CN112322510A - Rhodotorula mucilaginosa SYM-1 and screening method and application thereof - Google Patents
Rhodotorula mucilaginosa SYM-1 and screening method and application thereof Download PDFInfo
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Abstract
The invention discloses a rhodotorula mucilaginosa (A)Rhodotorula mucilaginosa) SYM-1 with the preservation number of CCTCC NO: M2020756. The invention also discloses rhodotorula mucilaginosa (A)Rhodotorula mucilaginosa) SYM-1 screening method and use thereof. Rhodotorula mucilaginosaBacteria (A), (B)Rhodotorula mucilaginosa) The SYM-1 strain can be used for biological prevention and treatment of aquaculture diseases or treatment of aquaculture water pollution, the SYM-1 strain has a strong bacteriostatic action on vibrio vulnificus, vibrio parahaemolyticus and vibrio anguillarum, and has a good degradation action on nitrite and ammonia nitrogen, the degradation rate is about 65% when the concentration of nitrite nitrogen is 50mg/L, and the degradation rate is about 97% when the concentration of ammonia nitrogen is 50mg/L, so that the SYM-1 strain has good development and application potentials.
Description
Technical Field
The present invention relates to a new strain, in particular, it relates to a rhodotorula mucilaginosa (A)Rhodotorula mucilaginosa) SYM-1; the invention also relates to a screening method and application of the strain SYM-1.
Background
The vibrio is one of the main pathogenic bacteria causing bacterial diseases in aquaculture, and the diseases caused by the vibrio have wide epidemic range and high morbidity, thereby causing great economic damage to the aquaculture industry. With the rapid development of economic globalization and foreign trade in China, the propagation speed of aquaculture diseases is accelerated due to frequent trading and circulation of aquatic products, and the infection of pathogenic vibrio diseases becomes a main cause of death of cultured fishes, shrimps and shellfishes. The prevention and treatment of bacterial diseases in aquaculture currently relies mainly on antibiotics. Although antibiotics have the advantages of quick response and good curative effect, the problems of water environment damage, drug resistance of cultured organisms and the like can be caused by blind use or excessive use. At present, biological control methods for controlling aquaculture diseases have attracted much attention.
With the prevalence of intensive culture modes with high density and high bait casting, organic matters, animal wastes and residual baits in water are accumulated continuously, so that the content of ammonia nitrogen is gradually beyond the bearing capacity of a water body, in addition, exogenous ammonia nitrogen entering the water body in the modes of sewage discharge, surface runoff, atmospheric sedimentation, pesticide residue and the like is increased day by day, the content of nitrite and ammonia nitrogen in the water body is too high and cannot be degraded, the problems of water body oxygen deficiency and the like can be caused, the physiological function of aquatic animals is disordered and the growth is influenced by light people, and the animals cannot breathe and die in serious cases. At present, pollution of nitrite and ammonia nitrogen in aquaculture water is mainly controlled by three methods, namely a physical method, a chemical method and a biological method, the physical method has complex treatment steps and higher cost, and is only suitable for degradation of low-concentration nitrite and ammonia nitrogen (Zhangetc. 2020); the chemical method for degrading nitrite and ammonia nitrogen needs to be assisted by related chemical substances, the required reaction time is long, and the problems of secondary pollution and the like (Voji Shuai and the like, 2020); however, the method of repairing the aquaculture water body by using the microorganism is increasingly emphasized because of the advantages of no toxic and side effects, short action time, no pollution and the like (Shida forest, etc., 2009).
Therefore, it is of great significance to research and obtain excellent bacterial strains for biological prevention and control of aquaculture diseases and treatment of aquaculture water pollution.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a new Rhodotorula mucilaginosa (A) against the deficiency of the prior artRhodotorula mucilaginosa) The SYM-1 strain has wide application range and good effect.
The technical problem to be solved by the present invention is achieved by the following technical means. The present invention relates to a kind of rhodotorula mucilaginosaRhodotorula mucilaginosa) SYM-1, characterized by: the preservation number is CCTCC NO: M2020756.
Rhodotorula mucilaginosa (A) related to the inventionRhodotorula mucilaginosa) SYM-1 strain, which has been deposited in China Center for Typical Culture Collection (CCTCC) at 11/18/2020, with a collection number of: CCTCC NO: M2020756. Telephone: 027) 87682319 fax (027) 87883833E-mail: cctcc @ whu. edu. cn address: wuhan, Wuhan university; and E, postcode: 430072.
the invention also discloses the rhodotorula mucilaginosa (A) as described aboveRhodotorula mucilaginosa) The screening method of SYM-1 is characterized by comprising the following steps:
(1) separation and purification of yeast
Collecting samples, respectively taking 5 mL of samples in 45 mLYEPD liquid culture medium, performing enrichment culture at 28 deg.C and 180 r/min for 48h, mixing the liquidAfter turbidity, the mixture was diluted with a gradient of 10 each-4、10-5、10-6、10-7Coating 4 diluted concentration 100 muL on a YEPD flat plate with the diameter of 9cm, culturing at the constant temperature of 28 ℃ for 48h, selecting yeast colonies on a PDA culture medium according to colony morphology characteristics, streaking three areas, separating and purifying to obtain single colonies, and inoculating the separated 19 yeast strains on a PDA inclined plane for storage;
(2) preparation of sterile yeast fermentation liquor
The yeast strain is activated on a PDA slant for 48 hours, the yeast cultured on a two-ring slant seed culture medium is inoculated into a 250 mL triangular flask containing 50 mL liquid YEPD culture medium, and shaking culture is carried out at 180 r/min for 24 hours to serve as seed liquid. Inoculating the seed solution into a triangular flask containing 60 mL YEPD liquid culture medium according to the inoculation amount of 10%, performing shaking culture at 28 deg.C and 180 r/min for 3d, centrifuging the fermentation liquid at 4 deg.C and 5000 r/min for 20 min, and filtering the supernatant with 0.22 μm bacterial filter to obtain sterile fermentation liquid;
(3) determination of bacteriostatic action
Measuring the diameter of the bacteriostatic circle by adopting an oxford cup method; in 19 separated yeast strains, HS-1, HN-1, SYM-1, HS-2 and BCH-1 have inhibition effect on vibrio vulnificus; the width of the bacteriostatic strip is 4.3 mm; RDN-1, HN-1, SYM-2, HS-2 and BCH-1 have inhibition effect on vibrio parahaemolyticus, wherein the widths of the bacteriostatic bands of the HS-2 strain and the SYM-1 strain are respectively 7.2 mm and 6.5 mm; HN-1, SYM-1, HS-2 and BCH-1 have inhibition effect on vibrio anguillarum;
selecting 7 strains of marine yeast sterile fermentation liquor with strong inhibition effect, determining the inhibition effect of the sterile fermentation liquor on vibrio vulnificus, vibrio anguillarum and vibrio parahaemolyticus, wherein the fermentation liquor of SYM-1, HN-1, HS-2 and BCH-14 strains has obvious inhibition effect on 3 kinds of vibrios, and the width of the inhibition zone of SYM-1 on the three kinds of vibrios is more than 2.5 mm;
(4) determination of ammonia nitrogen degradation rate
Activating and culturing the screened yeast strain with strong vibrio inhibiting effect on PDA slant for 2-3 d, washing off thallus Porphyrae with ammonia nitrogen degradation culture medium, and making into 5 × 108 CFU/mL bacterial suspension is inoculated into the container according to the inoculation amount of 10 percentInoculating 3 bottles of each strain in a 250 mL triangular flask of 60 mL ammonia nitrogen degradation culture medium for 3 times of repetition, and performing shake culture for 48 hours at 28 ℃ under the condition of 180 r/min; centrifuging the fermentation liquid at 4 deg.C and 5000 r/min for 20 min, collecting supernatant, and measuring OD of supernatant of different strains by indophenol blue spectrophotometry637Calculating the ammonia nitrogen concentration in the fermentation liquor according to a standard curve, and calculating the ammonia nitrogen degradation rate of different strains by taking an uninoculated culture medium as a reference; screening to obtain a strain SYM-1 with the degradation rate reaching 96.85% when the ammonia nitrogen concentration is 50 mg/L;
(5) determination of nitrite nitrogen degradation effect
Activating and culturing the screened yeast strain with strong vibrio inhibiting effect on PDA slant for 2-3 days, and washing off thallus Porphyrae with nitrite degradation culture medium to obtain 5 × 108The CFU/mL bacterial suspension is inoculated into a 250 mL triangular flask filled with 60 mL liquid nitrite degradation medium according to the inoculation amount of 10 percent, each bacterial strain is inoculated into 3 bottles for 3 times of repetition, and the shake culture is carried out for 48 hours under the conditions of 28 ℃ and 180 r/min. Centrifuging the fermentation liquid at 4 deg.C and 5000 r/min for 20 min, sampling supernatant 100 μ L, adding 6 mL Griess reagent, reacting at room temperature for 20 min, and measuring absorbance at 538 nm; calculating the nitrite nitrogen concentration in the fermentation liquor of different strains according to a standard curve, and calculating the nitrite nitrogen degradation rate of different strains by taking an uninoculated culture medium as a reference; screening to obtain a strain SYM-1 with the degradation rate of 64.66% when the concentration of nitrite nitrogen is 50 mg/L;
(6) identification of the species of the strains
Selecting a yeast strain SYM-1 with high degradation rate of degrading nitrite nitrogen and ammonia nitrogen and with a bacteriostatic action to carry out species identification; comparing the 18S rRNA gene sequence of SYM-1 with the sequence in NCBI, 18S rRNA of SYM-1 strain and Rhodotorula mucilaginosa (Rhodotorula mucilaginosa) The similarity of (a) is 100.0%; identification of the SYM-1 Strain as Rhodotorula mucilaginosa (Rhodotorula mucilaginosa）。
The invention also discloses the rhodotorula mucilaginosa (A)Rhodotorula mucilaginosa) Use of SYM-1, Rhodotorula mucilaginosa (B)Rhodotorula mucilaginosa) SYM-1 strain or its fermentation liquid can be used as bacteriostatic agent for inhibiting Vibrio. The vibrio to be inhibited is vibrio vulnificus, vibrio parahaemolyticus or vibrio anguillarum. Rhodotorula mucilaginosa (A) may also be addedRhodotorula mucilaginosa) The SYM-1 strain or the fermentation liquor thereof is used as a microbial inoculum for degrading ammonia nitrogen and nitrite nitrogen and is used in the biological control of aquaculture diseases or the treatment process of aquaculture water pollution.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a rhodotorula mucilaginosa (or rhodotorula mucilaginosa) which can be used for biological control of aquaculture diseases or treatment of aquaculture water pollutionRhodotorula mucilaginosa) The SYM-1 strain and the SYM-1 strain have strong bacteriostatic action on vibrio vulnificus, vibrio parahaemolyticus and vibrio anguillarum, have high degradation capability on nitrite and ammonia nitrogen degradation rate, have the degradation rate of about 65% when the concentration of nitrite nitrogen is 50mg/L and the degradation rate of about 97% when the concentration of ammonia nitrogen is 50mg/L, and have good development and application potentials.
Drawings
FIG. 1 is a graph showing the inhibitory effect of different yeast strains on various Vibrio species, in which: 1-HN-1, 2-HS-1, 3-QDS-1, 4-RDN-1, 5-SYM-1, 6-HS-2, 7-GH-1, 8-BCH-1, 9-SYM-3, 10-SYM-2, 11-0513F1-1, 12-0513S1, 13-0513F1-2, 14-0512S2, 15-0513S1, 16-HN-2, 17-YCD-1, 18-HN-3 and 19-HX-1;
FIG. 2 is a graph showing the inhibitory effect of different yeast strains on various Vibrio species, in which: 1-SYM-1, 2-HS-1, 3-RDN-1, 4-HN-1, 5-HS-2, 6-BCH-1, 7-SYM-2, CK-uninoculated medium;
FIG. 3 is a graph showing the standard working curve of ammonia nitrogen;
FIG. 4 is a graph of a standard operating curve for nitrite nitrogen;
FIG. 5 is a diagram of the morphological characteristics of the strain SYM-1;
FIG. 6 shows SYM-1 strain 18srRNA phylogenetic tree.
Rhodotorula mucilaginosa (A) related to the inventionRhodotorula mucilaginosa) SYM-1 strain, which has been deposited in China Center for Typical Culture Collection (CCTCC) at 11/18/2020, with a collection number of: CCTCC NO: M2020756. Telephone: 027) 87682319 fax (027) 87883833E-mail:cctcc @ whu. edu. cn address: wuhan, Wuhan university; and E, postcode: 430072.
Detailed Description
The following further describes particular embodiments of the present invention to facilitate further understanding of the present invention by those skilled in the art, and does not constitute a limitation to the right thereof.
Example 1 Rhodotorula mucilaginosa (Rhodotorula mucilaginosa) R-1 Strain experiments:
1 materials and reagents
1.1 test materials
Samples of seawater and sea mud are obtained from areas such as east, Tankou, Monikan island, and Haikou of river irrigation.
1.2 culture Medium
Yeast enrichment medium: YEPD liquid medium.
Yeast isolated medium: YEPD medium: 20 g of glucose (Sucrose), 3 g of Yeast powder (Yeast extract), 10g of tryptone, 20 g of agar powder and 1000ml of water, and the pH is adjusted to 7.0.
Yeast purification culture medium: PDA culture medium.
Vibrio activation medium: 2216E medium.
Ammonia nitrogen degradation culture medium: glucose 10g, (NH)4)2SO4 235.8g，NaCl 1g，MgSO4˙7H2O 1g，K2HPO4 1g，FeSO4˙7H2O 0.4g，Solution of trace elements1mL, 1000mL of water, pH 7.2-7.4. (Ammonia nitrogen concentration: 50 mg/L)
Nitrite degradation medium: glucose 10g, NaNO2 0.075 g，NaCl 1g，MgSO4˙7H2O 1g，K2HPO4 1g，FeSO4˙7H2O 0.4g，Solution of trace elements1mL, 1000mL of water, pH 7.2-7.4. (nitrite nitrogen concentration: 50 mg/L)
Solution of trace elements：EDTA 10g，ZnSO4 1.2g，CaCl2 1.5g，MnCl2˙4H20 1g，FeSO4˙H2O 2g，(NH4)6M˙7O24˙4H2O 1g，CuSO4˙5H2O 1g，CoCl2˙6H2O1 g, water 1000mL, pH 7.2-7.4.
1.3 reagents
(1) Preparation of solution A: 0.3622 g of sodium nitroferricyanide containing 2 crystal waters are weighed, dissolved in water and made into a sodium nitroferricyanide solution with a mass concentration of 1.25% by volume to 25 mL, and the solution is stored at 4 ℃ in a brown reagent bottle. 5.00 g of phenol is weighed, dissolved in 400 mL of water, added with 2.0 mL of prepared sodium nitroferricyanide solution, and the solution A is prepared with a constant volume of 500mL and stored at 4 ℃ in a brown reagent bottle.
(2) Preparation of solution B: 2.50 g of NaOH, 2.0 g of trisodium citrate and 3.5 mL of NaClO are weighed, dissolved in 400 mL of water, the volume is adjusted to 500mL to obtain solution B, and the solution B is stored in a brown reagent bottle at 4 ℃.
(3) Preparing a sulfanilic acid solution: dissolving 5.0 g of sulfanilic acid in 50 mL of concentrated hydrochloric acid, adding water to dilute to 300 mL, cooling, transferring to a 500mL volumetric flask, adding water to the marked line, and shaking up. Stored at 4 ℃ in brown reagent bottles.
(4) Preparing a naphthyl ethylenediamine hydrochloride solution: dissolving 0.5 g of naphthyl ethylenediamine hydrochloride in 100mL of water, transferring the solution into a 500mL volumetric flask, adding water to a constant volume of 500mL, and shaking up. Stored at 4 ℃ in brown reagent bottles.
(5) Griess reagent
Mixing a naphthyl ethylenediamine hydrochloride solution and a sulfanilic acid solution 2: 1 volume mixing.
1.4 pathogenic bacteria
Vibrio vulnificus (vibrio vulnificus ) Vibrio anguillarum (V.anguillarum)Vibrio anguillarum ) Vibrio parahaemolyticus: (Vibrio Parahemolyticus) Stored in the laboratory.
2.1 isolation and purification of Yeast
Collecting 5 mL of sample in 45 mLYEPD liquid culture medium, performing enrichment culture at 28 deg.C and 180 r/min for 48 hr, performing gradient dilution after the liquid is turbid, and respectively collecting 10-4、10-5、10-6、10-74 dilution concentrations of 100 muL are coated on a YEPD flat plate with the diameter of 9cm, the culture is carried out for 48h at the constant temperature of 28 ℃,according to the morphological characteristics of the colonies, selecting yeast colonies on a PDA culture medium, streaking the yeast colonies on three areas, separating and purifying the yeast colonies to form single colonies, and inoculating different separated strains of the yeast on a PDA inclined plane for storage.
2.2 determination of Vibrio inhibitory Effect of Marine Yeast
Adopting a method of facing plates containing bacteria. Culturing indicator bacteria such as Vibrio parahaemolyticus, Vibrio anguillarum, and Vibrio vulnificus on 2216E slant culture medium for 24 hr, washing thallus Porphyrae with 0.85% sterile physiological saline, and making into 5.0 × 106Taking 0.1 mL of bacterial suspension per mL, uniformly mixing with 2216E culture medium to prepare bacterial-containing plates, inoculating yeast bacterial dishes with the diameter of 5mm, uniformly placing 5 bacterial dishes on each plate, culturing at constant temperature of 28 ℃, and observing whether an obvious inhibition zone or coverage area appears around the bacterial dishes.
2.3 determination of bacteriostatic action of Yeast fermentation broth
2.3.1 preparation of sterile fermentation broth of Yeast
The yeast strain is activated on a PDA slant for 48 hours, the yeast cultured on a two-ring slant seed culture medium is inoculated into a 250 mL triangular flask containing 50 mL liquid YEPD culture medium, and shaking culture is carried out at 180 r/min for 24 hours to serve as seed liquid. Inoculating the seed solution into a triangular flask containing 60 mL YEPD liquid culture medium according to the inoculation amount of 10%, performing shaking culture at 28 deg.C and 180 r/min for 3d, centrifuging the fermentation liquid at 4 deg.C and 5000 r/min for 20 min, and filtering the supernatant with 0.22 μm bacterial filter to obtain sterile fermentation liquid.
2.3.2 determination of bacteriostatic Effect
The oxford cup method is adopted. Washing pathogenic bacteria to be tested cultured on 2216E culture medium slant for 24 hr with 2216E liquid culture medium to obtain solution with concentration of 106Taking 0.1 mL of bacterial suspension, uniformly coating the bacterial suspension by using a sterile coating rod, symmetrically arranging oxford cups at a position 1.5 cm away from the edge of a culture dish, injecting 200 mu L of sterile fermentation liquor of different strains into each oxford cup, and taking an equivalent YEPD culture medium as a control. Culturing at 28 deg.C for 24 h, and measuring the diameter of the zone of inhibition.
2.4 determination of Ammonia Nitrogen degradation by different Yeast strains
2.4.1 preparation of standard ammonia nitrogen working curve
And determining the ammonia nitrogen content in the solution by adopting an indophenol blue spectrophotometry. Weigh 235.8 mg (NH)4)2SO4Dissolving in water, fixing the volume to 100mL, preparing an ammonia nitrogen storage solution with the ammonia nitrogen concentration of 500 mg/L, taking the ammonia nitrogen storage solution for dilution, and preparing ammonia nitrogen standard solutions with the ammonia nitrogen concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50mg/L respectively. And (3) taking 100 mu L of ammonia nitrogen standard liquid with different concentrations, respectively adding 5 mL of the solution A and the solution B, fully mixing, putting into a water bath at 37 ℃ for color development for 20 min, taking out, cooling to room temperature, and measuring the 637 nm light absorption value. Using the concentration of ammonia nitrogen as the abscissa, OD637And drawing a standard curve for the ordinate, and establishing a regression equation.
2.4.2 determination of Ammonia Nitrogen degradation Rate
Activating and culturing the screened yeast strain with strong vibrio inhibiting effect on PDA slant for 2-3 d, washing off thallus Porphyrae with ammonia nitrogen degradation culture medium, and making into 5 × 108 The CFU/mL bacterial suspension is inoculated into a 250 mL triangular flask filled with 60 mL ammonia nitrogen degradation culture medium according to the inoculation amount of 10%, each bacterial strain is inoculated into 3 bottles for 3 times of repetition, and the shake culture is carried out for 48 hours under the conditions of 28 ℃ and 180 r/min. Centrifuging the fermentation liquid at 4 deg.C and 5000 r/min for 20 min, collecting supernatant, and measuring OD of supernatant of different strains by indophenol blue spectrophotometry637And calculating the ammonia nitrogen concentration in the fermentation liquor according to the standard curve, and calculating the ammonia nitrogen degradation rate of different strains by taking an uninoculated culture medium as a reference.
Ammonia nitrogen degradation rate/% = (control ammonia nitrogen concentration-treatment ammonia nitrogen concentration)/control ammonia nitrogen concentration) x 100%
2.5 determination of nitrite degrading ability of Yeast strains
2.5.1 making of nitrite nitrogen standard working curve
The content of nitrite nitrogen in the solution is determined by adopting a naphthyl ethylenediamine hydrochloride spectrophotometry (GB/T11889-1989). 0.375 g of sodium nitrite (NaNO) is weighed out2) Dissolving the nitrite nitrogen in 150 mL of water, transferring the solution into a 1000mL volumetric flask, fixing the volume to 1000mL, and shaking up to prepare 250 mg/L nitrite nitrogen storage solution (the solution is stored in a brown bottle and can be stored at 2-5 ℃ for one week). Nitrite nitrogen taking storageDiluting the solution, preparing nitrite nitrogen standard solution with concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50mg/L, adding 6 mL Griess reagent into 100 μ L of standard solution, reacting at room temperature for 20 min, and measuring absorbance at 538 nm. Using nitrite nitrogen concentration as abscissa, OD538And drawing a standard curve for the ordinate, and establishing a regression equation.
2.5.1 determination of nitrite nitrogen degradation by different Yeast strains
Activating and culturing the screened yeast strain with strong vibrio inhibiting effect on PDA slant for 2-3 days, and washing off thallus Porphyrae with nitrite degradation culture medium to obtain 5 × 108The CFU/mL bacterial suspension is inoculated into a 250 mL triangular flask filled with 60 mL liquid nitrite degradation medium according to the inoculation amount of 10 percent, each bacterial strain is inoculated into 3 bottles for 3 times of repetition, and the shake culture is carried out for 48 hours under the conditions of 28 ℃ and 180 r/min. Centrifuging the fermentation liquid at 4 deg.C and 5000 r/min for 20 min, sampling supernatant 100 μ L, adding 6 mL Griess reagent, reacting at room temperature for 20 min, and measuring absorbance at 538 nm. Calculating the nitrite nitrogen concentration in the fermentation liquor of different strains according to a standard curve, and calculating the nitrite nitrogen degradation rate of different strains by taking an uninoculated culture medium as a reference.
Nitrite nitrogen degradation rate/% = [ (control nitrite nitrogen concentration-treatment nitrite nitrogen concentration)/control nitrite nitrogen concentration ]. times.100%
2.6 identification of the species of the strains having the effects of bacteriostasis and high-efficiency degradation of nitrite nitrogen and ammonia nitrogen
Selecting yeast strains with high degradation rate of degrading nitrite nitrogen and ammonia nitrogen and bacteriostasis to perform species identification.
2.6.1 morphological observations
Inoculating the screened yeast strains to a PDA culture medium, culturing at the constant temperature of 28 ℃ for 48h, observing and recording the size, the shape and the color of colonies on the culture medium, selecting single colonies, and observing the color and the shape of the thalli under a microscope.
2.6218S rRNA sequence analysis
The yeast strain was sent to Shanghai Bioengineering services, Inc. to determine the 18S rRNA sequence of the strain. The obtained gene sequence is spliced and aligned with known sequences in a GenBank database by BLAST, and a phylogenetic tree of the 18S rRNA gene sequence of the strain is constructed by MEGA7.0 software.
3 results and analysis
3.1 separation and purification of Marine Yeast
A total of 22 samples were collected and 19 yeast strains were isolated, and the results are shown in Table 1.
Table 1 isolated 19 strains of yeast
3.2 determination of Vibrio inhibition by different strains of Marine Yeast
Among 19 separated yeast strains, HS-1, HN-1, SYM-1, HS-2 and BCH-1 have inhibition effect on vibrio vulnificus, wherein the inhibition effect of the HS-2 strain is strongest, and the width of an inhibition zone is 4.3 mm; RDN-1, HN-1, SYM-2, HS-2 and BCH-1 have inhibition effect on vibrio parahaemolyticus, wherein the HS-2 strain and the SYM-1 strain have stronger inhibition effect, and the widths of the inhibition zones are respectively 7.2 mm and 6.5 mm; HN-1, SYM-1, HS-2 and BCH-1 have inhibition effect on vibrio anguillarum, BCH-1 strain has strongest inhibition effect, and the width of the inhibition zone is 5.1 mm. The results are shown in FIG. 1 and Table 2.
TABLE 2 inhibitory Effect of different marine yeasts on 3 Vibrio species
3.1.3 determination of bacteriostatic action of Marine Yeast sterile fermentation broth
7 strains of marine yeast with strong inhibition are selected for fermentation, the inhibition of the aseptic fermentation liquor on vibrio vulnificus, vibrio anguillarum and vibrio parahaemolyticus is determined, the fermentation liquor of 4 strains such as SYM-1, HN-1, HS-2, BCH-1 and the like has obvious inhibition on 3 kinds of vibrios, the SYM-1 has the strongest inhibition, and the widths of the inhibition zones on the three kinds of vibrios are all more than 2.5 mm. The results are shown in FIG. 2 and Table 3.
Inhibition of surface 37 strain marine yeast bacteria-free fermentation liquor on 3 kinds of vibrio
3.2 determination of Ammonia Nitrogen degrading action of Yeast strains with bacteriostatic action
3.2.1 standard working curve for ammonia nitrogen
Taking the concentration of the standard ammonia nitrogen liquid as the abscissa and OD637And drawing a working curve as a vertical coordinate. In the set concentration range, OD is increased along with the increase of the concentration of ammonia nitrogen637Gradually increased and in positive correlation, and the regression equation is as follows: y =0.015x +0.0081, R2=0.9856, linear correlation is good, can be used for subsequent analysis.
3.2.2 determination of Ammonia Nitrogen degradation by different strains
Taking supernatant fluid after centrifuging fermentation liquor of different strains, and measuring OD637And the ammonia nitrogen concentration was calculated by the regression equation, and the results are shown in table 4. The 7 strains have certain degradation effect on ammonia nitrogen, wherein the degradation effect of the strain SYM-1 is strongest, the degradation rate reaches 96.85% at the ammonia nitrogen concentration of 50mg/L, and the degradation rates of HS-1, RDN-1 and HN-1 are 90.26%, 88.51% and 72.99% respectively, and the results are shown in Table 4.
TABLE 4 Ammonia nitrogen degradation effect of different strains
3.3 determination of nitrite degradation by Yeast strains with bacteriostatic action
3.3.1 drawing of Standard working Curve for nitrite Nitrogen
And (5) taking nitrite nitrogen standards with different concentrations, and measuring the light absorption value of 538 nm. And drawing a working curve by taking the standard nitrite nitrogen concentration as an abscissa and the light absorption value as an ordinate. In the set concentration range, OD is increased along with the increase of the concentration of the nitrite nitrogen538Gradually increased and in positive correlation, and the regression equation is as follows: y =0.054x +0.0974, R2=0.9949, has good linear correlation and can be used in nitroso stateAnd (4) calculating the nitrogen concentration.
3.3.2 determination of nitrite Nitrogen degradation of different Yeast strains
Determination of OD of centrifuged supernatants of fermentation broths of different strains538And calculating the concentration of the nitrite nitrogen by a regression equation, and the result is shown in table 5. After 48 hours of treatment, 7 strains all have certain degradation effects, wherein the best degradation effect is the strain SYM-1, the degradation rate is 64.66% when the concentration of nitrite nitrogen is 50mg/L, and the degradation rate is 37.48% when the strain HN-1 is used, and the results are shown in Table 5.
TABLE 5 nitrite degradation effect of different strains
3.4 identification of species of yeast strains inhibiting bacteria and efficiently degrading nitrite and ammonia nitrogen
According to the bacteriostasis test and the degradation effect on nitrite nitrogen and ammonia nitrogen, selecting a yeast strain SYM-1 for species identification.
3.4.1 morphological observations
The colony morphology of the strain SYM-1 on the PDA culture medium is circular and red, the colony is viscous and moist, and the thallus is in an ellipsoid shape, as shown in figure 5.
3.4.2 SYM-1 Strain 18S rRNA sequence analysis
Comparing the 18S rRNA gene sequence of SYM-1 with the sequence in NCBI, 18S rRNA of SYM-1 strain and Rhodotorula mucilaginosa (Rhodotorula mucilaginosa) (SEQ ID NO: EU978453.1) showed 100.0% similarity. Selecting 18S rRNA sequences of different strains with higher homology similarity in the same genus, and adopting Mega7.0 software to construct phylogenetic tree, SYM-1 and Rhodotorula mucilaginosa (of accession EU978453.1) ((Rhodotorula mucilaginosa) The relationships are closest, located in the same branch, and the results are shown in FIG. 6.
The SYM-1 strain was considered to be Rhodotorula mucilaginosa (combined with the results of morphological characterization)Rhodotorula mucilaginosa）。
4 conclusion and discussion
The invention separates 19 yeast strains from 22 seawater and sea mud samples such as seawater, sea mud and the like collected in the Hongkong harbor sea area, wherein 7 strains have the function of antagonizing vibrios, 19 strains all have the function of degrading ammonia nitrogen and nitrite, and 1 marine rhodotorula mucilaginosa SYM-1 which can efficiently degrade nitrite and ammonia nitrogen and antagonize vibrios is screened out. Has stronger bacteriostatic action on vibrio vulnificus, vibrio parahaemolyticus and vibrio anguillarum, simultaneously has good degradation action on nitrite and ammonia nitrogen, has the degradation rate of 64.66 percent when the concentration of nitrite nitrogen is 50mg/L and the degradation rate of 96.85 percent when the concentration of ammonia nitrogen is 50mg/L, and has better development and application potentials.
Claims (6)
1. A kind of Rhodotorula mucilaginosa (C)Rhodotorula mucilaginosa) SYM-1, characterized by: the preservation number is CCTCC NO: M2020756.
2. The Rhodotorula mucilaginosa strain of claim 1 (C.)Rhodotorula mucilaginosa) The screening method of SYM-1 is characterized by comprising the following steps:
(1) separation and purification of yeast
Collecting samples, respectively taking 5 mL of samples in 45 mLYEPD liquid culture medium, performing enrichment culture at 28 deg.C and 180 r/min for 48h, performing gradient dilution after the liquid is turbid, respectively taking 10-4、10-5、10-6、10-7Coating 4 diluted concentration 100 muL on a YEPD flat plate with the diameter of 9cm, culturing at the constant temperature of 28 ℃ for 48h, selecting yeast colonies on a PDA culture medium according to colony morphology characteristics, streaking three areas, separating and purifying to obtain single colonies, and inoculating the separated 19 yeast strains on a PDA inclined plane for storage;
(2) preparation of sterile yeast fermentation liquor
Activating a yeast strain on a PDA slant for 48 hours, inoculating yeast cultured on a two-ring slant seed culture medium into a 250 mL triangular flask containing 50 mL of a liquid YEPD culture medium, and performing shaking culture at 180 r/min for 24 hours to obtain a seed solution; inoculating the seed solution into a triangular flask containing 60 mL YEPD liquid culture medium according to the inoculation amount of 10%, performing shaking culture at 28 deg.C and 180 r/min for 3d, centrifuging the fermentation liquid at 4 deg.C and 5000 r/min for 20 min, and filtering the supernatant with 0.22 μm bacterial filter to obtain sterile fermentation liquid;
(3) determination of bacteriostatic action
Measuring the diameter of the bacteriostatic circle by adopting an oxford cup method; in 19 separated yeast strains, HS-1, HN-1, SYM-1, HS-2 and BCH-1 have inhibition effect on vibrio vulnificus; the width of the bacteriostatic strip is 4.3 mm; RDN-1, HN-1, SYM-2, HS-2 and BCH-1 have inhibition effect on vibrio parahaemolyticus, wherein the widths of the bacteriostatic bands of the HS-2 strain and the SYM-1 strain are respectively 7.2 mm and 6.5 mm; HN-1, SYM-1, HS-2 and BCH-1 have inhibition effect on vibrio anguillarum;
selecting 7 strains of marine yeast sterile fermentation liquor with strong inhibition effect, determining the inhibition effect of the sterile fermentation liquor on vibrio vulnificus, vibrio anguillarum and vibrio parahaemolyticus, wherein the fermentation liquor of SYM-1, HN-1, HS-2 and BCH-14 strains has obvious inhibition effect on 3 kinds of vibrios, and the width of the inhibition zone of SYM-1 on the three kinds of vibrios is more than 2.5 mm;
(4) determination of ammonia nitrogen degradation rate
Activating and culturing the screened yeast strain with strong vibrio inhibiting effect on PDA slant for 2-3 d, washing off thallus Porphyrae with ammonia nitrogen degradation culture medium, and making into 5 × 108 Inoculating the CFU/mL bacterial suspension into a 250 mL triangular flask filled with 60 mL ammonia nitrogen degradation culture medium according to the inoculation amount of 10%, inoculating each bacterial strain into 3 bottles for 3 times of repetition, and performing shake culture for 48 hours at 28 ℃ and 180 r/min; centrifuging the fermentation liquid at 4 deg.C and 5000 r/min for 20 min, collecting supernatant, and measuring OD of supernatant of different strains by indophenol blue spectrophotometry637Calculating the ammonia nitrogen concentration in the fermentation liquor according to a standard curve, and calculating the ammonia nitrogen degradation rate of different strains by taking an uninoculated culture medium as a reference; screening to obtain a strain SYM-1 with the degradation rate reaching 96.85% when the ammonia nitrogen concentration is 50 mg/L;
(5) determination of nitrite nitrogen degradation effect
Activating and culturing the screened yeast strain with strong vibrio inhibiting effect on PDA slant for 2-3 days, and washing off thallus Porphyrae with nitrite degradation culture medium to obtain 5 × 108The CFU/mL bacterial suspension is inoculated into a 250 mL triangular flask filled with 60 mL liquid nitrite degradation medium according to the inoculation amount of 10 percentInoculating 3 bottles of each strain, repeating for 3 times, and performing shake culture at 28 ℃ for 48h at 180 r/min; centrifuging the fermentation liquid at 4 deg.C and 5000 r/min for 20 min, sampling supernatant 100 μ L, adding 6 mL Griess reagent, reacting at room temperature for 20 min, and measuring absorbance at 538 nm; calculating the nitrite nitrogen concentration in the fermentation liquor of different strains according to a standard curve, and calculating the nitrite nitrogen degradation rate of different strains by taking an uninoculated culture medium as a reference; screening to obtain a strain SYM-1 with the degradation rate of 64.66% when the concentration of nitrite nitrogen is 50 mg/L;
(6) identification of the species of the strains
Selecting a yeast strain SYM-1 with high degradation rate of degrading nitrite nitrogen and ammonia nitrogen and with a bacteriostatic action to carry out species identification; comparing the 18S rRNA gene sequence of SYM-1 with the sequence in NCBI, 18S rRNA of SYM-1 strain and Rhodotorula mucilaginosa (Rhodotorula mucilaginosa) The similarity of (a) is 100.0%; identification of the SYM-1 Strain as Rhodotorula mucilaginosa (Rhodotorula mucilaginosa）。
3. Use of Rhodotorula mucilaginosa (Rhodotorula mucorginosa) SYM-1 according to claim 1, characterized in that the Rhodotorula mucilaginosa (Rhodotorula mucorginosa) SYM-1 strain or its fermentation broth is used as a bacteriostatic agent for inhibiting Vibrio.
4. Use of Rhodotorula mucilaginosa (Rhodotorula mucor) SYM-1 according to claim 3, characterized in that: the vibrio to be inhibited is vibrio vulnificus, vibrio parahaemolyticus or vibrio anguillarum.
5. Use of Rhodotorula mucilaginosa (Rhodotorula mucorginosa) SYM-1 according to claim 1, characterized in that the Rhodotorula mucilaginosa (Rhodotorula mucorginosa) SYM-1 strain or its fermentation broth is used as an agent for degrading ammonia nitrogen and nitrite nitrogen.
6. The use of Rhodotorula mucilaginosa (Rhodotorula mucopolysaccharides) SYM-1 according to claim 5 is characterized in that the Rhodotorula mucilaginosa (Rhodotorula mucilaginosa) SYM-1 strain or its fermentation broth is used in the biological control of aquaculture diseases or in the treatment of aquaculture water pollution.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845401A (en) * | 2009-11-24 | 2010-09-29 | 东北农业大学 | Rhodotorula mucilaginose strain and application thereof in degradation of butachlor |
| CN108865908A (en) * | 2018-07-16 | 2018-11-23 | 湖南科技大学 | A kind of high Rhodotorula mucilaginose of resistance to antimony DJHN070401 and its separating screening method and application |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN108865908A (en) * | 2018-07-16 | 2018-11-23 | 湖南科技大学 | A kind of high Rhodotorula mucilaginose of resistance to antimony DJHN070401 and its separating screening method and application |
Non-Patent Citations (1)
| Title |
|---|
| 李正等: "黏红酵母对牙鲆肠黏液黏附及肠道定植规律的研究", 《安徽农业科学》 * |
Cited By (1)
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|---|---|---|---|---|
| CN115572687A (en) * | 2022-11-24 | 2023-01-06 | 清上(苏州)环境科技有限公司 | Rhodotorula mucilaginosa strain and application thereof in sewage treatment |
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