CN117363498B - Wick ham yeast CYW-7 and application thereof - Google Patents
Wick ham yeast CYW-7 and application thereof Download PDFInfo
- Publication number
- CN117363498B CN117363498B CN202311553851.4A CN202311553851A CN117363498B CN 117363498 B CN117363498 B CN 117363498B CN 202311553851 A CN202311553851 A CN 202311553851A CN 117363498 B CN117363498 B CN 117363498B
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- yeast
- phosphorus
- wick
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/32—Yeast
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
- B09B3/60—Biochemical treatment, e.g. by using enzymes
-
- 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
- C02F3/347—Use of yeasts or fungi
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
- C12P5/023—Methane
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/105—Phosphorus compounds
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2303/00—Specific treatment goals
- C02F2303/02—Odour removal or prevention of malodour
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Mycology (AREA)
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Abstract
The invention relates to a Wick ham yeastWickerhamomyces sp.) The CYW-7 has the preservation number of CGMCC No.28485, can efficiently remove odor and phosphorus, has good capabilities of releasing silicon, dissolving phosphorus, dissolving potassium, producing siderophores and IAA, also produces protease and cellulase, has antagonism on the corn leaf spot germ, can be applied to the treatment of eutrophic polluted water, improves the hydrolysis efficiency and deodorization of raw materials by anaerobic fermentation of organic solid wastes, can be used for research and development of novel bio-organic fertilizers, improves the plant yield and disease resistance, improves the quality and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of environmental microorganisms, and particularly relates to a Wilkham yeast CYW-7 and application thereof.
Background
In recent years, due to rapid development of industrialization, agricultural modernization and city, the discharged nutrient substances are increased, so that a large amount of organic solid wastes such as livestock and poultry manure, crop straws, kitchen wastes and the like are generated, and the improper treatment is extremely easy to cause non-point source pollution and even pollute water bodies. The eutrophication phenomenon of water body is serious because of the large amount of fertilizer and pesticide application and the large amount of various wastes. Therefore, both solid waste treatment and eutrophic water treatment are hot spots in current environmental research.
Yeast, like bacteria, has a set of intracellular and extracellular enzyme systems for decomposing macromolecular substances into small molecular substances which are easily utilized by cellular metabolism. Yeast belongs to heterotrophic organisms and is facultative anaerobe, and under the condition of oxygen, sugar is decomposed into carbon dioxide and water; under the condition of oxygen deficiency, sugar is decomposed into alcohol and carbon dioxide. And the yeast is harmless and easy to grow, and can survive in air, soil, water and animal bodies.
Yeast is one of the most important biological factors in the geochemical circulation process, is an extremely precious strain resource, has the characteristics of good osmotic pressure resistance, acid resistance, high metabolic efficiency and the like, has good enzyme system in vivo, and can adapt to various special environments. But because the yeast is far less abundant than bacteria, either in number or in kind. And saccharomycetes are mostly used in food industry and medical care industry, and application research in sewage and organic solid waste treatment process has been ignored for a long time.
Disclosure of Invention
The invention aims to provide Wick ham yeast CYW-7 which can efficiently gather phosphorus and deodorize, has good capabilities of releasing silicon, dissolving phosphorus, dissolving potassium, producing siderophores and IAA, also produces protease and cellulase, has antagonism on corn big spot germs, treats eutrophic polluted water by using the same, improves the hydrolysis efficiency and deodorization of organic solid waste anaerobic fermentation raw materials, and can be used for producing novel bio-organic fertilizer to improve plant yield and disease resistance.
The invention adopts the following technical scheme:
weikeham yeast @ (Chinese character)Wickerhamomycessp.) CYW-7 with a preservation number of CGMCC No.28485. The strain is preserved in China general microbiological culture Collection center (China general microbiological culture collection center) with an address of Beijing, 2023, 9 and 19 days.
Further, it has deodorizing and phosphorus accumulating capabilities.
Further, it has the ability to release silicon, dissolve phosphorus, dissolve potassium, produce siderophores and produce IAA.
Further, it has the ability to produce proteases and cellulases.
Further, it has antagonism to the phoma zeylanicum.
An application of the Wilkham yeast CYW-7 in removing phosphorus in water body.
An application of the Wilkham yeast CYW-7 in improving the hydrolysis efficiency and deodorization of raw materials by organic solid waste fermentation.
The application of the Wilkham yeast CYW-7 in preparing a biological organic fertilizer, a biological control microbial agent, a sewage treatment agent or a solid waste treatment agent.
Wherein the fungus content of Wick ham yeast CYW-7 in sewage treatment agent and solid waste treatment agent is not less than 2×10 8 individual/mL; the content of the biocontrol microbial agent is not less than 1.0X10 9 The content of the organic fertilizer is not less than 0.5X10 8 Each/g.
The invention has the beneficial effects that:
(1) The Wilkham yeast CYW-7 can be used for treating eutrophic water, improving the dephosphorization efficiency of wastewater and effectively reducing malodor pollution.
(2) The Wilkham yeast CYW-7 can be applied to anaerobic fermentation of agricultural organic solid waste, so that the hydrolysis efficiency of raw materials can be improved, and the biogas yield can be improved by more than 18.2%.
(3) The invention can be used for producing the bio-organic fertilizer by utilizing the Wick ham yeast CYW-7, thereby improving the yield, disease resistance and quality of plants.
(4) Can provide high-quality strain resources for the development of biological dephosphorization, microecological deodorant, compost and anaerobic fermentation microbial inoculum, and has practical economic and social ecological benefits and wide application prospect.
Drawings
FIG. 1 is a micrograph of a Welch Hance yeast CYW-7 cell according to the invention.
FIG. 2 is a phylogenetic tree of the Wilkham yeast CYW-7 of the invention.
Detailed Description
The invention is further described below with reference to examples and figures. The scope of the invention is not limited to the examples, and any modifications within the scope of the claims are within the scope of the invention.
EXAMPLE 1 separation and preservation of Wick Hanm Yeast CYW-7
The Wick Hanm yeast CYW-7 is obtained by separating activated sludge from a sewage treatment plant in Hebei province through enrichment culture, passage, blue white spot primary screening and differential dyeing particle dyeing double screening.
The specific method comprises the following steps: accurately weighing a 1 g sludge sample, adding the sludge sample into a 100 mL enrichment culture medium, and carrying out shaking culture at a temperature of 28 ℃ and 180 r/min for 2-3 d to obtain domesticated bacteria liquid. After 5 times of continuous passage and stabilization, single colonies with different forms are obtained by adopting a gradient dilution plate method, and are subjected to repeated lineation and purification, transferred to an inclined plane to be cultured for 48 hours at 28 ℃ and stored in a refrigerator at 4 ℃ for standby.
And (3) performing primary screening of phosphorus accumulating bacteria on the pure bacterial colonies by adopting a blue-white spot screening method. The method comprises the following steps: and inoculating the strain obtained by separation and purification on an enrichment medium plate into a glucose-MOPS phosphorus limiting and rich phosphorus plate, culturing for 2 d at 28 ℃, and observing the growth condition of the blue and white spots. Colonies which are blue on glucose-MOPS phosphorus-limiting and phosphorus-rich plates are picked for preservation.
And (3) re-screening the strain obtained by the primary screening by adopting a different-dyeing particle dyeing method. The different-dyed particles are dyed by adopting an Albert dyeing method, the different-dyed particles are black, and other parts of the thalli are green.
The culture medium is adopted:
enrichment medium: second stepSodium sulfate 5g, beef extract 3g, peptone 10g,NaCl 5g, KH 2 PO 4 0.02 g，H 2 O 1000 mL，pH7.0~7.2。
glucose-MOPS medium (10×): MOPS 8.370 g,tricine 0.717 g,30 mL sterile water, 10mol/L KOH is used for adjusting the pH value to 7.4, and the volume is fixed to 44 mL;0.01 mol/L freshly prepared FeSO 4 1 mL; the solutions were added in the following order: NH (NH) 4 Cl（1.9 mol/L）5 mL， K 2 SO 4 （0.276 mol/L）1 mL，CaCl 2 ·2H 2 O（0.02 mol/L）0.025 mL，MgCl 2 ·6H 2 O (2.5 mol/L) 0.21 mL, naCl (5 mol/L) 10 mL; the microelement mixture is 0.02, mL, glucose is 0.1, g, and the microelement thiamine solution is 1, mL, the volume is fixed to 100, mL, and the solution is filtered by a sterile filter for standby.
Phosphorus-limited medium: 25mL of 10 Xglucose-MOPS medium was placed in a medium containing 0.0087g K 2 HPO 4 200 mL agar aqueous medium.
Phosphorus-rich medium: 25mL of 10 Xglucose-MOPS medium was placed in a medium containing 0.173. 0.173g K 2 HPO 4 200 mL agar aqueous medium.
PDA medium: 200g of potato, 20g of glucose, 1L of distilled water and natural pH (15-20 g of agar is needed to be added into a solid culture medium).
Experiments show that the strain CYW-7 grows on a PDA culture medium, and has round colony, milky white color, neat edge, moist and smooth surface, and is sticky and easy to pick up. The thallus is elliptic and budding and reproduction are carried out, as shown in figure 1.
26S rDNA D1/D2 sequencing of the strain CYW-7: and selecting bacterial liquid in logarithmic growth phase, extracting strain genome DNA by using a Tiangen company yeast genome DNA extraction kit, and performing PCR amplification by using the bacterial liquid as a template.
The amplification primers are as follows: NL1 (5'-GCA TAT CAA TAAGCG GAG GAA AAG-3') and NL4 (5'-GGT CCG TGT TTC AAG ACG G-3').
PCR reaction procedure: the pre-denaturation at 98℃is carried out for 5 mm, the cycle is carried out for 35 s at 95℃and 35 s at 55℃and 40 s at 72℃and 35 cycles are carried out for 8 min.
PCR product sequencing was performed by Jin Weizhi Biotechnology Co., ltd, and the 26S rDNA D1/D2 sequence of the strain CYW-7 was shown as SEQ ID No. 1. Phylogenetic tree was constructed with DNAMAN software after sequencing was completed (see fig. 2).
Based on strain morphology and 26S rDNA sequence analysis, the strain CYW-7 was identified as Wick Hanm Yeast @Wickerhamomycessp.). The strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) at 9 months and 19 days of 2023, and has the address of China academy of sciences of China, national academy of sciences of China, no. 3, and the preservation number of: CGMCC No.28485.
Example 2 dephosphorization Properties of Wick Hanm Yeast CYW-7 at different initial phosphorus concentrations
After activation of Wick Hanm Yeast CYW-7, the concentration is uniformly adjusted to 1X 10 9 CFU/mL is inoculated into phosphorus-rich liquid culture media with initial phosphorus mass concentrations of 2, 5, 8, 10, 15 and 20 mg/L respectively according to the volume ratio of 1 percent, and the culture medium is subjected to shaking culture at the temperature of 28 ℃ for 180 r/min for 48-72 hours until bacterial liquid becomes turbid. Centrifuging the fermentation liquor 8000 r/min for 10 min, taking supernatant, passing through a 0.22 μm filter membrane to measure the mass concentration of phosphorus in the culture liquor before and after culturing, and calculating the phosphorus removal rate of each strain.
The measuring method comprises the following steps:
(1) Total phosphorus: the total phosphorus is measured by a molybdenum-antimony-scandium spectrophotometry in GB 11893-1989.
(2) The method for calculating the dephosphorization rate comprises the following steps:
phosphorus removal rate= (control total phosphorus amount-supernatant total phosphorus amount)/control total phosphorus amount x 100%.
The test results are shown in Table 1, and the phosphorus removal rate tends to decrease with increasing initial phosphorus concentration, but the phosphorus removal rate reaches 96.32% when the initial phosphorus concentration is 2 mg/L and reaches 85.70% when the initial phosphorus concentration is 5 mg/L.
TABLE 1 Effect of different initial phosphorus concentrations on the phosphorus removal effect of the strain CYW-7
。
EXAMPLE 3 determination of the deodorant Capacity of Wilkham Yeast CYW-7
Taking 18 mL Wick ham yeast CYW-7 zymophyte liquid, wherein the thallus concentration is 1 multiplied by 10 6 CFU/mL, in a 2L beaker, and 500mg/L ammonia 2 mL was added; then, a 50/mL small beaker containing 20 mL of 0.005N sulfuric acid absorbent was placed in the large beaker, and the beaker was covered with a double-layered plastic film for sealing. With equal amount of sterile water as control, repeating for 3 times, and culturing in a 28 deg.C incubator. 24 After h, the small beaker was removed and the ammonia concentration was measured.
The method for measuring ammonia comprises the following steps: nashi reagent colorimetry.
The ammonia removal rate calculation formula:
ammonia removal rate= (ammonia concentration in control absorption liquid-ammonia concentration in treated absorption liquid)/ammonia concentration in control absorption liquid x 100%.
As a result, it was found that within 24 h, the deamination rate of Wick ham yeast CYW-7 was 60% or more.
EXAMPLE 4 determination of silicon and phosphorus and Potassium Release Capacity of Wick Hanm Yeast CYW-7
The strain CYW-7 seed plate is prepared, inoculated on a silicon-releasing, phosphorus-dissolving and potassium-dissolving detection culture medium, cultured in a constant temperature incubator at 28 ℃ for 3 d, and then the growth condition and whether a transparent ring exists or not are observed. The calculation method comprises the following steps: solvency = transparent circle diameter D/colony diameter D.
Silicon release medium: sucrose 5.0. 5.0g, na 2 HPO 4 2.0 g、MgSO 4 ·7H 2 O 0.5 g、FeCl 3 0.005 g、CaCO 3 0.1 g, magnesium trisilicate 1.0g, water 1L, agar 15-20 g and pH 7.0-7.4.
Phosphate solubilizing activity assay medium (NBRIP medium): glucose 10g, ca 3 (PO 4 ) 2 5 g、(NH 4 ) 2 SO 4 0.5 g、MgSO 4 ·7H 2 O 0.25 g、KCl 0.2 g、MgCl 2 ·6H 2 O5 g, 0.4% bromophenol blue 6 mL, water 1L, agar 15~20 g,pH 7.0~7.2.
Potassium-dissolving culture medium: sucrose 10, g, na 2 HPO 4 1 g、(NH 4 ) 2 SO 4 0.5 g、MgSO 4 ·7H 2 O1 g, yeast powder 0.2 g, naCl 0.1 g and CaCO 3 0.1 g、FeCl 3 0.005 g, potassium feldspar 1 g, water 1L, 15-20 g of agar and pH 7.00.
The measurement result shows that: in the silicon releasing, phosphorus dissolving and potassium releasing culture medium, obvious transparent rings appear around bacterial colonies of the bacterial strain CYW-7, and the D/D value of the silicon releasing culture medium is larger than 1.3, the D/D value of the NBRIP culture medium is larger than 1.6, and the D/D value of the potassium releasing culture medium is larger than 1.5, which indicates that the bacterial strain has stronger silicon releasing, phosphorus dissolving and potassium releasing capabilities.
EXAMPLE 5 Wilkham yeast CYW-7 siderophore and IAA assay
Siderophore production capability: preparing a bacterial strain CYW-7 seed plate, inoculating a part of bacterial colonies by a needle, inoculating the bacterial colonies on a CAS detection medium, and culturing at 30 ℃ for 3 d, wherein yellow-green halos appear at the periphery of the bacterial colonies, which indicates that the bacterial strain CYW-7 has the capability of producing ferrites.
IAA secretion performance: preparing a bacterial strain CYW-7 seed flat plate, inoculating part of bacterial colonies into a PDA liquid culture medium (containing 100 mg/L of L-tryptophan), placing the bacterial strain into a shaking table at 28 ℃ and 180 r/min for shaking culture for 3-5 d, taking 50 mu L of supernatant after 8000 r/min centrifugation, adding 50 mu L of Salkowski colorimetric solution, dripping the Salkowski colorimetric solution onto a white porcelain plate, and developing for 30 min in a dark place, and observing color change, wherein the liquid turns red, so that the bacterial strain CYW-7 can secrete IAA, and the darker the color is the greater the secretion intensity.
The results show that the strain CYW-7 has the capability of producing IAA and siderophores. IAA is a plant growth hormone, which has promoting effect on the top bud end formation of plant branches or buds, seedlings and the like; the siderophores are compounds secreted by bacteria, fungi and the like and are used for being transferred outside cells to exchange substances and combined with free iron ions in the environment, so that the iron is conveyed into the cells, the trace element requirements of plants are met, and the growth of the plants is promoted. Therefore, it is indirectly demonstrated that the strain CYW-7 has the capability of promoting plant growth.
EXAMPLE 6 detection of cellulase and protease produced by Wilkham yeast CYW-7
The strain CYW-7 seed plate is prepared, inoculated on a detection culture medium, cultured in a constant temperature incubator at 28 ℃ for 3D, the growth condition and whether transparent rings are arranged around the colony are observed, the diameter (D) of the transparent rings and the diameter (D) of the colony are measured respectively, and the D/D value is calculated.
Cellulase detection medium: CMC-Na 5.0g, K 2 HPO 4 1.0g，MgSO 4 0.5g，(NH 4 ) 2 SO 4 2.0g of NaCl 0.5g, beef extract 0.5g, peptone 1.0g, congo red 1.5g, agar 15g, distilled water 1000mL, and pH 7.2-7.4.
Protease detection medium: agar powder 12 g, skim milk powder 10g, distilled water 1000mL, and natural pH.
The results show that clear circles are generated around the CYW-7 strain, wherein the D/D value on the cellulase detection medium plate is greater than 1.8, and the D/D value on the protease detection medium plate is greater than 2.5, which indicates that the strain has the capability of producing cellulase and protease.
EXAMPLE 7 antagonism of Wilkham's yeast CYW-7 against Spot-corn pathogen
The corn northern leaf blight is an important disease which damages the corn producing area in China and seriously affects the yield and quality of corn. Spot-germ of cornSetosphaeria turcica): belongs to the genus Helminthosporium of the phylum Deuterina, the order Cellularales, and is an important ascomycete which infects corn and causes harm. The pathogen penetrates the epidermis of the host plant by using turgor pressure and physical mechanical force through generating a special cell structure-attachment cell, so as to complete the infection process.
Whether the Wikiwim yeast CYW-7 has antagonism on the corn big spot bacteria is determined by a plate facing method: pathogenic fungi fungus blocks are inoculated at the center of the PDA plate, bacterial strains CYW-7 are inoculated at equal intervals at two sides of the fungus blocks, and the culture is carried out at the constant temperature of 28 ℃ for 48 hours, and the process is repeated for 3 times. As a result, it was found that the Rhizoctonia cerealis was unable to grow normally in the vicinity of the Wilkham yeast CYW-7, indicating that the strain CYW-7 has an obvious antagonistic effect on Rhizoctonia cerealis.
The antibacterial activity of the Wick ham yeast CYW-7 on the corn leaf spot bacteria is measured by a growth rate method, namely, the CYW-7 is cultivated by a PDA liquid culture medium for 48 h, centrifugated, the supernatant is taken to be sterilized by a sterile filter membrane of 0.22 mu m, a certain amount of sterile filtrate is added into the PDA culture medium of 40-45 ℃, and the mixture is poured into a culture dish of 9 cm after being uniformly mixed, so that the sterile filtrate is not added as a blank control. After the culture medium is solidified, the center of each plate is inoculated with a corn big spot germ block, and the culture is repeated for 3 times at 28 ℃. Colony diameters were measured by the crisscross method, and inhibition rates were calculated. The result shows that the antibacterial rate of the Wilkham yeast CYW-7 on the corn leaf spot bacteria reaches more than 80 percent.
Antibacterial ratio (%) = (control bacterial colony diameter-treated bacterial colony diameter)/(control bacterial colony diameter-bacterial mass diameter) ×100%.
Examples 8 to 10 dephosphorization effects of Wick Hanm Yeast CYW-7 in synthetic wastewater
After the bacterial strain CYW-7 is activated, the concentration is uniformly regulated to be 5 multiplied by 10 8 CFU/mL was inoculated into the wastewater synthesis medium (Table 2) at a volume ratio of 1% -5%, respectively, and shaking culture was performed at 28℃at 180 r/min at 48. 48 h. Taking a proper amount of bacterial liquid, centrifuging at 8000 rpm for 10 min, filtering with a 0.22 μm filter membrane, measuring the phosphorus content of the supernatant according to a total phosphorus measurement method, comparing with the total phosphorus value of the culture liquid without bacterial inoculation, and calculating the phosphorus removal rate (the method is the same as in example 2).
Table 2 synthetic wastewater formulation
。
The test results are shown in Table 3. Adding 1% -5% of Wick ham yeast CYW-7 into the phosphorus-containing wastewater, wherein the phosphorus removal rate is in an increasing trend along with the increase of the added yeast amount, and the phosphorus removal rate is more than 75%.
TABLE 3 determination of phosphorus removal efficiency of Wick Hanm Yeast CYW-7
。
Example 11 simulation treatment test of Wick Hanm Yeast CYW-7 on Black, odorous and eutrophic Water
And (3) expanding culture of strains: after 36 h of the bacterial strain CYW-7 is activated and cultured, the culture solution is taken and centrifuged, wet thalli are collected, and the thalli are washed for 2 times by sterile water, thus obtaining the thalli with the concentration of 5 multiplied by 10 8 CFU/mL of bacterial suspension.
Outdoor vat simulation treatment test:
6 polyvinyl chloride large barrels of 25 and L are taken, 20 and L test water samples are filled, no bacteria are added as control treatment, and each treatment is performed three times in parallel. After the inoculation liquid is added, the inoculation amount is 5%, and the change condition of the water quality index is dynamically monitored (a test water sample is taken from 10 cm parts under the water surface).
Table 4 comparison of Water quality index of simulated eutrophicated Black and odorous Water body before and after treatment
。
The specific results of the test are shown in Table 4, the phosphorus concentration is reduced from 7.55 mg/L to 1.08 mg/L, the removal rate is up to 85.70%, and the malodor is substantially eliminated.
Example 12 influence of Wilkham yeast CYW-7 on anaerobic fermentation of agricultural wastes
The strain CYW-7 is cultured in PDA liquid culture medium at 28 deg.c and 180 rpm in shaking culture 48 h to detect and regulate thallus concentration to 0.5×10 8 CFU/mL。
Adopting a 250 mL anaerobic bottle, taking 50% cow dung and 50% corn straw powder as raw materials, and adopting a Wen Quanhun anaerobic fermentation process, wherein 2 treatments are provided: treatment one: blank control; and (2) treatment II: adding 3% of Wick ham yeast CYW-7 zymophyte liquid. The organic load TS is 12% (TS is also called dry matter concentration, refers to that a certain amount of fermentation liquid is placed in a baking oven at 100-105 ℃ and is baked to constant weight, the baked matter accounts for the percentage of the total weight), biogas slurry in a normal fermentation biogas digester is used as an inoculum, the inoculum size is 20-30% (volume ratio), and the fermentation temperature is: medium temperature 35±2 ℃, fermentation time 30 d, 5 replicates per treatment. The specific results are shown in Table 5. After the agricultural waste corn stalk and livestock manure are used as raw materials for anaerobic fermentation, after Wikkaim yeast CYW-7 is added, the total volatile acid (VFA) is improved by 23.7% compared with the control, and the biogas yield is improved by 18.2% compared with the control, which is obviously higher than the control, so that the hydrolysis efficiency of the raw materials can be obviously promoted and the methane production efficiency of the anaerobic fermentation of the raw materials can be obviously improved by adding the strain.
TABLE 5 biogas production
。
Example 13 Effect of Wilkham yeast CYW-7 on soil and plant growth
The strain CYW-7 is cultured in PDA liquid culture medium at 28 deg.c and 180 rpm in shaking culture 48 h to detect and regulate thallus concentration to 0.5×10 8 CFU/mL。
According to 20 mL.kg -1 Mixing with matrix, wherein the matrix is formed by compounding part of garden soil (40%), iron tailing sand (20%) and vermiculite (40%). The control was prepared by mixing the same volume of sterile water into the matrix and performing the corn potting test. Three seedlings growing well in each pot are reserved after the seeds germinate in a greenhouse at 25 ℃, and the overground indexes and root growth conditions of soil and plants (corns) are measured after the seedlings grow 40 d. 3 replicates were run for each set of experiments.
TABLE 6 influence of Wilkham yeast CYW-7 on maize plant growth and soil fertility
。
The results show (see table 6): the inoculated treatment groups had positive growth promoting effects on maize plants compared to the non-inoculated CK treatment, with the plant height, root length, stem thickness, chlorophyll content, available silicon, available potassium and available phosphorus being increased by 15.6%, 30.4%, 22.5%, 25.3%, 29.8%, 33.7% and 70.2% respectively compared to the control. The addition of the microbial inoculum CYW-7 can effectively promote the growth of corn plants and the increase of effective fertilizer components in soil.
The present invention is described in detail with reference to the above embodiments. It should be noted that the above embodiments are merely illustrative of the invention. Numerous alternatives and modifications of the present invention will be devised by those skilled in the art without departing from the spirit and nature of the invention, which should be construed as being within the scope of the present invention.
Claims (6)
1. Weikeham yeast @ (Chinese character)Wickerhamomycessp.) CYW-7, characterized in that the preservation number is CGMCC No.28485.
2. Use of the wilms yeast CYW-7 of claim 1 for dephosphorization of water.
3. Use of the wilms yeast CYW-7 of claim 1 for increasing the efficiency of anaerobic fermentation hydrolysis of corn stalks and livestock manure as raw materials.
4. Use of the wilms yeast CYW-7 of claim 1 for deodorizing black and odorous eutrophic water bodies.
5. Use of the wilms yeast CYW-7 of claim 1 in the preparation of a bio-organic fertilizer, a bio-control microbial agent, a sewage treatment agent or a solid waste treatment agent.
6. The use according to claim 5, wherein the fungus content of the Wick yeast CYW-7 in the sewage treatment agent and the solid waste treatment agent is not less than 2X 10 8 individual/mL; the content of the biocontrol microbial agent is not less than 1.0X10 9 The content of the organic fertilizer is not less than 0.5X10 8 Each/g.
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| 防治蔬菜灰霉病解淀粉芽胞杆菌的分离鉴定及抑菌特性;吕钊;张丽萍;程辉彩;尹淑丽;张根伟;赵宝华;;农药;20130710(07);第490-493页 * |
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