CN103114066B - Diethylamine degradation pseudomonas strain EYA-2 and application thereof - Google Patents
Diethylamine degradation pseudomonas strain EYA-2 and application thereof Download PDFInfo
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- CN103114066B CN103114066B CN201310083191.8A CN201310083191A CN103114066B CN 103114066 B CN103114066 B CN 103114066B CN 201310083191 A CN201310083191 A CN 201310083191A CN 103114066 B CN103114066 B CN 103114066B
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- diethylamine
- eya
- pseudomonas
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Abstract
The invention provides degrading bacteria and immobilized cells for eliminating diethylamine pollution, and belongs to the high technical field of biology. A used strain is Gram staining reaction negative bacteria EYA-2, and identified as pseudomonas (Pseudomonas sp.). Main biological characteristics are that the strain EYA-2 is G-, logarithmic growth phase thallus is rod-shaped, a bacterial colony is of a round white bulge, nitrate can be reduced, the oxidase reaction is positive, indole is not produced, spores are not generated, and an aerobic growth is carried out. Diethylamine can be taken as the sole carbon source and nitrogen source for the growth, and is completely mineralized to produce ammonia nitrogen. Under the condition of a laboratory shake flask, the degradation rate of 100mg/L diethylamine by the strain reaches 100%, and the problem that diethylamine is difficultly biodegraded in wastewater treatment is solved.
Description
Technical field
The present invention relates to environment is biological technical field, particularly, relates to strain diethylamine degraded pseudomonas strains EYA-2 and an application thereof.
Background technology
Organic amine product is widely used in the industries such as agricultural chemicals, medicine, rubber, synthetic dyestuff, oil, process hides, washing composition and organic synthesis, inorganic chemical industry.Organic amine product category is many, and each product all can have remaining raw material and intermediate product to enter in waste water in process of production, the most high nitrogen low-carbon (LC) of these materials, and poisonous and harmful, is difficult to biological degradation, thereby has brought difficulty to the processing of this type of wastewater from chemical industry.
Diethylamine is the synthetic important source material of organic solvent and chemical industry, the catalyzer of the poly-carbonic acid vinegar phosgenation of conduct in synthetic resins is manufactured, the stopper of tetrafluoroethylene and thiofide; Also can be used as food preservatives, emulsifying agent; Still manufacture the raw material of dyestuff, sterilant simultaneously.Diethylamine (diethylamine) is a kind of fatty amine, molecular formula C
4h
11n, has strong ammonia stink, irritant, corrodibility, inflammable, volatile.Diethylamine is extensively present in the factory effluents such as organic synthesis, agricultural chemicals, dyestuff, adopts physico-chemical process at present for the processing containing diethylamine waste water more, and its cost is higher, and easily causes secondary pollution; And bioremediation technology can be degraded and transform all kinds of different organic pollutants because of microorganism itself and had huge potentiality.Therefore the efficient bacterium that, screening obtains removing diethylamine from physical environment is significant.
Summary of the invention
The object of the invention is to for the practical problems in production practice and demand, utilize method of microorganism to carry out the pollutent in degrading waste water, be applicable to modern waste water treatment process, make discharged wastewater met the national standard, reduce and produce and use cost simultaneously.
The invention provides a kind of diethylamine degradation bacteria strains EYA-2, its bacterial strain is a strain gramstaining reaction negative bacterium, in
on January 15th, 2013be preserved in Chinese microorganism strain and preserve management committee's common micro-organisms center (being called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number
cGMCC NO.7140, through identify, its sequence be Rhodopseudomonas (
pseudomonas sp.).Main Biological is G
-, logarithmic phase thalline is shaft-like, and bacterium colony is circular yellow crowned, and bacterium colony is dry, can reduce nitrate, the oxydase reaction positive, edwardsiella hoshinae, without gemma, aerobic growth.
The invention provides the application of pseudomonas EYA-2 in diethylamine degraded.
The immobilized spherule that the present invention also provides described bacterial strain EYA-2 to prepare.
The present invention also provides a kind of bacterial strain EYA-2 to be prepared into the method for immobilized spherule, comprises the following steps:
1) be inoculated in LB liquid nutrient medium in 30 ℃ with a small amount of bacterial classification of transfering loop picking on slant medium, under 160r/min condition, shaking culture is to logarithmic phase;
2) by above-mentioned cultured bacterium liquid centrifugal 2min under the condition of 8000r/min, outwell supernatant liquor, add the sterilized water of same volume, shake up the centrifugal 2min of rear 8000r/min, so wash twice, with the sterilized water suspension of same volume, make thalline liquid.
3) 4% sodium alginate soln of getting 0.7ml thalline liquid and 20ml mixes in normal temperature, then with syringe, mixed solution is splashed into the 3%CaCl under normal temperature
2solution obtains calcium alginate gel immobilized spherule after 4 ℃ of crosslinked calcification 6h, aseptic washing be stored in for twice 4 ℃ for subsequent use.
LB substratum described in step 1) is: NaCl 10.0g/L, peptone 10.0g/L, yeast powder 5.0g/L, pH7.0.
The present invention also provides the application of described immobilized spherule in diethylamine degraded.
The application of described immobilized spherule in diethylamine degraded, be specially: first immobilized spherule aeration 12h in waste water is activated, 28 ± 2 ℃ of water temperatures in reactivation process, aeration rate is at 1.2L/min, then be under 28 ± 2 ℃, the pH value suitable condition that is 6.8-7.2 in temperature, at diethylamine influent concentration lower than 1200mg/L in the situation that, every liter of diethylamine waste water takes the immobilized spherule of 175ml to process, simultaneously, hydraulic detention time is 54h, the clearance of diethylamine, more than 97%, has good removal effect in this case.
Diethylamine degradation bacteria strains EYA-2 of the present invention can be take diethylamine as sole carbon source, nitrogenous source is grown, and its thorough mineralising is produced to ammonia nitrogen.Under the shaking flask condition of laboratory, the degradation rate of 100mg/L diethylamine is reached to 100%.The present invention also provides the prepared immobilized spherule of a kind of bacterial strain EYA-2, and laboratory biological degradation experimental result shows, immobilized spherule reaches 95.75% to the degradation rate of 100mg/L diethylamine.It is low, easy to use that the immobilized spherule of the degradation bacteria EYA-2 that this invention is produced has production cost, the advantage that removal effect is good, and the diethylamine in can degrading waste water, promotes the use of in the wastewater treatment that is adapted at containing diethylamine.The present invention is for preserving the ecological environment, and to protect mankind healthy reduces cost for wastewater treatment and have great importance.
Accompanying drawing explanation
Fig. 1 is diethylamine degradation bacteria strains EYA-2 bacterium colony picture;
Fig. 2 bacterial strain EYA-2 thalline gramstaining photo (1000x);
The degradation effect of Fig. 3 liquid chromatographic detection immobilized spherule to diethylamine;
The degradation effect of immobilized spherule to diethylamine when the different influent concentration of Fig. 4 liquid chromatographic detection.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
Separation and the evaluation of embodiment 1 diethylamine degradation bacteria strains EYA-2
Get diethylamine enrichment bacterium liquid 1.0ml, add in 9.0ml sterilized water, fully mix and be made into 10
-1pregnant solution, then draw that 1.0ml prepares 10
-1pregnant solution add in 9.0ml sterilized water, fully mix and be made into 10
-2pregnant solution, by that analogy, pregnant solution is carried out to gradient dilution.The diluent 0.1ml that draws each gradient coats on the inorganic salt solid medium containing 100mg/L diethylamine, and its formula is: every liter containing 1.5 g K
2hPO
4, 0.5 g KH
2pO
4, 0.2 g MgSO
4× 7H
2o, 1.0 g NaCl, 1.0 g (NH
4)
2sO
4, 20g agar, 7.0,30 ℃ of cultivations of pH 7 days.After 7 days from above inorganic salt solid medium picking list bacterium colony, in the LB of 5ml liquid nutrient medium, cultivate after 24 hours, centrifugal 2min under the condition of 8000r/min, remove supernatant liquor, add the sterilized water of 5ml to shake up, still centrifugal 2min under the condition of 8000r/min, according to said method uses after twice of aseptic washing, adds 5ml sterilized water this bacterium that suspends.Draw this bacterium liquid of 1ml and add in the inorganic salt liquid substratum that 100ml diethylamine concentration is 100mg/l, its formula is: every liter containing 1.5 g K
2hPO
4, 0.5 g KH
2pO
4, 0.2 g MgSO
4× 7H
2o, 1.0 g NaCl, 1.0 g (NH
4)
2sO
4, pH 7.0, in 160r/min, shaking culture after 3 days under 30 ℃ of conditions, surveys its degradation effect with high performance liquid chromatography.A strain bacterial strain higher degradation efficiency is preserved, carried out subsequent experimental.Fig. 1 is shown in by its bacterium colony picture, its thalline gramstaining photo (1000x) as shown in Figure 2, be accredited as Rhodopseudomonas (
pseudomonas sp.); Called after: EYA-2.Main Biological is G
-, logarithmic phase thalline is shaft-like, bacterium colony is circular white crowned, can reduce nitrate, and the oxydase reaction positive, edwardsiella hoshinae, without gemma, aerobic growth.Can be take diethylamine as sole carbon source, nitrogenous source grows, and its thorough mineralising produced to ammonia nitrogen, by Inspection Certificate, under the shaking flask condition of laboratory, free bacteria reaches 100% to the degradation rate of 100mg/L diethylamine.
Embodiment 2 bacterial strain EYA-2 are prepared into the method for immobilized spherule
1) be inoculated in LB liquid nutrient medium in 30 ℃ with a small amount of bacterial classification of transfering loop picking on slant medium, under 160r/min condition, shaking culture is to logarithmic phase;
2) by above-mentioned cultured bacterium liquid centrifugal 2min under the condition of 8000r/min, outwell supernatant liquor, add the sterilized water of same volume, shake up the centrifugal 2min of rear 8000r/min, so wash twice, with the sterilized water suspension of same volume, make thalline liquid.
3) 4% sodium alginate soln of getting 0.7ml thalline liquid and 20ml mixes in normal temperature, then with syringe, mixed solution is splashed into the 3%CaCl under normal temperature
2solution obtains calcium alginate gel immobilized spherule after 4 ℃ of crosslinked calcification 6h, aseptic washing be stored in for twice 4 ℃ for subsequent use.
LB substratum described in step 1) is: NaCl 10.0g/L, peptone 10.0g/L, yeast powder 5.0g/L, pH7.0.
The application method of embodiment 3 immobilized spherules in diethylamine degraded
First immobilized spherule aeration 12h in waste water is activated, 28 ± 2 ℃ of water temperatures in reactivation process, aeration rate is at 1.2L/min, then it is 28 ± 2 ℃ in temperature, pH value is under the suitable condition of 6.8-7.2, at diethylamine influent concentration lower than 1200mg/L in the situation that, every liter of diethylamine waste water takes the immobilized spherule of 175ml to process, simultaneously, hydraulic detention time is 54h, the clearance of diethylamine is more than 97% in this case, there is good removal effect, specifically as shown in Figure 3 and Figure 4, wherein Fig. 3 has shown in the situation of diethylamine influent concentration lower than 1200mg/L, every liter of diethylamine waste water takes the immobilized microorganism bead of 175ml to process, simultaneously, hydraulic detention time is 54h, the clearance of diethylamine is more than 97%, and Fig. 4 has shown the removal situation for the diethylamine of different influent concentrations, the clearance of diethylamine reduces along with the rising of influent concentration, in the time that the diethylamine concentration of intaking is in the scope lower than 1200mg/l, diethylamine has higher clearance, can reach more than 97%.
Claims (2)
- One strain diethylamine degraded pseudomonas ( pseudomonas sp.) bacterial strain EYA-2, its deposit number is cGMCC NO.7140.
- 2. the application of pseudomonas EYA-2 in diethylamine degraded described in claim 1.3. contain the immobilized spherule of pseudomonas EYA-2 claimed in claim 1.4. described in claim 1, pseudomonas EYA-2 is prepared into the method for immobilized spherule, comprises the following steps:1) be inoculated in LB liquid nutrient medium in 30 ℃ with a small amount of bacterial classification of transfering loop picking on slant medium, under 160r/min condition, shaking culture is to logarithmic phase;2) by above-mentioned cultured bacterium liquid centrifugal 2min under the condition of 8000r/min, outwell supernatant liquor, add the sterilized water of same volume, shake up the centrifugal 2min of rear 8000r/min, so wash twice, with the sterilized water suspension of same volume, make thalline liquid;3) 4% sodium alginate soln of getting 0.7ml thalline liquid and 20ml mixes in normal temperature, then with syringe, mixed solution is splashed into the 3%CaCl under normal temperature 2solution obtains calcium alginate gel immobilized spherule after 4 ℃ of crosslinked calcification 6h, aseptic washing be stored in for twice 4 ℃ for subsequent use.5. method according to claim 4, is characterized in that, the LB substratum described in step 1) is: NaCl 10.0g/L, peptone 10.0g/L, yeast powder 5.0g/L, pH7.0.6. the application of the immobilized spherule described in claim 4 or 5 in diethylamine degraded.7. the application of immobilized spherule according to claim 6 in diethylamine degraded, be specially: first immobilized spherule aeration 12h in waste water is activated, 28 ± 2 ℃ of water temperatures in reactivation process, aeration rate is at 1.2L/min, then be under 28 ± 2 ℃, the pH value suitable condition that is 6.8-7.2 in temperature, at diethylamine influent concentration, lower than 1200mg/L in the situation that, every liter of diethylamine waste water takes the immobilized spherule of 175ml to process, meanwhile, hydraulic detention time is 54h.
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