus roseus (R) according to claim 1Rhodococcusrhodochrous) The application of the strain XHRR1 in purifying ammonia in aquaculture water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711026615.1A CN107828679B (en) | 2017-10-27 | 2017-10-27 | Rhodococcus roseus strain XHRR1 for purifying ammonia in aquaculture water and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711026615.1A CN107828679B (en) | 2017-10-27 | 2017-10-27 | Rhodococcus roseus strain XHRR1 for purifying ammonia in aquaculture water and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107828679A CN107828679A (en) | 2018-03-23 |
| CN107828679B true CN107828679B (en) | 2021-04-27 |
Family
ID=61650874
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711026615.1A Active CN107828679B (en) | 2017-10-27 | 2017-10-27 | Rhodococcus roseus strain XHRR1 for purifying ammonia in aquaculture water and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107828679B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111471611B (en) * | 2020-03-10 | 2022-09-27 | 中国水产科学研究院南海水产研究所深圳试验基地 | Rhodococcus ruber HDRR1 for purifying inorganic nitrogen and phosphorus in tail water of seawater pond culture and application thereof |
| CN111471612B (en) * | 2020-03-10 | 2022-09-30 | 中国水产科学研究院南海水产研究所深圳试验基地 | Rhodococcus ruber HDRR2Y for purifying inorganic nitrogen and phosphorus in seawater pond culture tail water and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001054380A (en) * | 1999-08-13 | 2001-02-27 | Daicel Chem Ind Ltd | New nitrilase and production and 2-hydroxy-4- methylthiobutylic acid |
| CN103667108A (en) * | 2013-10-17 | 2014-03-26 | 浙江省环境保护科学设计研究院 | Rhodococcus corynebacterioides strain and application thereof in printing and dyeing wastewater treatment |
| CN104140935A (en) * | 2013-05-08 | 2014-11-12 | 中国科学院生态环境研究中心 | Denitrification rhodococcus and rhodococcus microbial agent production method and application |
| CN104628225A (en) * | 2015-01-09 | 2015-05-20 | 张玉兰 | Treatment method of ammonia-nitrogen-containing industrial wastewater |
| CN104673710A (en) * | 2014-12-31 | 2015-06-03 | 浙江至美环境科技有限公司 | Rhodococcus sp. strain and application thereof |
| CN104694447A (en) * | 2015-04-06 | 2015-06-10 | 朱凌玮 | Complex microbial inoculum for treating sewage and application method thereof |
| CN106635927A (en) * | 2017-02-10 | 2017-05-10 | 哈尔滨明慧生物技术开发有限公司 | Composite fungicide for treating sludge and sludge treatment method |
-
2017
- 2017-10-27 CN CN201711026615.1A patent/CN107828679B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001054380A (en) * | 1999-08-13 | 2001-02-27 | Daicel Chem Ind Ltd | New nitrilase and production and 2-hydroxy-4- methylthiobutylic acid |
| CN104140935A (en) * | 2013-05-08 | 2014-11-12 | 中国科学院生态环境研究中心 | Denitrification rhodococcus and rhodococcus microbial agent production method and application |
| CN103667108A (en) * | 2013-10-17 | 2014-03-26 | 浙江省环境保护科学设计研究院 | Rhodococcus corynebacterioides strain and application thereof in printing and dyeing wastewater treatment |
| CN104673710A (en) * | 2014-12-31 | 2015-06-03 | 浙江至美环境科技有限公司 | Rhodococcus sp. strain and application thereof |
| CN104628225A (en) * | 2015-01-09 | 2015-05-20 | 张玉兰 | Treatment method of ammonia-nitrogen-containing industrial wastewater |
| CN104694447A (en) * | 2015-04-06 | 2015-06-10 | 朱凌玮 | Complex microbial inoculum for treating sewage and application method thereof |
| CN106635927A (en) * | 2017-02-10 | 2017-05-10 | 哈尔滨明慧生物技术开发有限公司 | Composite fungicide for treating sludge and sludge treatment method |
Non-Patent Citations (4)
| Title |
|---|
| High Cell Density Culture of Rhodococcus rhodochrous by pH-Stat Feeding and Dibenzothiophene degradation;Hiroyuki Honda et al;《Journal of Fermentaion and Bioengineering》;19981231;第85卷(第3期);第334-338页 摘要，材料与方法，结果和讨论部分 * |
| 基于PCR-DGGE 技术辅助筛选氨氮降解菌株;张智超 等;《浙江农业学报》;20170215;第29卷(第2期);第286-290页 摘要，材料与方法，结果与分析，讨论部分 * |
| 微生物腈水合酶的发酵及性质研究;曾亮;《中国优秀博硕士学位论文全文数据库 (硕士) 基础科学辑》;20050315;A006-153 * |
| 氯化铵对/黄海1号0中国对虾免疫相关酶类的影响;哈承旭 等;《渔业科学进展》;20090228;第30卷(第1期);第34-40页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107828679A (en) | 2018-03-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9102552B2 (en) | Production of cyanobacterial or algal biomass using chitin as a nitrogen source | |
| CN110656066B (en) | Acinetobacter strain for shortcut nitrification and denitrification and application thereof | |
| CN104845920A (en) | Marine zobellella sp. and application thereof | |
| CN112551692B (en) | Halomonas with aerobic denitrification and heterotrophic sulfur oxidation functions and application thereof | |
| CN108865940B (en) | Heterotrophic nitrification-aerobic denitrification bacillus and composite bacterial preparation thereof | |
| CN115386520B (en) | Rhodococcus pyridine-philic RL-GZ01 strain and application thereof | |
| CN107723264B (en) | Seawater nitrate reducing bacteria strain XHNA1 for purifying inorganic nitrogen in aquaculture water and application thereof | |
| CN107828679B (en) | Rhodococcus roseus strain XHRR1 for purifying ammonia in aquaculture water and application thereof | |
| CN111471611B (en) | Rhodococcus ruber HDRR1 for purifying inorganic nitrogen and phosphorus in tail water of seawater pond culture and application thereof | |
| US20150329397A1 (en) | Process of Treating Buchu Mercaptan Production Wastewater Using Microalgae and Chitin as a Nitrogen Source | |
| CN114717149B (en) | South-sea deep-sea fish-source heterologous alkane-eating bacterium AXMZ1 and application thereof | |
| CN109280631B (en) | Sulfadimidine degrading bacterium S-2 and application thereof | |
| CN111471612B (en) | Rhodococcus ruber HDRR2Y for purifying inorganic nitrogen and phosphorus in seawater pond culture tail water and application thereof | |
| JP4707251B2 (en) | Activated sludge and wastewater treatment method | |
| Banerjee et al. | Immobilized periphytic cyanobacteria for removal of nitrogenous compounds and phosphorus from shrimp farm wastewater | |
| CN114292798B (en) | Anaerobic denitrifying strain and application thereof in riverway water body remediation | |
| KR101007843B1 (en) | Root surface colonial bacterium, massilia sp. dh-1(kacc91486p), which be highly capable of degrading nitrogen and phosphorus in the polluted water source, its culturing method, fixing method to aquatic plant and their utilizing method in the water | |
| CN108102946B (en) | Cyrtomium oxysulfide strain XHCT1 for purifying ammonia in aquaculture water and application thereof | |
| CN110407338B (en) | Low-temperature denitrification and dephosphorization bacteria and application thereof | |
| CN111378603B (en) | Paracoccus angularis LFPH1 for purifying inorganic nitrogen and phosphorus in seawater pond culture water body and application thereof | |
| Navrátilová et al. | Characterization of Rhodococcus wratislaviensis strain J3 that degrades 4-nitrocatechol and other nitroaromatic compounds | |
| Suman et al. | Simultaneous biomass production and mixed-origin wastewater treatment by five environmental isolates of Cyanobacteria | |
| CN116904349B (en) | Adhesive sword bacteria with aerobic denitrification capability and application thereof | |
| CN113322199B (en) | Meiji reef lagoon fish source hydrocarbon-removing marinobacterium MHMJ1 and application thereof | |
| CN107674846A (en) | One plant of Rhodopseudomonas palustris RP1 and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220222 Address after: 510300 No. 231 West Xingang Road, Guangzhou, Guangdong, Haizhuqu District Patentee after: SOUTH CHINA SEA FISHERIES Research Institute CHINESE ACADEMY OF FISHERY SCIENCES Patentee after: Guangzhou Xinhai Lisheng Biotechnology Co., Ltd Address before: 510300 No. 231 West Xingang Road, Guangzhou, Guangdong, Haizhuqu District Patentee before: SOUTH CHINA SEA FISHERIES Research Institute CHINESE ACADEMY OF FISHERY SCIENCES Patentee before: Chaozhou Zhengcheng Agricultural Technology Co., Ltd |