CN114395484B - Humicola insolens KC0924g, microbial inoculum produced by same and application thereof - Google Patents
Humicola insolens KC0924g, microbial inoculum produced by same and application thereof Download PDFInfo
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Abstract
The invention discloses a Humicola KC0924g strain, a microbial inoculum produced by the same and application thereof, wherein the strain is identified as Humicola and is preserved in China Center for Type Culture Collection (CCTCC) NO: M2020752. The strain KC0924g has good degradation effect on enrofloxacin and other common fluoroquinolone antibiotics. The strain KC0924g can also degrade malachite green and recessive malachite green which are forbidden drugs in fishery. The degradation microbial inoculum prepared by the strain can degrade enrofloxacin and malachite green residues in loose sediment in a culture pond in a short time, the degradation rate is over 80 percent, and meanwhile, the acute biotoxicity in the sediment is obviously reduced. The strain has good application value in repairing the culture environment polluted by enrofloxacin and malachite green residues, and can effectively reduce the adverse effects of the residual enrofloxacin and malachite green residues on the aquaculture environment, aquatic products and human health.
Description
Technical Field
The invention belongs to the field of microorganism-mediated polluted environment restoration, and in particular relates to a bacterial agent prepared from degradation of residue of enrofloxacin and malachite green rot and used for producing and application of the bacterial agent
Background
The veterinary antibiotics have remarkable effects in the intensive breeding industry, and can prevent and treat animal diseases and promote animal growth. Only 20% -30% of the antibiotics for animals are absorbed and utilized by feeding objects, and the rest of antibiotics are directly discharged along with excrement or culture wastewater in a raw medicine form and are permeated into soil and water environment. Overuse of antibiotics and residual contamination exacerbate food and ecological safety hazards.
Enrofloxacin, which has the structure shown in figure 1, is also called enfluoroquinoline carboxylic acid and ethyl ciprofloxacin, belongs to third-generation fluoroquinolone antibiotics, has the characteristics of low price, broad antibacterial spectrum, quick drug effect, long time effect, small toxic and side effects and the like, and can be used for preventing and treating infectious diseases of animals and can also be used as a feed additive for promoting the growth of cultured animals. The application of enrofloxacin ensures the development of the breeding industry and effectively promotes the income increase of farmers and the development of rural economy. However, due to the lack of knowledge of scientific operations and the driving of economic benefits, the phenomenon of abuse of enrofloxacin in the farming industry is ubiquitous, leading to its residues in food, environmental samples and even human bodies which are frequently detected. More serious problems are that enrofloxacin can enter the human body along with the food chain, thereby threatening the health of the human body. The antibiotics remaining in the environment directly affect the microbial community structure and population number, and inhibit the ecological functions of the microbial community, such as material circulation. Prolonged and repeated exposure to sublethal concentrations of antibiotics promotes physiological changes in bacterial communities, leading to the emergence of drug resistance genes (antibiotic resistance genes, ARGs). The global spread and spread of ARGs induces the formation of "superbacteria" that cause the antibiotic to gradually lose therapeutic effect. Furthermore, there is growing evidence that the adverse effects of enrofloxacin exposure extend from common side effects to immune and metabolic diseases associated with the human microbiome, such as childhood obesity. At present, enrofloxacin residues in the environment are mainly removed by physical and chemical methods, such as photocatalytic degradation, electrochemical oxidation-reduction technology, zero-valent bimetal nanoparticle adsorption method, ozone oxidation method, microwave radiation method and the like, and the methods are often high in energy consumption and high in cost. In contrast, the microbial remediation method has the characteristics of no secondary pollution, low cost and the like, and is an ideal method for eliminating enrofloxacin pollution in the environment.
At present, few reports about enrofloxacin degradation strain screening are made at home and abroad, and a representative degradation strain is Gloeophyllum striatum and mixed flora enriched in artificial wetland or tidal flat plant rhizosphere soil samples. As reported enrofloxacin degradation strains are few, further development and application of the enrofloxacin polluted environment microbial remediation technology are restricted. Therefore, the enrofloxacin high-efficiency degradation strain is domesticated and screened, the degradation characteristic is researched, and the enrofloxacin high-efficiency degradation strain is applied to bioremediation of the enrofloxacin polluted environment, so that the method has important ecological significance.
Malachite green is an artificially synthesized triphenylmethane dye, and has been widely used for environmental disinfection, saprolegniasis treatment, parotitis treatment and the like in aquaculture due to low cost and good insect expelling and sterilizing effects. Malachite green is easy to metabolize and convert into recessive malachite green with more stable chemical property and stronger toxicity, and has slow metabolism and long residual time in organisms and environments. Research shows that malachite green and its metabolite, recessive malachite green, are all carcinogenic, teratogenic and mutagenic. Currently, malachite green is classified as a forbidden drug in many countries, including china. Because malachite green has been widely used, a part of the cultivation environment is polluted, and most of the cultivation environment is reflected by farmers, malachite green medicines are not used in the cultivation process, but are detected in products, and presumably caused by historical residues in the environment. Therefore, there is a need for efficient, economical and green microbial remediation techniques for the remediation of residual malachite green and recessive malachite green in a farming environment.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the prior art, the invention provides a strain of Humicola (Humicola sp.) KC0924g, which can effectively degrade enrofloxacin and fluoroquinolone antibiotics and can treat residual pollution of malachite green and recessive malachite green. The invention also aims to provide a method for producing the degradation bacterial agent based on 0924g of the degradation bacterial strain KC and application thereof to repair the bottom mud of the culture pond polluted by enrofloxacin and malachite green residues. The degradation microbial inoculum prepared by the strain can degrade the enrofloxacin remained in the loose bottom mud of the culture pond in a short time.
The technical scheme is as follows: in order to achieve the above object, the present invention provides a strain of Humicola (Humicola sp.) KC0924g, which has been deposited in the chinese collection of typical cultures, deposit address: the preservation time of the Chinese Wuhan is 11 months and 16 days in 2020, and the preservation number is CCTCC NO: M2020752.
The invention relates to application of Humicola sp KC0924g in degrading enrofloxacin.
Wherein, the KC0924g is applied to degrading enrofloxacin in loose bottom mud of a culture pond.
The invention relates to application of Humicola (sp.) KC0924g in degrading fluoroquinolone antibiotics.
The invention relates to application of Humicola sp KC0924g in degradation of malachite green and recessive malachite green.
Wherein, the KC0924g is applied to degrading malachite green in loose bottom mud of a culture pond.
The degradation microbial inoculum produced by the Humicola sp KC0924g.
The preparation method of the degradation microbial inoculum is characterized by comprising the following steps:
(1) Inoculating 0924g of bacterial strain KC onto a PDA culture medium, culturing until spores are mature, eluting spores, and obtaining primary seed liquid;
(2) Inoculating the primary seed liquid into a seed tank containing a PD culture medium, and culturing in the seed tank to obtain a secondary seed liquid;
(3) And (3) inoculating the secondary seed liquid to a solid fermentation culture medium, and drying and crushing the fermented solid material at low temperature to obtain the degradation microbial inoculum.
Wherein the concentration of spores in the primary seed liquid in the step (1) is 9.0X10 6 ～1.2×10 7 cfu mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The inoculation in the step (2) is performed according to the volume of the fermentation medium of 0.8-1.2%, the culture temperature is 25-28 ℃, and the fermentation time is 24-36 h. Wherein, in the step (3), the solid state fermentation temperature is 26-28 ℃, the ambient humidity is 50-85%, and the fermentation time is 3-5 d; drying the fermented solid material by a low-temperature dryer until the water content reaches 5-12%, crushing to 40-60 meshes by a crusher, and packaging to obtain the finished product.
Preferably, the concentration of spores in the primary seed liquid in the step (1) is 10 7 cfu mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The inoculation in the step (2) is performed according to 1% of the volume of the fermentation medium, the culture temperature is 28 ℃, and the fermentation time is 24 hours; the solid fermentation culture medium in the step (3) comprises a solid fermentation substrate and a nutrient solution, the water content of the solid fermentation substrate is 65-75wt%,
preferably, the solid fermentation substrate comprises rice hull powder (20 meshes) and wheat bran (40 meshes); wherein, the mass ratio of the rice hull powder (20 meshes) to the wheat bran (40 meshes) is 2:3;
the nutrient solution comprises honey, peptone, magnesium sulfate and calcium chloride; according to the mass percentage, 1 percent of honey, 0.5 percent of peptone, 0.2 percent of magnesium sulfate, 0.05 percent of calcium chloride and the balance of deionized water.
The solid state fermentation temperature in the step (3) is 26-28 ℃, the ambient humidity is 50-85%, and the fermentation time is 3-5 d; supplementing a nutrient solution every 24 hours, and uniformly spraying the nutrient solution according to the adding amount of 1% by weight; drying the fermented solid material by a low-temperature dryer until the water content reaches 5-12%, crushing to 40-60 meshes by a crusher, checking, and grading, metering and packaging to obtain the finished product.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
the invention separates and screens to obtain enrofloxacin degradation bacterial strain KC0924g, which is used for inorganic substancesThe concentration of the salt culture solution is 1mg L -1 The degradation rate of enrofloxacin after being treated for 72 hours reaches 91.2 percent. The strain has good degradation effect on other common fluoroquinolone antibiotics (ciprofloxacin, norfloxacin and ofloxacin). In addition, the strain KC0924g can degrade malachite green and recessive malachite green which are forbidden in fishery, and the degradation rates of malachite green and recessive malachite green in an inorganic salt culture medium are respectively 99.9% and 87.5% within 48 hours. The degradation microbial inoculum prepared by the strain can degrade enrofloxacin and malachite green residues in loose sediment of a culture pond in a short time, the degradation rate is more than 80%, and compared with the acute biotoxicity in the sediment of a control group treatment group, the degradation microbial inoculum has obvious reduction. The results show that the strain has good application value in repairing the environment of the loose soil of the culture pond polluted by enrofloxacin and malachite green residues.
The degradation microbial inoculum can be produced by using general fermentation equipment in the solid state fermentation industry, has the advantages of low production cost, convenient use and good removal effect, and is suitable for repairing sediment polluted by enrofloxacin in aquaculture plants and the surrounding area; the invention has important significance for protecting ecological environment and human health.
Drawings
FIG. 1 is a molecular structural formula of enrofloxacin;
FIG. 2 is an HPLC chart of the strain KC0924g for degrading enrofloxacin (A, enrofloxacin CK; B, enrofloxacin + strain KC0924 g);
FIG. 3 shows photographs of strain KC0924g of the present invention (A, front photographs of colonies; B, rear photographs of colonies; C, photomicrographs of hyphae after dyeing with phenol cotton blue lactate; D, photomicrographs of hyphae and chlamydospores after dyeing with phenol cotton blue lactate);
FIG. 4 is a phylogenetic tree of rDNA-ITS sequences in strain KC0924g;
FIG. 5 is a graph of degradation of enrofloxacin over time in a basal salt medium;
FIG. 6 shows the degradation rate of strain KC0924g for other fluoroquinolone antibiotics and malachite green, recessive malachite green;
FIG. 7 shows the color change of the strain KC0924g for degrading malachite green (A, malachite green CK; B, malachite green+inactivated strain KC0924g; C, malachite green+strain KC0924 g).
FIG. 8 is a graph showing the trend of the effect of the substrate sludge sample leaching liquor on the luminosity of the luminescent bacteria (Vibrio fischeri) at various time points.
Detailed Description
The invention is further described below with reference to the drawings and examples.
Malachite green and recessive malachite green used in the embodiment of the invention are purchased from chemical reagent limited company of national medicine group; deuterated malachite green and deuterated recessive malachite green were purchased from dr.
Enrofloxacin, ciprofloxacin, norfloxacin and ofloxacin are purchased from alexidine.
Example 1
Isolation and identification of strain KC0924 g:
collecting sediment of a culture pond (sediment sample is detected by enrofloxacin) planted with submerged plant herba Sonchi Oleracei (Vallisneria spiralis), placing 10g of sediment sample in 100mL, adding 1mg L -1 Inorganic salt liquid medium (NH) of enrofloxacin 4 NO 3 1.0g，K 2 HPO 4 1.5g，KH 2 PO 4 0.5g，MgSO 4 0.2g of NaCl 1.0g, water 1L, pH 7.0-7.2), at 28℃and 160rpm for 7d, is transferred to the addition of 2mg L in an inoculum size of 5% by volume -1 Inorganic salt liquid culture medium of enrofloxacin, wherein the concentration of enrofloxacin is increased by 1mg L each time of transferring -1 Until 4 successive transfers. And (3) determining the degradation effect of the enrichment solution on enrofloxacin by using a high performance liquid chromatography to obtain the enrichment solution with the degradation effect. The enrichment solution with degradation effect is prepared by mixing 10 volumes -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Gradient dilution, 200. Mu.L of the enrichment solution of each dilution gradient was applied to a solution containing 5mg L -1 Nutrient agar medium plates (peptone 10g, beef powder 3g, sodium chloride 5g, agar 15g, pH 7.3.+ -. 0.2) of enrofloxacin and containing 5mg L -1 PDA culture medium plate of enrofloxacin (peeled potato 200g, glucose 20)g, agar 15-20g, distilled water 1L, pH nature) at 28℃for 7d. Bacterial or fungal single colonies (the morphology is directly judged by naked eyes) grown on the plates are respectively picked up, and the bacterial colonies are inoculated on a plate containing 5mg L -1 Broth of enrofloxacin (peptone 10g, beef powder 3g, sodium chloride 5g, pH 7.3.+ -. 0.2), fungal colonies were inoculated to a broth containing 5mg L -1 Culturing the strain in PD medium (peeled potato 200g, glucose 20g, distilled water 1L, pH natural) of enrofloxacin to logarithmic phase, inoculating 1mg L with 1% by volume of the inoculum -1 Culturing in inorganic salt culture medium of enrofloxacin at 28deg.C at 160r/min for 3d, and detecting degradation effect by high performance liquid chromatography. The strain with degradation effect on enrofloxacin is the degradation strain.
Sample pretreatment method: the 1mL sample was pipetted into a 1.5mL centrifuge tube and centrifuged at 12000rpm for 5min. Filtering impurities from the supernatant with a bacterial filter of 0.22 μm, and filling into a sample bottle for detection.
The sample detection method comprises the following steps:
HPLC-fluorescence detector assay
The model of the high performance liquid chromatograph is Agilent 1100/1200HPLC, the chromatographic column is Agilent Eclipseplus C, the specification is 250mm multiplied by 4.6mm multiplied by 5 mu m, and the column temperature is 40 ℃; mobile phase: 0.05M phosphoric acid-triethylamine in water/acetonitrile (18:82, v/v); the flow rate is 1.0mL min -1 The sample injection amount is 20 mu L; for enrofloxacin, ciprofloxacin and norfloxacin, the fluorescence detector excitation wavelength is 280nm, and the emission wavelength is 450nm; for ofloxacin, the fluorescence detector excitation wavelength is 283nm and the emission wavelength is 490nm.
From the enriched liquid, 1 enrofloxacin degradation strain was obtained by verification by HPLC-fluorescence detector detection (FIG. 2), and designated KC0924g. The strain was used for 1mg L in 72h -1 The degradation rate of enrofloxacin is up to 91.2%. Bacterial strain KC0924g formed divergently wrinkled colonies on PDA medium with white, velvet-like front hyphae attached to the substrate surface (fig. 3A); the reverse side was yellow brown to black with pigmentation (fig. 3B); the hyphae end branches (fig. 3C) and spores are spherical (fig. 3D).
The rDNA-ITS fragment of strain KC0924g is amplified by adopting universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), the length is 613bp, and the gene sequence is shown as SEQ ID No. 1. The sequencing result is compared with GenBank database in NCBI, and the result shows that the similarity of the strain KC0924g with Humicola (Humicola sp.) strain is highest, and the similarity with Humicola sp.L-2 is 98.67%. Based on morphological characteristics of strain KC0924g and rDNA-ITS sequence evolution developmental tree analysis (FIG. 4), it was initially identified as Humicola sp, designated Humicola sp KC0924g. The strain KC09254g is delivered to China center for type culture collection (CCTCC for short) for preservation, wherein the preservation time is 11 months and 16 days in 2020, and the preservation number is CCTCC NO: M2020752.
Example 2
Degradation effect of bacterial strain KC0924g on enrofloxacin in inorganic salt medium:
colonies grown on PDA plates were repeatedly rinsed with sterile water to obtain spore suspension (1.0X10) of strain KC0924g 7 cfu mL -1 ). Inoculating the spore suspension into 100mL PDA culture medium with the inoculation amount of 5%vol, and culturing at 28 ℃ and 160rpm for 2d by shaking to obtain fresh bacterial liquid, namely seed liquid. Adding 1mg L of the final concentration into the inorganic salt culture medium -1 Inoculating seed liquid of strain KC0924g according to 5% volume ratio of inoculation amount; at the same time, adding 1mg L of the final concentration into the inorganic salt culture medium -1 Inoculating inactivated strain KC0924g seed solution with the same volume ratio as control, placing in a constant temperature shaking table at 28 ℃ and 160rpm for culture, and taking samples at 6h,12h,24h (1 d), 72h (3 d), 120h (5 d) and 168h (7 d) respectively. Degradation of enrofloxacin by strain KC0924g was detected by high performance liquid chromatography using the method for verifying degradation effect in example 1, and degradation rate was calculated, as shown in FIG. 5, by strain KC0924g to 1mg L in 72h (3 d) -1 The degradation rate of enrofloxacin was 91.2% and the degradation rate did not change much over time (168 h, 92.0%).
Example 3
Degradation effects of strain KC0924g on other fluoroquinolone antibiotics (ciprofloxacin, norfloxacin and ofloxacin) in inorganic salt medium:
seed solution of strain KC0924g was obtained by the method of example 2. Adding 1mg L of the final concentration into the inorganic salt culture medium -1 Ciprofloxacin, norfloxacin and ofloxacin, and inoculating seed solution of strain KC0924g according to the inoculation amount of 5% volume ratio; simultaneously, 1mg L of final concentration is respectively added into the inorganic salt culture medium -1 Ciprofloxacin, norfloxacin and ofloxacin, inoculating inactivated strain KC0924g seed solution at 5% volume ratio as control, and culturing in a constant temperature shaker at 28deg.C at 160rpm for 72h (3 d). Taking a degradation sample of 3d, detecting the degradation condition of the strain KC0924g on ciprofloxacin, norfloxacin and ofloxacin by adopting a high performance liquid chromatography method for verifying the degradation effect in the embodiment 1, and calculating the degradation rate, wherein the concentration of the strain KC0924g on 1mg L in 72h is shown in FIG. 6 -1 The degradation rates of ciprofloxacin, norfloxacin and ofloxacin were 86.4%,70.2% and 46.4%, respectively.
Example 4
Degradation effect of bacterial strain KC0924g on malachite green and recessive malachite green in inorganic salt culture medium:
seed solution of strain KC0924g was obtained by the method of example 2. Adding 1mg L of the final concentration into the inorganic salt culture medium -1 The seed solution of the strain KC0924g is respectively inoculated according to the inoculation amount of 5 percent of volume ratio of malachite green and recessive malachite green; simultaneously, the final concentration of 1mgL is respectively added into the inorganic salt culture medium -1 Malachite green and recessive malachite green, and inoculating inactivated strain KC0924g seed solution according to 5% volume ratio, and culturing in a constant temperature shaker at 28deg.C and 160rpm for 48 hr. As malachite green is blue-green, the degradation condition can be primarily judged through color change. As shown in fig. 7, malachite green in the treated group faded to colorless within 48 hours (fig. 7C) as compared to the control group (fig. 7A and B). The method comprises the steps of measuring residues of malachite green and recessive malachite green in inorganic salts by an HPLC-MS/MS method and calculating degradation rate, wherein the specific method is as follows:
sample pretreatment method:
10mL of sample is centrifuged at 8000rpm for 5min, 100. Mu.L of supernatant is diluted into 10mL of water, and 300. Mu.L of solution with concentration of 0.2. Mu.mL is added -1 Uniformly mixing deuterated malachite green and deuterated recessive malachite green, taking 1mL of sample to pass through a PRS solid phase extraction column, activating (2 mL of acetonitrile and 2mL of water), loading, leaching (2 mL of water and 2mL of acetonitrile) the PRS column, and finally adopting 3mL of acetonitrile: ammonium acetate (v: v=1:1), 1mL of eluent was taken and filtered, and checked on the machine.
The sample detection method comprises the following steps:
chromatographic column: thermo C18 chromatographic column (100 mm. Times.2.1 mm); flow rate: 0.25mL/min; sample injection amount: 10. Mu.L; the mobile phase consisted of acetonitrile and 0.2% ammonium acetate and the gradient elution procedure is shown in table 1.
TABLE 1 gradient elution conditions
Ion source: electrospray ion source (ESI); ionization mode: a positive ion; scanning mode: multiple Reaction Monitoring (MRM); ion spray voltage: 5500V; ion transport tube temperature: 500 ℃; sheath air flow: 30mL/min; auxiliary air flow: 9mL/min; collision gas: argon gas.
Under natural conditions, malachite green is easily reduced to form more toxic recessive malachite green. The result shows that the degradation rate of the strain KC0924g to malachite green in the inorganic salt culture medium within 48 hours is 99.9 percent and the recessive malachite green is not accumulated in the degradation system; the degradation rate of the strain KC0924g to the recessive malachite green in the inorganic salt culture medium within 48h is 87.5%.
Example 5
Preparation of degradation inoculant of strain KC0924 g:
(1) Inoculating degradation strain KC0924g onto PDA culture medium, culturing until spores are mature, eluting spores to obtain primary seed solution, wherein the concentration of spores in the primary seed solution is 1.0X10 7 cfu mL -1 ；
(2) Inoculating the primary seed liquid into a seed tank containing a PD (potential difference) culture medium according to 1% of the volume of the fermentation culture medium, and culturing in the seed tank at 28 ℃ for 24 hours to obtain a secondary seed liquid;
(3) Inoculating the secondary seed liquid to a solid fermentation culture medium, and drying and crushing the fermented solid material at low temperature to obtain a degradation microbial inoculum; the solid state fermentation medium in the step (3) comprises a solid state fermentation substrate and a nutrient solution (the mass ratio is 3:7), the water content is about 70wt%, the solid state fermentation temperature is 28 ℃, the ambient humidity is 70%, and the fermentation time is 5d; supplementing a nutrient solution every 24 hours, and uniformly spraying and adding the nutrient solution according to the weight ratio of 1% of the whole fermentation system; drying the fermented solid material by a low-temperature dryer until the water content reaches 8%, crushing to 60 meshes by a crusher, checking, and grading, metering and packaging to obtain the finished product.
Wherein the solid fermentation matrix comprises rice hull powder (20 meshes) and wheat bran (40 meshes); wherein, the mass ratio of the rice hull powder (20 meshes) to the wheat bran (40 meshes) is 2:3; the nutrient solution comprises honey, peptone, magnesium sulfate and calcium chloride; according to the mass percentage, 1 percent of honey, 0.5 percent of peptone, 0.2 percent of magnesium sulfate, 0.05 percent of calcium chloride and the balance of water.
Example 6
Preparation of degradation inoculant of strain KC0924 g:
(1) Inoculating degradation strain KC0924g onto PDA culture medium, culturing until spores are mature, eluting spores to obtain primary seed solution, wherein the concentration of spores in the primary seed solution is 1.2X10 7 cfu mL -1 ；
(2) Inoculating the primary seed liquid into a seed tank containing a PD (potential difference) culture medium according to 1.2% of the volume of the fermentation culture medium, and culturing in the seed tank at 28 ℃ for 24 hours to obtain a secondary seed liquid;
(3) Inoculating the secondary seed liquid to a solid fermentation culture medium, and drying and crushing the fermented solid material at low temperature to obtain a degradation microbial inoculum; the solid state fermentation medium in the step (3) comprises a solid state fermentation substrate and a nutrient solution (the mass ratio is 3:7), the water content is about 70wt%, the solid state fermentation temperature is 26 ℃, the ambient humidity is 85%, and the fermentation time is 5d; supplementing a nutrient solution every 24 hours, and uniformly spraying and adding the nutrient solution according to the weight ratio of 1% of the whole fermentation system; drying the fermented solid material by a low-temperature dryer until the water content reaches 12%, crushing to 60 meshes by a crusher, checking, and grading, metering and packaging to obtain the finished product.
Wherein the solid fermentation matrix comprises rice hull powder (20 meshes) and wheat bran (40 meshes); wherein, the mass ratio of the rice hull powder (20 meshes) to the wheat bran (40 meshes) is 2:3; the nutrient solution comprises honey, peptone, magnesium sulfate and calcium chloride; according to the mass percentage, 1 percent of honey, 0.5 percent of peptone, 0.2 percent of magnesium sulfate, 0.05 percent of calcium chloride and the balance of water.
Example 7
Preparation of degradation inoculant of strain KC0924 g:
(1) Inoculating degradation strain KC0924g onto PDA culture medium, culturing until spores are mature, eluting spores to obtain primary seed solution, wherein the concentration of spores in the primary seed solution is 9.0X10 6 cfu mL -1 ；
(2) Inoculating the primary seed liquid into a seed tank containing a PD (potential difference) culture medium according to the volume of 0.8% of the fermentation culture medium, and culturing in the seed tank at 28 ℃ for 36h to obtain a secondary seed liquid;
(3) Inoculating the secondary seed liquid to a solid fermentation culture medium, and drying and crushing the fermented solid material at low temperature to obtain a degradation microbial inoculum; the solid state fermentation medium in the step (3) comprises a solid state fermentation substrate and a nutrient solution (the mass ratio is 3:7), the water content is about 70wt%, the solid state fermentation temperature is 28 ℃, the ambient humidity is 50%, and the fermentation time is 3d; supplementing a nutrient solution every 24 hours, and uniformly spraying and adding the nutrient solution according to the weight ratio of 1% of the whole fermentation system; drying the fermented solid material by a low-temperature dryer until the water content reaches 5%, crushing to 40 meshes by a crusher, checking, and grading, metering and packaging to obtain the finished product.
Wherein the solid fermentation matrix comprises rice hull powder (20 meshes) and wheat bran (40 meshes); wherein, the mass ratio of the rice hull powder (20 meshes) to the wheat bran (40 meshes) is 2:3; the nutrient solution comprises honey, peptone, magnesium sulfate and calcium chloride; according to the mass percentage, 1 percent of honey, 0.5 percent of peptone, 0.2 percent of magnesium sulfate, 0.05 percent of calcium chloride and the balance of water.
Example 8
Determination of degradation effect of bacterial strain KC0924g degrading microbial inoculum on enrofloxacin and malachite green in bottom mud of culture pond
Weighing 500g of pond sediment subjected to air drying and sieving, and adding enrofloxacin and malachite green to ensure that the concentration of enrofloxacin and malachite green in the sediment is 1mg kg -1 Adding KC0924g degrading bacterial agent prepared in example 5 with an inoculum size of 1% by mass, mixing uniformly, arranging substrate sludge without KC0924g degrading bacterial agent and with the same volume of inactivated KC0924g degrading bacterial agent as a control in treatment groups, placing in a 28 ℃ incubator for constant temperature culture, keeping the water content at 60%, carrying out parallel experiments for 3 times in each group, and collecting substrate sludge samples by adopting a five-point sampling method.
The residual amounts of enrofloxacin and malachite green in the bottom mud were measured by sampling at 28d, the residual amounts were measured by high performance liquid chromatography-mass spectrometry, and the average degradation rate was calculated for a plurality of times, and the results are shown in table 2.
Sediment samples in the treatment group and the control group were collected at 1d, 3d, 5d, 10d, 17d and 28d, respectively, and leach solutions were prepared as follows: 2.5g of sediment sample and 2.5mL of Microtox diluent are weighed, homogenized and sonicated for 20min, and subjected to standing leaching for 24h, and centrifuged at 10000rpm for 5min, and the supernatant is sediment leaching solution. Toxicity detector using luminous bacteria (vibrio fischeri)
FX analyzer, MODERNWATER) to measure the change in the influence of the sediment leaching liquor on the luminosity of the luminescent bacteria at each time point, and measure the acute biotoxicity caused by the residue of the traditional Chinese medicine in the sediment sample to be measured. FIG. 8 is a graph showing the trend of the effect of the substrate sludge sample leaching liquor on the luminosity of the luminescent bacteria (Vibrio fischeri) at various time points.
TABLE 2 degradation Effect of the KC0924g degrading microbial inoculant on enrofloxacin and malachite green residues in substrate sludge
As seen from Table 2, the concentration of enrofloxacin and malachite green in the sediment was 1mg kg -1 When the strain KC0924g has a degradation rate of 80.5% to enrofloxacin and 86.8% to malachite green without accumulation of recessive malachite green. As can be seen from fig. 8, the influence of the sediment leaching solution in the control group on the luminosity of the luminescent bacteria (vibrio freudenreichii) does not fluctuate much with time, and the luminosity of the sediment leaching solution in the 28 th is increased by 5% relative to that in the sediment leaching solution in the 1 st; the sediment extract in the treatment group showed an increasing trend of luminosity to luminescent bacteria (Vibrio freudenreichii) over time, and the luminosity of the sediment extract of 28d to luminescent bacteria (Vibrio freudenreichii) was increased by 21% relative to that of the sediment extract of 1 d. The results show a significant reduction in acute biotoxicity in the sediment compared to the control group. In conclusion, KC0924g degrading microbial inoculum can effectively repair enrofloxacin and malachite green pollution residues in the bottom mud of the culture pond.
Sequence listing
<110> Shanghai aquatic institute (Shanghai aquatic technical popularization station)
<120> A strain of Humicola insolens KC0924g, and microbial inoculum and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 613
<212> DNA
<213> Humicola sp.)
<400> 1
ttttttttcc tttccttccg gcttatgata tgcttaagtt cagcgggtct tcctacctga 60
tccgaggtca accttgggta aataaggctt ttaacggccg gaacccgcag cacgcccaga 120
gcgagatgta actactacgc tcggagtgac agcgagcccg ccactgcatt tcagggcctg 180
cggcagccgc aggtccccaa cacaagcccg gggcttgatg gttgaaatga cgctcgaaca 240
ggcatgcccg ccagaatact ggcgggcgca atgtgcgttc aaagattcga tgattcactg 300
aattctgcaa ttcacattac ttatcgcatt tcgctgcgtt cttcatcgat gccagaacca 360
agagatccgt tgttgaaagt tttgacttat tcagtacaga agactcagag aggccataca 420
atgcaatagt ttggttttcc tccggcgggc gcccgcgagg gccgaggggc gccgggaagg 480
acccgggccg cccgccgaag caacggttta ggtaacgttc acaatggttt agggagtttt 540
gcaactctgt aatgatccct ccgctggttc accaacggag accttgttac gattttttac 600
ttccaaaatc gtc 613
Claims (11)
1. Humicola insolensHumicola sp.) KC0924g, which is preserved in China center for type culture Collection with a preservation time of 11 months and 16 days in 2020, and a preservation number of CCTCC NO: M2020752.
2. A process according to claim 1Humicola sp.) KC0924g for use in degrading enrofloxacin.
3. The use according to claim 2, wherein the KC0924g is used for degrading enrofloxacin in loose bottom mud of a culture pond.
4. A process according to claim 1Humicola sp.) KC0924g for use in degrading fluoroquinolone antibiotics.
5. A process according to claim 1Humicolasp.) KC0924g for use in degrading malachite green and malachite green recessive.
6. The use according to claim 5, wherein KC0924g is used for degrading malachite green in loose bottom mud of a culture pond.
7. A method of producing a plant of the genus Humicola according to claim 1Humicola sp.) KC0924g.
8. A method for preparing the degradation microbial agent of claim 7, comprising the steps of:
(1) Inoculating 0924g of KC onto a PDA culture medium, culturing until spores are mature, eluting spores, and obtaining primary seed liquid;
(2) Inoculating the primary seed liquid into a seed tank containing a PD culture medium, and culturing in the seed tank to obtain a secondary seed liquid;
(3) And (3) inoculating the secondary seed liquid to a solid fermentation culture medium, and drying and crushing the fermented solid material at low temperature to obtain the degradation microbial inoculum.
9. The method according to claim 8, wherein the concentration of spores in the primary seed liquid in the step (1) is 9.0X10 6 ~1.2×10 7 cfu mL -1 The method comprises the steps of carrying out a first treatment on the surface of the And (2) inoculating according to 0.8-1.2% of the volume of the fermentation medium, wherein the culture temperature is 25-28 ℃, and the fermentation time is 24-36 h.
10. The preparation method according to claim 8, wherein the solid state fermentation temperature in the step (3) is 26-28 ℃, the ambient humidity is 50-85%, and the fermentation time is 3-5 d; drying the fermented solid material by a low-temperature dryer until the water content reaches 5-12%, crushing to 40-60 meshes by a crusher, and packaging to obtain the finished product.
11. Use of the degrading bacterial agent of claim 7 in degrading enrofloxacin and malachite green.
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