CN107058136B - Geotrichum strain Fh5 and application thereof - Google Patents
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Abstract
The invention discloses Geotrichum (Geotrichum) Fh5 which is preserved in China general microbiological culture Collection center (CGMCC) in 5, 4 and 2017, and the preservation number is CGMCC No. 13777. The Geotrichum strain Fh5 can effectively degrade ammonia nitrogen with the concentration of 500mg/L in sewage, percolate and ammonia gas in odor, and in the degradation process, nitrite nitrogen and nitrate nitrogen are not accumulated, nitrification and denitrification can be synchronously performed, so that the degradation rate of the ammonia nitrogen in the sewage, the percolate or the odor reaches 100%, and the residual total nitrogen is 10 mg/L.
Description
Technical Field
The invention belongs to the technical field of biological ammonia nitrogen removal of sewage, and particularly relates to geotrichum Fh5 and application thereof.
Background
The traditional ammonia removal process is completed by a nitration reaction and a denitrification reaction. Although the conventional nitrification and denitrification processes play a certain role in the denitrification of wastewater, there are still some problems, such as the nitrification process is completed under aerobic conditions, the denitrification reaction requires certain organic matters, the entire denitrification process requires a large amount of energy, and so on. Because the traditional ammonia removal process has some defects, some scholars at home and abroad improve the research of the traditional ammonia removal process, and in recent years, novel ammonia removal processes such as anaerobic ammonia oxidation, short-range nitrification and denitrification, simultaneous nitrification and denitrification and the like are researched, so that the novel processes greatly shorten the time of the ammonia removal process, shorten the reaction and greatly reduce the energy consumption. The novel anaerobic ammonia oxidation process can greatly reduce the oxygenation energy consumption of nitration reaction, and an exogenous electron donor of denitrification reaction is omitted, and the anaerobic ammonia oxidation bacteria can convert ammonia nitrogen contained in sewage into nitrogen gas through biochemical reaction in an anoxic environment to remove the nitrogen gas, so that the novel anaerobic ammonia oxidation process has great significance to global nitrogen circulation and plays an important role in sewage treatment.
The traditional theory holds that the ammonia removal process comprises two stages of nitration reaction and denitrification reaction, firstly NH4 +Oxidation of-N to NO by microorganisms2 -N (nitrosation) and further oxidation to NO3 -N (nitration), then NO3 -Reduction of-N to N by microbial action2(denitrification).
Conventional theory and thinking that nitrification requires a minimum of two bacteria to be performed together, the first stage of nitrification is NH4 +Oxidation of-N to NO by nitrosobacteria2 - N, the second stage is NO2 - Oxidation of-N to NO by nitrifying bacteria3 -N, the process is completed by nitrosobacteria and nitrobacteria. The conventional theory also holds that the nitrification process and the denitrification process of the whole ammonia removal process are completed by requiring two kinds of bacteria, namely nitrifying bacteria and denitrifying bacteria at minimum.
Disclosure of Invention
The invention aims to solve the problems and provides a geotrichum Fh5 and application thereof. The technical scheme of the invention is realized as follows.
According to the first aspect of the invention, the inventor selects and breeds a Geotrichum Fh5 which is classified and named as Geotrichum Geotrichum, is preserved in China general microbiological culture Collection center (CGMCC), has the preservation number of CGMCC No.13777, has the preservation date of 2017, 5 and 4 days, and has the preservation unit address: the microbial research institute of the Chinese academy of sciences, zip code 100101, west road No.1, north chen, chaoyang, china, and beijing.
According to a second aspect of the present invention, there is provided a biodegradation agent comprising the strain Geotrichum Fh 5.
According to a third aspect of the present invention, there is provided the use of the strain Geotrichum Fh5 and/or a biodegradation agent for degrading ammoniacal nitrogen of an object to be detected.
Wherein the substance to be detected is one or more of sewage, percolate and odor.
According to a fourth aspect of the present invention, there is provided a method of using the strain of Geotrichum Fh5 and/or the biodegradable agent, comprising the step of inoculating the Geotrichum Fh5 and/or the biodegradable agent into the specimen in a proportion of 1% by volume of the specimen to the Geotrichum Fh 5.
According to the fifth aspect of the invention, the preparation method of the biodegradation agent is provided, which comprises a screening culture step and a fermentation step of single geotrichum Fh5, wherein in the screening culture step: the culture temperature is 25-35 ℃, the pH value is 6.5-8.5, the rotating speed is 150-200 r/min, and the fermentation time is 36-72 hours. In the fermentation step: the fermentation temperature is 25-30 ℃, the pH value is 6.5-8.5, the rotation speed is 150-200 r/min, and the fermentation time is 36-72 hours.
Wherein in the screening culture step, the screening culture medium comprises the following components: 9.51g of brown sugar, 4.09g of sodium citrate and K2HPO414g，KH2PO46g，(NH4)2SO42.36g，MgSO4·7H20.2g of O, 2mL of trace elements and 1000mL of water.
In the fermentation culture step, the components of the fermentation culture medium are as follows: 9.51 to 19.02g of brown sugar, 4.09 to 8.17g of sodium citrate and K2HPO414g，KH2PO46g，(NH4)2SO42.36～4.72g，MgSO4·7H20.2g of O, 2mL of trace elements and 1000mL of water.
Wherein, the carbon/nitrogen ratio of the screening culture medium and the fermentation culture medium is 9-11: 1, and high-efficiency degradation can be achieved.
Wherein, when the ratio of brown sugar to sodium citrate in the screening culture medium and the fermentation culture medium is 3-5: 1, the ammonia nitrogen degradation effect is optimal. The combination of brown sugar and sodium citrate also plays a role in adjusting the pH value of the culture medium on the basis of providing a carbon source, so that the pH values of the screening culture medium and the fermentation culture medium are both 6.5-8.5.
Wherein the trace element components in the screening culture medium and the fermentation culture medium are EDTA & 2Na 5-57.1 g and ZnSO4·7H2O 3.9g，CaCl2·2H2O 7g，MnCl2·4H2O 5.1g，FeSO4·7H2O 5g，(NH4)6Mo7O24·4H2O1.1g，CuSO4·5H2O 1.6g，CoCl2·6H2O1.6 g, 1000mL of water.
The Geotrichum strain Fh5 can effectively degrade ammonia nitrogen with the concentration of 500mg/L in sewage and percolate and ammonia gas in odor, and in the degradation process, nitrite nitrogen and nitrate nitrogen are not accumulated, nitrification and denitrification can be synchronously performed, the degradation rate of ammonia nitrogen reaches 100%, and the residual total nitrogen in the sewage, the percolate or the odor is 10 mg/L.
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Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:
FIG. 1 shows an individual morphometric microscope picture of Geotrichum Fh5 strain according to an embodiment of the present invention;
FIG. 2 shows a phylogenetic tree of the Geotrichum Fh5 strain according to an embodiment of the present invention.
Detailed Description
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
To more clearly illustrate the geotrichum Fh5 and its use according to the invention, it will be further elucidated by way of a specific example.
Example 1: separation and purification of Geotrichum Fh5
Collecting garbage of refuse composting farm in south palace of Beijing CityInoculating 10ml percolate into 250ml triangular flask containing 100ml sterilized enrichment culture medium, shaking, performing shake culture at 28 deg.C and rotation speed of 160r/min for 7 days, and supplementing (NH) every 1 day4)2SO4To eliminate the unavailable NH4 +-N. And (3) coating 1ml of the bacterial liquid in the enrichment medium on a separation medium plate, carrying out inverted culture in a 28 ℃ incubator, and selecting a single bacterial colony growing on the plate to carry out repeated separation and purification to obtain a single bacterial strain named Fh 5.
Wherein, the enrichment medium comprises the following components: 11.9-19.02 g of brown sugar, K2HPO414g，KH2PO46g，(NH4)2SO42.36～4.72g，MgSO4·7H20.2g of O, 2mL of trace elements (EDTA-2 Na 5-57.1 g, ZnSO)4·7H2O 3.9g，CaCl2·2H2O 7g，MnCl2·4H2O 5.1g，FeSO4·7H2O 5g，(NH4)6Mo7O24·4H2O 1.1g，CuSO4·5H2O1.6g，CoCl2·6H2O1.6 g, water 1000mL), water 1000 mL; the composition of the separation and purification culture medium is as follows: 11.9-19.02 g of brown sugar, K2HPO414g，KH2PO46g，(NH4)2SO42.36～4.72g，MgSO4·7H20.2g of O, 2mL of trace elements (EDTA-2 Na 5-57.1 g, ZnSO)4·7H2O 3.9g，CaCl2·2H2O 7g，MnCl2·4H2O 5.1g，FeSO4·7H2O5g，(NH4)6Mo7O24·4H2O 1.1g，CuSO4·5H2O 1.6g，CoCl2·6H2O1.6 g, water 1000mL), agar 20g, water 1000 mL.
And (3) strain identification:
1. colony characteristics: the bacterial colony is in a milky white plush shape, the growth speed on a flat plate is high, the bacterial colony is in planar diffusion and is flat, the emission lines of surrounding hyphae grow, the thallus is wet and smooth, the front and back colors are the same, the hyphae are easy to pick out, the growth in a liquid culture medium is high, and the culture medium is turbid.
2. Individual morphological characteristics: as shown in FIG. 1, Fh5 mycelium is fungus with transverse septa, and after the mycelium matures, the mycelium breaks into single or chain-shaped long cylindrical node spores.
3. Phylogenetic analysis of strains: selecting a single colony of purified Fh5, inoculating the single colony in a screening culture medium, carrying out shake culture for 24 hours at the temperature of 30 ℃ and under the condition of a shaking table of 180r/min, then using a bacterial liquid as a template to prepare an ITS sequence of a PCR amplification strain, carrying out sequencing, and using molecular evolution genetic analysis software to prepare a sequencing result as a development tree thereof, and specifically as shown in figure 2, finding that the sequence homology of the Fh5 and the registered Geotrichum is the highest and reaches 99 percent, thereby determining that the Fh5 of the composite bacterial agent is Geotrichum.
4. And (3) molecular identification: the ITS sequence of Fh5 determined is as SED ID NO: 1, the DNA sequence is aligned on http:// archive-dtd.ncbi.nlm.nih.gov/website, and based on homology analysis, Geotrichum sp.Fh 5 is a new member of Geotrichum, which is named Geotrichum sp.Fh 5.
Example 2: preparation of biodegradable X1
The method comprises the following steps:
screening and culturing: and selecting a single Fh5 colony after purification, inoculating the single Fh5 colony into a screening culture medium, controlling the temperature, adjusting the pH to 6.5, rotating the speed, and fermenting for 72 hours to prepare the biological bacteria degradation agent mother liquor M1.
Wherein the screening culture medium comprises brown sugar 9.51g, sodium citrate 4.09g, and K2HPO414g，KH2PO46g，(NH4)2SO42.36g，MgSO4·7H20.2g of O, and trace elements (EDTA-2 Na 57.1g, ZnSO)4·7H2O 3.9g，CaCl2·2H2O 7g，MnCl2·4H2O 5.1g，FeSO4·7H2O 5g，(NH4)6Mo7O24·4H2O 1.1g，CuSO4·5H2O1.6g，CoCl2·6H2O1.6 g, water 1000mL)2mL, water 1000 mL.
Fermentation culture: adding the biological bacteria degradation agent mother liquor M1 into a fermentation culture medium according to the proportion of 1%, controlling the temperature, the pH value to be 6.5, rotating speed to be 150rpm, and fermenting for 72 hours to prepare the biological bacteria degradation agent X1.
Wherein the fermentation medium comprises brown sugar 9.51g, sodium citrate 4.09g, and K2HPO414g，KH2PO46g，(NH4)2SO42.36g，MgSO4·7H20.2g of O, and trace elements (EDTA-2 Na 57.1g, ZnSO)4·7H2O 3.9g，CaCl2·2H2O 7g，MnCl2·4H2O 5.1g，FeSO4·7H2O 5g，(NH4)6Mo7O24·4H2O 1.1g，CuSO4·5H2O1.6g，CoCl2·6H2O1.6 g, water 1000mL)2mL, water 1000 mL.
Example 3: preparation of biodegradable X2
The method comprises the following steps:
screening and culturing: and selecting a single colony of purified Fh5, inoculating the single colony into a screening culture medium, controlling the temperature at 30 ℃, adjusting the pH to 8.5, rotating at 200rpm, and fermenting for 36 hours to prepare the biological bacteria degradation agent mother liquor M2.
Wherein the screening culture medium comprises brown sugar 9.51g, sodium citrate 4.09g, and K2HPO414g，KH2PO46g，(NH4)2SO42.36g，MgSO4·7H20.2g of O, and trace elements (EDTA-2 Na 5g, ZnSO)4·7H2O 3.9g，CaCl2·2H2O 7g，MnCl2·4H2O 5.1g，FeSO4·7H2O 5g，(NH4)6Mo7O24·4H2O 1.1g，CuSO4·5H2O 1.6g，CoCl2·6H2O1.6 g, water 1000mL)2mL, water 1000 mL.
Fermentation culture: adding the biological bacteria degradation agent mother liquor M2 into a fermentation culture medium according to the proportion of 1%, controlling the temperature at 35 ℃, the pH value at 8.5, the rotating speed at 200rpm, and the fermentation time at 36 hours, and preparing the biological bacteria degradation agent X2.
Wherein the fermentation medium comprises brown sugar 19.02g, sodium citrate 8.17g, and K2HPO414g，KH2PO46g，(NH4)2SO44.72g，MgSO4·7H20.2g of O, and trace elements (EDTA-2 Na 5g, ZnSO)4·7H2O 3.9g，CaCl2·2H2O 7g，MnCl2·4H2O 5.1g，FeSO4·7H2O 5g，(NH4)6Mo7O24·4H2O 1.1g，CuSO4·5H2O 1.6g，CoCl2·6H2O1.6 g, water 1000mL)2mL, water 1000 mL.
Experimental example 1: application of geotrichum Fh5
Carrying out ammonia nitrogen index determination on a sample taken back by a large tower of a Majia building biological deodorization tower in Beijing: the ammonia nitrogen content of the tower odor washing liquid is 504mg/L, the pH value is 5.36, and the ammonia gas content at the gas outlet is 2.34mg/m3(ii) a Adding the geotrichum Fh5 of example 1 and sewage in the tower into a Nangong tower according to the proportion of 1%, culturing for 3d in an aeration environment at 32 ℃, degrading ammonia nitrogen in a tower odor washing liquid to 0, wherein the pH value is 6.43, and the ammonia content at an air outlet is 0.
Experimental example 2: application of biodegradation agent
Carrying out ammonia nitrogen index determination on a sample taken back by a small tower of a Majia building biological deodorization tower in Beijing: the ammonia nitrogen content of the odor washing liquid of the small tower is 471mg/L, the pH value is 6.24, and the ammonia gas content at the gas outlet is 3.13mg/m3(ii) a Adding the biodegradation agent X1 of example 2 into a Nangong small tower according to the volume ratio of 1% of contained geotrichum Fh5 and sewage in the tower, culturing for 3d in an aeration environment at 32 ℃, wherein the ammonia nitrogen degradation efficiency of the odor washing liquid of the small tower reaches 100%, the pH value is 7.07, and the ammonia content at the air outlet is 0.25mg/m3。
Experimental example 3: application of biodegradation agent
Performing index determination on a water sample taken back from a bacterial liquid pool of an Ashbya waste composting farm in Beijing and ammonia gas at an air inlet and an air outlet collected by a biological deodorization tower: the ammonia nitrogen content is 521mg/L, the pH value is 7.45, and the ammonia gas content at the air inlet of the biological deodorization tower is 10.13mg/m3The ammonia gas content at the gas outlet is 8.66mg/m3(ii) a Adding the biological degradation agent X2 obtained in the example 3 and sewage in the bacterial liquid into an asonavir liquid pool according to the mass ratio of 1%, culturing for 3d in an aeration environment at 30 ℃, degrading ammonia nitrogen to 0, wherein the degradation efficiency reaches 100%, the pH value is 7.26, and the ammonia gas content at an air inlet is 10.13mg/m3But the ammonia content at the gas outlet was 0.
In conclusion, the Geotrichum japonicum Fh5 disclosed by the invention can degrade the ammonia nitrogen content of 500mg/L, the degradation efficiency reaches 100%, and the total nitrogen residual amount is only 10 mg/L. Effectively relieves the sewage stink and stink pollution caused by ammoniacal nitrogen, has short treatment time in production, but has particularly obvious application effect.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
SEQUENCE LISTING
<110> Beijing epoxy environmental protection science and technology development Co., Ltd
<120> geotrichum strain Fh5 and application thereof
<160>1
<170>PatentIn version 3.5
<210>1
<211>374
<212>DNA
<213> Geotrichum (Geotrichum)
<400>1
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gagaaacaac gctcaaacaa gtatactttg ggggataccc caaagtgcaa tgtgcgttca 180
aaaactgatg attcacttct gcaattcaca agaaatatcg cgtttcgctg cgttcttcat 240
cgatacgaga accaagagat ccattgttaa aagttttaat tatttttgtt ttgattgtat 300
gatttttgtt tgctgtggta aattcacaaa tatttataat tcataatgat ccttccgcag 360
gttcacctac ggaa 374
Claims (5)
1. A Geotrichum (Geotrichum) Fh5 is characterized in that the Geotrichum is preserved in China general microbiological culture Collection center (CGMCC) in 2017, 5 and 4 months, and the preservation number is CGMCC No. 13777.
2. A biodegradation agent, comprising the Geotrichum fhi 5 according to claim 1.
3. Use of the geotrichum Fh5 of claim 1 and/or the biodegrading agent of claim 2 for degrading ammoniacal nitrogen in an object to be detected.
4. Use of the Geotrichum Fh5 and/or a biodegradation agent according to claim 3 for the degradation of ammoniacal nitrogen in an object to be detected,
the substance to be detected is one or more of sewage, percolate and odor.
5. The method of using the geotrichum Fh5 and/or the biodegradation agent of claim 2, comprising the steps of:
the geotrichum Fh5 and/or the biodegradation agent are inoculated into the substance to be detected in a proportion of 1% of the volume of the substance to be detected of the geotrichum Fh 5.
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| CN110272834B (en) * | 2019-05-23 | 2020-11-10 | 浙江工业大学 | Odorless microbial agent for kitchen waste treatment and preparation method and application thereof |
| CN110407333B (en) * | 2019-07-23 | 2021-06-15 | 中国科学院南京地理与湖泊研究所 | Horizontal subsurface flow wetland denitrification enhancement method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999028244A1 (en) * | 1997-12-01 | 1999-06-10 | Fungi-Gulp Pty. Ltd. | Curtain assembly for waste treatment |
| CN101434907A (en) * | 2008-12-18 | 2009-05-20 | 天津大学 | Microbial preparation for processing refuse leachate and preparation |
| CN102250769A (en) * | 2011-06-15 | 2011-11-23 | 江苏新琦环保有限公司 | Biological agent for organic waste composting and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999028244A1 (en) * | 1997-12-01 | 1999-06-10 | Fungi-Gulp Pty. Ltd. | Curtain assembly for waste treatment |
| CN101434907A (en) * | 2008-12-18 | 2009-05-20 | 天津大学 | Microbial preparation for processing refuse leachate and preparation |
| CN102250769A (en) * | 2011-06-15 | 2011-11-23 | 江苏新琦环保有限公司 | Biological agent for organic waste composting and preparation method thereof |
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