CN106434453A - Microbial flora capable of rapidly degrading estrogen and analogs in chicken manure - Google Patents
Microbial flora capable of rapidly degrading estrogen and analogs in chicken manure Download PDFInfo
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- CN106434453A CN106434453A CN201610876461.4A CN201610876461A CN106434453A CN 106434453 A CN106434453 A CN 106434453A CN 201610876461 A CN201610876461 A CN 201610876461A CN 106434453 A CN106434453 A CN 106434453A
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- comamonas testosteroni
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- 229940011871 estrogen Drugs 0.000 title claims abstract description 32
- 239000000262 estrogen Substances 0.000 title claims abstract description 32
- 241000287828 Gallus gallus Species 0.000 title claims abstract description 27
- 210000003608 fece Anatomy 0.000 title claims abstract description 27
- 239000010871 livestock manure Substances 0.000 title claims abstract description 27
- 230000000813 microbial effect Effects 0.000 title abstract description 10
- 230000000593 degrading effect Effects 0.000 title abstract description 3
- 241000589518 Comamonas testosteroni Species 0.000 claims abstract description 35
- 230000015556 catabolic process Effects 0.000 claims abstract description 27
- 238000006731 degradation reaction Methods 0.000 claims abstract description 27
- 150000001875 compounds Chemical class 0.000 claims abstract description 18
- 241001620353 Comamonas testosteroni ATCC 11996 Species 0.000 claims abstract description 14
- 241000589562 Brucella Species 0.000 claims abstract description 8
- 241000607598 Vibrio Species 0.000 claims abstract description 8
- 244000005700 microbiome Species 0.000 claims description 13
- 230000001629 suppression Effects 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 230000003637 steroidlike Effects 0.000 claims description 12
- 101710172715 Hydrolase 3 Proteins 0.000 claims description 10
- 239000012530 fluid Substances 0.000 claims description 9
- 235000015097 nutrients Nutrition 0.000 claims description 9
- -1 hydroxyl sterol Chemical class 0.000 claims description 8
- 206010047400 Vibrio infections Diseases 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 claims description 5
- 229960002064 kanamycin sulfate Drugs 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- 101150076489 B gene Proteins 0.000 claims description 3
- 238000003209 gene knockout Methods 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 230000009467 reduction Effects 0.000 claims description 3
- 241000589519 Comamonas Species 0.000 claims description 2
- 229930182558 Sterol Natural products 0.000 claims description 2
- 230000003647 oxidation Effects 0.000 claims description 2
- 238000007254 oxidation reaction Methods 0.000 claims description 2
- 235000003702 sterols Nutrition 0.000 claims description 2
- 150000002576 ketones Chemical class 0.000 claims 1
- 210000001550 testis Anatomy 0.000 claims 1
- 230000036541 health Effects 0.000 abstract description 4
- 238000003912 environmental pollution Methods 0.000 abstract description 3
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 7
- 229960005309 estradiol Drugs 0.000 description 7
- 229930182833 estradiol Natural products 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000002131 composite material Substances 0.000 description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 102000038379 digestive enzymes Human genes 0.000 description 2
- 108091007734 digestive enzymes Proteins 0.000 description 2
- 230000001076 estrogenic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 244000005706 microflora Species 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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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
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F11/00—Treatment of sludge; Devices therefor
- C02F11/02—Biological treatment
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/0105—3Alpha-hydroxysteroid 3-dehydrogenase (B-specific) (1.1.1.50)
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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01051—3 (or 17)-Beta-hydroxysteroid dehydrogenase (1.1.1.51)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01213—3Alpha-hydroxysteroid 3-dehydrogenase (A-specific) (1.1.1.213)
-
- 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
- C02F2101/30—Organic compounds
- C02F2101/305—Endocrine disruptive agents
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Abstract
The invention discloses microbial flora capable of rapidly degrading estrogen and analogs in chicken manure and belongs to the technical field of microbial engineering. The microbial flora comprises equal amounts of Comamonas testosteroni ATCC11996, KF1-Fe (Comamonas testosteroni ATCC49202), Ca3, Cb7, M6, M2, A3, vibrio H5 and Brucella S19-1. The environment adaptability of strains and the degradation efficiency of the estrogen and analogs are improved under the coordinate symbiosis action of compound microbial flora, and the risk caused by environmental pollution to ecology and human health is effectively reduced.
Description
Technical field
The invention belongs to microbial engineering field, especially relating to one can be with degrading estrogen and homologue thereof
Microorganism species.
Background technology
In environment, the main source of estrogen is the intensive metaplasia of Chemical Manufacture, pharmacy industry, agricultural, forestry and animal husbandry
Produce.In the half a century in past, animal husbandry have become as estrogen in environment primary pollution source (Maser E.et al.,
J.Steroid Biochem.Mol.Biol.,121,633-640,2010).The World Health Organization (WHO) suggestion in 2003 is drunk
Estrogen in water and homologue thereof should receive grams per liter less than 1.The estradiol that China Shenzhen and Hong Kong determine area water source contains
Amount, result considerably beyond WHO standard (Gelsleichter J.et al., Chemosphere., 63,1506-1522,
2006).Estrogen excessive in diet can make the incidence of disease of female tumor increase；Make under semen volume and sperm concentration
Fall.Threaten future world population balance (Andersson A.M.et al., Eur.J.Endocrinol., 140,477-485,
1999), therefore this subject under discussion by the most attention of developed country.Nineteen fifty-one, German scholar is first at Barcelona, ESP
Secondary separate the Gram-negative bacteria Comamonas being capable of degradation of steroid compounds (estrogen belongs to steroidal compounds)
testosteroni(ATCC11996)(Talalay P.et al.,Nature,170,620-621,1952).Research proves：
Comamonas testosteroni environmental sound.Although major part participate in steroid compound metabolism enzyme gene by
Clone, but metabolic pathway still unintelligible complete (Maser E.et al., Biochem.Biophys.Res.Commun., 272,
622-628,2000).The enzyme of subparticipation steroid compound metabolism is induced enzyme, i.e. in bacterial body not or only seldom
Scale reaches, but can be induced by substrate or product, thus promotes the raising of enzyme gene expression, and then increases bacterium to sterides compound
The degradation capability (Xiong G.et al., Chem.Biol.Interact., 178,215-220,2009) of thing.By to induction
The research of mechanism, it has been found that special operon that some regulation steroid compound metabolic enzyme genes are expressed, suppression
With activation (Maqbool F.et al., Life Sci., 3205,30048-30055,2015).By engineered method
Knock out some suppression son, by strong promoter insert activate subbase because of upstream all may improve Comamonas testosteroni
The ability of degradation of steroid compound.At present both at home and abroad mainly from the angle of single bacterial strain, study its mechanism of degradation and
Removal to estrogen in environment.Therefore need badly in the middle of prior art and want a kind of novel technical scheme to solve this problem.
Content of the invention
The technical problem to be solved is：There is provided a kind of can estrogen and homologue thereof in fast degradation chicken manure
Microorganism species, by the collaborative synbiosis of complex microbial community improve the adaptive capacity to environment of bacterial classification and estrogen and
The degradation efficiency of homologue, the ecology that effectively reduction environmental pollution brings and the risk of health.
In a kind of energy fast degradation chicken manure, the microorganism species of estrogen and homologue thereof, is characterized in that：Including equivalent
Comamonas testosteroni (Comamonas testosteroni ATCC11996), Ca3, Cb7, M6, M2, A3, KF1-Fe
(Comamonas testosteroni ATCC49202) vibrios H5 and Brucella sp S19-1,
Described Comamonas testosteroni (Comamonas testosteroni ATCC11996) its deposit number is
1.3167；
After described Ca3 is the suppression sub-A gene knockout of steroidal compounds hydrolase 3 α-HSD in Comamonas testosteroni
The dissociant obtaining；
Described Cb7 is after the sub-1 B gene of suppression of steroidal compounds hydrolase 3 α-HSD in Comamonas testosteroni knocks out
The dissociant obtaining；
Described M6 is that the sub-PhaR of suppression of hydrolase 3, the 17 β-HSD of steroidal compounds in Comamonas testosteroni knocks out
The dissociant obtaining；
Described M2 be steroidal compounds in Comamonas testosteroni hydrolase 3,17 β-HSD in sigma factor suppression son
RseA knocks out the dissociant obtaining；
Described A3 is that in Comamonas testosteroni, strong promoter tac inserts the variant that the upstream of 3 α-HSD genes obtains.
KF1-Fe is Comamonas testosteroni (Comamonas testosteroni ATCC49202).
Described Comamonas testosteroni and KF1-Fe are cultivated by LB fluid nutrient medium.
Described Ca3, Cb7, M6 and M2 are cultivated by kanamycin sulfate LB fluid nutrient medium.
Described H5 and S19-1 is by cultivating containing the LB fluid nutrient medium that concentration is 2%NaCl.
Described vibrios H5 is with Brucella sp S19-1 and separates from the Germany Baltic Sea.
By above-mentioned design, the present invention can bring following beneficial effect：A kind of can female in fast degradation chicken manure swash
Element and the microorganism species of homologue thereof, the environment being improved bacterial classification by the collaborative synbiosis of complex microbial community adapts to energy
Power and the degradation efficiency of estrogen and homologue thereof, the ecology that effectively reduction environmental pollution brings and the risk of health.
In Ca3 and in two bacterial strains of Cb7 a crucial steroidal compounds digestive enzyme 3 α-HSD (oxidation of hydroxyl sterol/
Reductase) expression improve nearly ten times, thus improve the digestive enzyme ability to steroidal compounds；
A3 can improve bacterium hydrolysis steroidal compounds ability；
It is resistant to height from the littoral isolated in the Germany Baltic Sea and ooze (NaCl：5%) and the vibrios H5 of environmental sound
With Brucella sp S19-1, not only there is the ability of degradation of steroid compound, additionally it is possible to tolerance height oozes environment.The osmotic pressure of chicken manure is past
High toward comparing, it is thus possible to be more suitable for the yeasting of chicken manure.
Brief description
Below in conjunction with the drawings and specific embodiments, the present invention is further illustrated：
Fig. 1 be the present invention a kind of can in fast degradation chicken manure the microorganism species of estrogen and homologue thereof to female in chicken manure
Glycol degradation situation column schematic diagram.
Fig. 2 be the present invention a kind of can in fast degradation chicken manure the microorganism species of estrogen and homologue thereof to female in chicken manure
Hormone homologue degraded situation column schematic diagram.
Detailed description of the invention
In a kind of energy fast degradation chicken manure, the microorganism species of estrogen and homologue thereof, is characterized in that：Including equivalent
Comamonas testosteroni (Comamonas testosteroni ATCC11996), Ca3, Cb7, M6, M2, A3, KF1-Fe
(Comamonas testosteroni ATCC49202), vibrios H5 and Brucella sp S19-1,
Described Comamonas testosteroni (Comamonas testosteroni ATCC11996) its deposit number is
1.3167；
Described Ca3 is sterides compound in Comamonas testosteroni (Comamonas testosteroni ATCC11996)
The dissociant obtaining after the sub-A gene knockout of suppression of thing hydrolase 3 α-HSD；
Described Cb7 is sterides compound in Comamonas testosteroni (Comamonas testosteroni ATCC11996)
The dissociant that the sub-1 B gene of suppression of thing hydrolase 3 α-HSD obtains after knocking out；
Described M6 is sterides compound in Comamonas testosteroni (Comamonas testosteroni ATCC11996)
The sub-PhaR of suppression of hydrolase 3, the 17 β-HSD of thing knocks out the dissociant obtaining；
Described M2 is sterides compound in Comamonas testosteroni (Comamonas testosteroni ATCC11996)
In hydrolase 3, the 17 β-HSD of thing, the sub-RseA of suppression of sigma factor knocks out the dissociant obtaining；
Described A3 is strong promoter in Comamonas testosteroni (Comamonas testosteroni ATCC11996)
Tac inserts the variant that the upstream of 3 α-HSD genes obtains.
KF1-Fe is Comamonas testosteroni (Comamonas testosteroni ATCC49202).
Described Comamonas testosteroni and KF1-Fe are cultivated by LB fluid nutrient medium.
Described Ca3, Cb7, M6 and M2 are cultivated by kanamycin sulfate LB fluid nutrient medium.
Described H5 and S19-1 is by cultivating containing the LB fluid nutrient medium that concentration is 2%NaCl.
Described vibrios H5 is with Brucella sp S19-1 and separates from the Germany Baltic Sea.
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is certainly
Routine biochemistry reagent shop is commercially available.Quantitative test in following example, is respectively provided with four times and repeats experiment, and result is made even
Average± standard deviation (SD), p≤0.05.
Estradiol standard items：Buy in Sigma-Aldrich company.
The preparation of estradiol storing solution：Weigh 133mg estradiol standard items to be dissolved in the absolute ethyl alcohol of 10mL, obtain
The estradiol solution of 50mM, is stored in 4 DEG C of refrigerators.
LB culture medium：Yeast extract 5g, tryptone 10g, sodium chloride 10g, NaOH adjust pH to 7.0, and distilled water is fixed
Hold to 1L.121 DEG C, 20min autoclaving.
Kanamycin sulfate：Buy in Scientific research special company.
Trifluoroacetic acid aqueous solution：Buy in Fisher scientific company.
The cultivation of embodiment the 1st, bacterial classification and the preparation of flora
Comamonas testosteroni (Comamonas testosteroni ATCC11996), KF1-Fe, Ca3, Cb7, M6,
Nine bacterial classifications of M2, A3, H5 and S19-1, take 200 μ L bacterium solution respectively from each bacterial classification mother liquor, add the LB culture medium of 2mL.Wherein
The LB culture medium of Ca3, Cb7, M6, M2 and A3 adds the kanamycin sulfate of 40 μ g/mL；In the LB culture medium of H5 and S19-1
Add the NaCl of 2%.By above-mentioned nine kinds of bacterium solution at 27 DEG C, 180r/min shaking table is cultivated, overnight.Respectively with LB culture medium by above-mentioned
Bacterium solution after nine kinds of cultivations adjusts OD600nm=1.The bacterium solution taking same volume respectively mixes, and i.e. obtains composite flora.
The response characteristic test of estrogen in embodiment the 2nd, composite flora degraded chicken manure：
(1) degraded in flora for the chicken manure：
It is 0 by composite bacteria liquid and LB culture medium with volume ratio:1；1:99；1:9；1:0 mixes, thus prepares OD
Value is respectively 0；0.01；0.1；The composite bacteria liquid of 1.
Take chicken manure 1g respectively and join OD value respectively 0；0.01；0.1；In the composite bacteria liquid of 1, additionally add artificially
The estradiol solution of 0.5mM, at 27 DEG C, 180r/min shaking table is cultivated 10 days.
(2) extraction of estrogen and homologue in chicken manure：
Owing to estrogen and homologue thereof are soluble in chloroform, therefore we use chloroform extraction method to extract chicken manure or through micro-
Estrogen and homologue thereof in chicken manure after biological flora degraded.It is i.e. separately added into 4mL sterilized water and 1mL chloroform, and be transferred to
In centrifuge tube, vibrate 10 minutes.Centrifuge 10 minutes under the conditions of 4000r/min.Take off phase 500 μ L, under the conditions of 13000r/min
Centrifugal 10 minutes, take off phase clarified solution 300 μ L, vacuum drying, to be measured.
(3) it is dissolved in acetonitrile by above-mentioned through the sample that chloroform is obtained by extraction：Water=1：In 1 (volume ratio), use efficient liquid phase
Chromatogram (HPLC, Waters e2695) measures estrogen degraded situation.Detector is UV (2988Water Detector), chromatogram
Post is C18 post (5 μm of particle diameters, 4.6mm × 150mm, Waters), and flowing is acetonitrile mutually：Water=1：1 (volume ratio), detects wavelength
For 225nm, flow velocity is 1mL/min, column temperature 25 DEG C, sample size 5 μ L.
Calculated the degradation rate of estrogen by high performance liquid chromatography result.Every kind of comparison is all repeated 4 times.Result such as Fig. 1
Shown in, show that complex microbial community can be with the estrogen in efficient degradation chicken manure sample.Through the microflora degradation of 10 days, artificially
The degradation rate of the high concentration estradiol (0.5mM) adding is up to 70%.In chicken manure, original estrogen homologue then can be complete
Degradable is as shown in Figure 2.
The above results illustrates, complex microbial community of the present invention can significantly improve the estrogen degradation rate in chicken manure, in fall
The Ecological Environment Risk aspect that the steroid hormone that low aquaculture causes pollutes has important using value.And the present invention has
Low cost, the advantage that simple to operate, degradation efficiency is high, Environmental compatibility is good, reduce use chemical reagent, avoid secondary pollution,
The biological prosthetic aspect polluting environment at estrogenic chemicals has a good application prospect.Therefore can be used for new estrogenic fall
Solve the merchandized handling of microbial inoculum.
Claims (5)
1. can the microorganism species of estrogen and homologue thereof in fast degradation chicken manure, it is characterized in that：Including the testis of equivalent
Ball ketone comamonas (Comamonas testosteroni ATCC11996), KF1-Fe (Comamonas
Testosteroni ATCC11996), Ca3, Cb7, M6, M2, A3, vibrios H5 and Brucella sp S19-1,
Described Comamonas testosteroni (Comamonas testosteroni ATCC11996) its deposit number is 1.3167；
Described Ca3 is the pressing down of steroidal compounds hydrolase 3 α-HSD hydroxyl sterol oxidation/reduction enzyme in Comamonas testosteroni
The dissociant obtaining after system A gene knockout；
Described Cb7 is to obtain after the sub-1 B gene of suppression of steroidal compounds hydrolase 3 α-HSD in Comamonas testosteroni knocks out
Dissociant；
Described M6 is that the sub-PhaR of suppression of hydrolase 3, the 17 β-HSD of steroidal compounds in Comamonas testosteroni knocks out and obtains
Dissociant；
Described M2 be steroidal compounds in Comamonas testosteroni hydrolase 3,17 β-HSD in the sub-RseA of suppression of sigma factor
Knock out the dissociant obtaining；
Described A3 is that in Comamonas testosteroni, strong promoter tac inserts the variant that the upstream of 3 α-HSD genes obtains.
KF1-Fe is Comamonas testosteroni (Comamonas testosteroni ATCC49202).
2. according to claim 1 a kind of can the microorganism species of estrogen and homologue thereof in fast degradation chicken manure, its
Feature is：Described Comamonas testosteroni and KF1-Fe are cultivated by LB fluid nutrient medium.
3. according to claim 1 a kind of can the microorganism species of estrogen and homologue thereof in fast degradation chicken manure, its
Feature is：Described Ca3, Cb7, M6, M2 and A3 are cultivated by kanamycin sulfate LB fluid nutrient medium.
4. according to claim 1 a kind of can the microorganism species of estrogen and homologue thereof in fast degradation chicken manure, its
Feature is：Described H5 and S19-1 is by cultivating containing the LB fluid nutrient medium that concentration is 2%NaCl.
5. according to claim 1 a kind of can the microorganism species of estrogen and homologue thereof in fast degradation chicken manure, its
Feature is：Described vibrios H5 is with Brucella sp S19-1 and separates from the Germany Baltic Sea.
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| CN112941088A (en) * | 2021-02-04 | 2021-06-11 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Gene related to brucella virulence and application thereof in evaluation of brucella virulence and preparation of attenuated brucella |
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