CN102211823B - Method for degrading steroid pollutants in water body - Google Patents
Method for degrading steroid pollutants in water body Download PDFInfo
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- CN102211823B CN102211823B CN 201110095251 CN201110095251A CN102211823B CN 102211823 B CN102211823 B CN 102211823B CN 201110095251 CN201110095251 CN 201110095251 CN 201110095251 A CN201110095251 A CN 201110095251A CN 102211823 B CN102211823 B CN 102211823B
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Abstract
The invention provides a method for degrading steroid pollutants in a water body. The method comprises the following steps of: preparing a degrading bacterial suspension from a comamonas testoseroni modified engineering strain P8; inoculating the degrading bacterial suspension to the water body containing the steroid pollutants, wherein the concentration of the steroid pollutants is more than or equal to 0.1ng/L, the pH value is 7.0+/-0.5, and the temperature is between 25 to 30 DEG C; and after inoculation, stirring or oscillating a water body system to ensure that the steroid pollutants in the water body are degraded, wherein the degradation rate after 48+/-1 hours can reach 68.32 to 70.22 percent, the density of the inoculated engineering strain is reduced exponentially, and the amount of living cells becomes less and less as time goes by and finally reaches 10 to 100cfu/L. The method has the advantages of simplicity in operation, low cost, high degradation efficiency, strong adaptability and the like.
Description
Technical field
The invention belongs to biological technical field.The present invention relates to the microbial environment restorative procedure, more specifically relate to a kind of degradation method of steroid pollutants in water body.
Background technology
Environmental hormone (environmental hormone EH) refers to human some toxic chemical substance that is discharged in the environment in production, life process, hormone in they and the humans and animals body has similar chemical structure, bringing into play similar functions of hormones, they enter in the body, upset the normal secretion of hormone, the physiology program of humans and animals is got muddled, cause the function generation obstacles such as reproduction, immunity.Body, offspring or population are caused the xenobiotics of reversibility or non-reversibility biological effect, be also referred to as " exogenous endocrine disrupter ".Steroid hormone (Steroid hormone) is the main class in the environmental hormone.In numerous environmental hormone materials, the hormonal activity of steroid compound is much higher than other environmental hormone class materials, will demonstrate stronger interference secretion under extremely low concentration conditions.The pollution of steroid hormone mainly refers to the estrogenic pollution of steroid, natural estrogen (mainly being E2 and E1) and synthetic oestrogenic hormon (EE2) are the essential substance that has endocrine disrupting in the water surrounding, i.e. oestrone, estradiol, trihydroxy-oestrin etc.
Comamonas testosteroni (
Comamonas testosteroni) be a kind of gram negative bacillus of strictly aerobic, non-fermentation, the flagellum of extremely growing thickly is arranged, belong to Comamonas (
Comamonas) a kind.Distribute at occurring in nature very extensive, this bacterium has the ability of the multiple difficult degradation compound of degraded, can be with aromatic compound and steroid compound as sole carbon source and the energy.
Comamonas testosteroni degrading enzyme gene 3 α hydroxysteroid dehydrogenases (3 α-HSD) in degraded digestion steroid substances, play a crucial role, study the Regulation Mechanism of this gene, determine that tentatively 3 α-HSD is one of initial enzyme in many C19-C27 steroid catabolic pathways.But utilize the rarely seen report of research of steroid substances in the Comamonas testosteroni strain pollution degradation water body.
Summary of the invention
The object of the present invention is to provide a kind of degradation method of steroid pollutants in water body, to solve the pollution problem of such material in the water body.
To achieve these goals, the technical scheme taked of the degradation method of steroid pollutants in water body of the present invention is as follows:
The engineering strain P8 of Comamonas testosteroni improvement is made bacteria suspension, be inoculated in the water body that contains the steroid pollutent, make the steroid pollutants in water body degraded; Described engineering strain P8 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preservation date on January 25th, 2008, preserving number is CGMCC NO.2365.
The concrete steps of described degradation method are as follows:
(1) microbial culture: take out and be stored in-80 ℃ engineering strain P8, draw 200 μ L and join in the sterilizing test tubes that 4 mL LB liquid nutrient mediums are housed, at 30 ℃, constant-temperature table 12～16h under the 180 r/min culture condition; The bacterial strain of activation is transferred to and contains in the antibiotic LB liquid nutrient medium, and wherein microbiotic is for adding 60 μ g/mL Amp, and at 30 ℃, constant-temperature table is cultivated 12～16h under the 180 r/min conditions;
(2) preparation of bacteria suspension: engineering strain P8 regulates the OD of its bacterium liquid after cultivating
600Value is for 0.95-1.05, centrifugal 5 min under 2000 rpm/min then, and collecting thalline, to be suspended in weight ratio be to make bacteria suspension among 0.85% the NaCl;
(3) the pH value of adjusting polluted-water is 7.0 ± 0.5,25～30 ℃ of the temperature of polluted-water; Press 0.5% inoculation bacteria suspension of polluted-water volume, stir simultaneously or vibrate, make the steroid contaminant degradation in the water body.
Concentration need 〉=the 0.1ng/L of steroid pollutants in water body wherein.
The engineering strain P8 that the present invention uses is
TeiRThe gene efficient expression bacterium, this bacterial strain is at wild strain
Comamonas testosteroni teiRThe upstream of gene has added
TacStrong promoter, high efficient expression
TeiRGene meanwhile can be induced in a large number
3a-HSD/CRExpression; Can carry out the orientation regulation and control from the transcriptional control level, improve Comamonas testosteroni to the degradation capability of the Enviromental pollutants such as sterid and polynuclear aromatics.
Degradation method of the present invention 48 ± 1 h behind inoculation P8 bacterial strain can reach 68.32%～70.22% to the degradation rate of steroid pollutent.And inoculation engineering bacteria density is index decreased, and As time goes on viable cell quantity is fewer and feweri, finally can reach 10-100 cfu/L.
Advantage of the present invention is that cost is low, simple to operate, degradation efficiency is high, strong adaptability, the degraded that is applicable to environmental classes sterol pollutent in various natural water bodies and the artificial water is removed, and is having a good application prospect aspect the biological restoration of steroid contaminate environment.
Description of drawings
Fig. 1 is that engineering strain P8 is to the degraded of oestrone;
Fig. 2 is that engineering strain P8 is to the degraded of estradiol;
Fig. 3 is that engineering strain P8 is to the degraded of trihydroxy-oestrin.
Embodiment
Following specific embodiments of the invention further describe the present invention, but the present invention is not limited only to this.
1. microbial culture: take out and be stored in-80 ℃ engineering bacteria bacterial classification, draw 200 μ L and join in the sterilizing test tubes that 4 mL LB liquid nutrient mediums are housed, at 30 ℃, constant-temperature table spends the night under the 180 r/min culture condition.The bacterial strain of activation is transferred to contain in the antibiotic LB liquid nutrient medium and (adds 60 μ g/mL Amp), at 30 ℃, constant-temperature table incubated overnight under the 180 r/min conditions.
The preparation of bacterial suspension: the engineering strain of incubated overnight, regulate the OD of its bacterium liquid
600Value is centrifugal 5 min of 1.0,2000 rpm/min, collects thalline and is suspended among 0.85% the NaCl and makes bacteria suspension.
Polluted-water to steroid enriching pollutants minimum concentration 〉=0.1ng/L of detecting, regulating its pH value is 7.0 ± 0.5, get the bacteria suspension of respective volume by 0.5% of polluted-water volume, the inoculation engineering bacteria also stirs or oscillation system, makes the steroid pollutants in water body degraded.
2. the degraded of steroid substances in the Fuzhou Section of Minjiang River polluted-water
In May, 2010 is sewage draining exit water sampling 5 L near the bridge of Fuzhou Section of Minjiang River flood mountain, throw in the P8 bacterial strain by above-mentioned testing sequence steroid substances in the water body is degraded, the content of estradiol, oestrone, trihydroxy-oestrin in 12 h sampling detection water body.Such as Fig. 1, Fig. 2 and shown in Figure 3, engineering bacteria P8 can reach 68.32%～70.22% to the degradation rate of steroid pollutent main component (oestrone, estradiol, trihydroxy-oestrin) behind 48 ± 1 h.
Claims (1)
1. the application of Comamonas testosterone engineering bacteria in the degraded steroid pollutants in water body, it is characterized in that: the engineering strain P8 of Comamonas testosteroni improvement is made bacteria suspension, be inoculated in the water body that contains the steroid pollutent, make the steroid pollutants in water body degraded; Described engineering strain P8 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preservation date on January 25th, 2008, preserving number is CGMCC NO.2365; The concrete steps of described degraded are as follows:
(1) microbial culture: take out and be stored in-80 ℃ engineering bacteria bacterial classification, draw 200 μ L and join in the sterilizing test tubes that 4 mL LB liquid nutrient mediums are housed, at 30 ℃, constant-temperature table spends the night under the 180 r/min culture condition;
The bacterial strain of activation is transferred to and contains in the antibiotic LB liquid nutrient medium, add 60 μ g/mL Amp, at 30 ℃, constant-temperature table incubated overnight under the 180 r/min conditions;
The preparation of bacterial suspension: the engineering strain of incubated overnight, regulate the OD of its bacterium liquid
600Value is centrifugal 5 min of 1.0,2000 rpm/min, collects thalline and is suspended among 0.85% the NaCl and makes bacteria suspension;
Polluted-water to steroid enriching pollutants minimum concentration 〉=0.1ng/L of detecting, regulating its pH value is 7.0 ± 0.5, get the bacteria suspension of respective volume by 0.5% of polluted-water volume, the inoculation engineering bacteria also stirs or oscillation system, makes the steroid pollutants in water body degraded; Described steroid pollutent is oestrone, estradiol, trihydroxy-oestrin.
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| CN 201110095251 CN102211823B (en) | 2011-04-15 | 2011-04-15 | Method for degrading steroid pollutants in water body |
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| CN 201110095251 CN102211823B (en) | 2011-04-15 | 2011-04-15 | Method for degrading steroid pollutants in water body |
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| CN106434453A (en) * | 2016-10-08 | 2017-02-22 | 吉林工商学院 | Microbial flora capable of rapidly degrading estrogen and analogs in chicken manure |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006271380A (en) * | 2005-03-04 | 2006-10-12 | Institute Of Physical & Chemical Research | Method for producing steroid metabolism product using gene disruption strain of bacterium of genus comamonas |
| CN101302489A (en) * | 2008-05-08 | 2008-11-12 | 福建农林大学 | Comamonas testosterone engineering bacteria and use thereof |
| CN101434929A (en) * | 2008-12-25 | 2009-05-20 | 福建农林大学 | Comamonas testosterone engineering bacteria |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006271380A (en) * | 2005-03-04 | 2006-10-12 | Institute Of Physical & Chemical Research | Method for producing steroid metabolism product using gene disruption strain of bacterium of genus comamonas |
| CN101302489A (en) * | 2008-05-08 | 2008-11-12 | 福建农林大学 | Comamonas testosterone engineering bacteria and use thereof |
| CN101434929A (en) * | 2008-12-25 | 2009-05-20 | 福建农林大学 | Comamonas testosterone engineering bacteria |
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