CN103497918B - Bromoxynil degrading bacterium and application thereof - Google Patents
Bromoxynil degrading bacterium and application thereof Download PDFInfo
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- CN103497918B CN103497918B CN201310475449.9A CN201310475449A CN103497918B CN 103497918 B CN103497918 B CN 103497918B CN 201310475449 A CN201310475449 A CN 201310475449A CN 103497918 B CN103497918 B CN 103497918B
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Abstract
The invention discloses a bromoxynil degrading bacterium and application thereof, and belongs to the field of high biotechnologies. A bacterial strain of the bromoxynil degrading bacterium is a Gram staining reaction negative bacterium BX03, and belongs to Burkholderia bacterial genus (Burkholderia sp.) is identified as Burkholderia sp.. The bromoxynil degrading bacterium is mainly biologically characterized in that the bromoxynil degrading bacterium is Gram-negative, is in the shape of a short spar and is free of spores, and flagella grow at the ends of the bromoxynil degrading bacterium. Bromoxynil can be used as the only carbon source and the only nitrogen source to grow the bacterium, and the bacterium can be mineralized. The bromoxynil degrading bacterium and the application have the advantages that 95% of 100mg/L of the bromoxynil can be degraded by the bacterial strain within 24 hours under a flask shaking condition in a laboratory, and accordingly the problem of difficulty in biologically degrading bromoxynil during waste water treatment can be solved.
Description
Technical field
The present invention relates to technical field of environmental microorganism, particularly, relate to a kind of bromoxynil degradation bacteria and application thereof.
Background technology
A large amount of uses of weedicide cause tremendous influence to environment.First the use of chemical herbicide is while effective management of weeds, also reduce the activity of some enzymes in the quantity of Soil Microorganism and soil to a certain extent, thus affect soil quality and feeder capability, and the larger impact of formulation rate is larger.Secondly, weedicide can pollute underground water, thus affects the health of the mankind.
Bromoxynil is with its octanoate, sodium salt, and the form of sylvite is widely used as cauline leaf process contact killing type weedicide after selectivity bud abroad.Absorb primarily of blade, by each process of inhibited photosynthesis, make tissue necrosis rapidly.This agent is mainly used in the crop fields such as wheat, barley, rye, corn, Chinese sorghum, flax, prevents and kill off puncture, amaranth, Herba Silenes conoideae, the broadleaf weedss such as black nightshade, Siberian cocklebur, Herba Salsolae Collinae, corn gromwell, Herba seu Flos Convolvuli arvensis, corn-bind.Wheat consumption is every mu of 22.5 ~ 35g(effective ingredient); Chinese sorghum, corn are every mu of 18.7 ~ 30g; Every mu, flax is no more than 18.7g.Bromoxynil shelf-stable, and other weedicides generally do not react, except alone, all right and multiple weedicide mixture, expands herbicidal spectrum.
Biological degradation is the main path that occurring in nature bromoxynil is removed, domestic at present still do not have the research of bromoxynil efficient degrading bacteria to report, therefore, from the environment that bromoxynil pollutes, isolate the microorganism strains of efficient degradation bromoxynil, utilize the bromoxynil in the effect repairing environment of microorganism to pollute and there is great theory significance and potential application prospect.
Summary of the invention
The object of the invention is to for the practical problems in production practice and demand, a kind of bromoxynil degradation bacteria is provided.
Another object of the present invention is to provide the application of this bacterium.
Bromoxynil degradation bacteria strains BX03 provided by the invention is a strain gramstaining reaction negative bacterium, be preserved in Chinese microorganism strain on April 9th, 2013 and preserve management committee's common micro-organisms center (abbreviation CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature is bulkholderia cepasea Burkholderia sp., and deposit number is CGMCC NO.7438.
Bromoxynil degradation bacteria BX03 Main Biological be G ?, the raw flagellum of end, rod-short, without gemma, mobility.
Bromoxynil degradation bacteria BX03 feature of cultivating two days later in LB substratum is: bacterium colony is rounded, smooth moistening, umbo shape, neat in edge, for light yellow.The composition of described LB substratum is: yeast extract paste 5.00g/L, peptone 10.00g/L, NaCl10.00g/L, pH7.0.
The invention provides the microbial inoculum that described bromoxynil degradation bacteria BX03 produces.
Present invention also offers a kind of method that described bromoxynil degradation bacteria BX03 produces microbial inoculum, comprise the steps: inclined-plane kind-shaking flask kind-seeding tank-production tank-product, packaging formulation is liquid bacterial agent or solid absorption microbial inoculum.
Detailed implementation step is:
1) activation culture: the test tube kind of bromoxynil degradation bacteria BX03 be inoculated in LB substratum, shaking culture is to logarithmic phase;
2) seed liquor preparation: by above-mentioned cultured bacterial classification by 10% inoculum size inoculate into 500 liters of seeding tanks, be cultured to logarithmic phase.Seeding tank culture medium prescription used is: glucose 0.8%, (NH
4)
2sO
41%, K
2hPO
40.2%, MgSO
40.05%, NaCl0.01%, CaCO
30.3%, yeast extract paste 0.02%, pH value 7.2 ?7.5.
3) microbial inoculum preparation: tank cultivation and fermentation is produced in the inoculum size access of seed liquor being pressed 10%, and the rear nutrient solution that fermented is microbial inoculum.Produce tank used medium identical with seed tank culture base.
Described step 2) in produce sterile air in the culturing process of tank in seeding tank and step 3) air flow be 1:0.6-1.2, stirring velocity is 180-240 rev/min, culture temperature is 30-35 DEG C, and whole technical process incubation time is 48-60 hour.
The invention provides a kind of degradation bacteria eliminated bromoxynil and pollute, can produce by the general fermentation equipment of fermentation industry, and this bacterium can be sole carbon source and nitrogenous source with bromoxynil grows, laboratory biological degradation experiment result shows, reaches 95% to the degradation rate of 100mg/L bromoxynil.
It is low, easy to use that the degradation bacterial agent that this invention is produced has production cost, and the advantage that removal effect is good, is adapted at promoting the use of in the wastewater treatment containing bromoxynil.The present invention is for preserving the ecological environment, and the protection mankind's is healthy, reduces cost for wastewater treatment and has great importance.
Accompanying drawing explanation
Fig. 1 is degradation bacteria BX03 thalline violet staining photo under microscope;
Fig. 2 is bacterial strain BX03 bacterium colony photo;
Fig. 3 is that immobilized spherule and free state microbial inoculum are to the degradation experiment result of bromoxynil.
Biomaterial preservation information
Bromoxynil degradation bacteria strains BX03, Classification And Nomenclature is bulkholderia cepasea Burkholderia sp., be 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, deposit number is CGMCC NO.7438.
Embodiment
Following examples for illustration of 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 amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
The separation andpreconcentration of embodiment 1 bromoxynil degradation bacteria BX03
Getting the active sludge 5.0ml that obtains from the purification tank for liquid waste of certain the insecticide factory's production plant producing bromoxynil, to add bromoxynil concentration be that during the 100ml inorganic salt liquid of 100mg/L is cultivated, its formula is: often liter contains 1.50g K
2hPO
4, 0.50g KH
2pO
4, 0.20g MgSO
47H
2o, 1.00g NaCl, 1.00g (NH
4)
2sO
4, pH7.0, in 160r/min, shaking culture under 30 DEG C of conditions, every 5 days by 5% inoculum size be transferred in fresh minimal medium, continuously switching 5 times.
Get above bromoxynil enrichment bacterium liquid, carry out gradient dilution.Diluent is coated on the inorganic salt solid medium containing 100mg/L bromoxynil, and culture medium prescription is: often liter containing 1.5g K
2hPO
4, 0.5g KH
2pO
4, 0.2g MgSO
47H
2o, 1.0g NaCl, 18g agar, pH=7.0, cultivates a couple of days for 30 DEG C.Therefrom picking list bacterium colony, utilizes HPLC to verify its degradation effect, by a strain bacterial strain higher for degradation efficiency with 30% glycerine be stored in-20 DEG C of refrigerators.Fig. 2 is shown in by its bacterium colony picture, according to 16SrDNA qualification result, after nucleotide sequence listed in itself and GenBank is carried out tetraploid rice, is accredited as bulkholderia cepasea (Burkholderia sp.); Called after: BX03.Main Biological is G-, the raw flagellum of end, rod-short, without gemma, and mobility.Can be that sole carbon source and nitrogenous source grow with bromoxynil, and by its mineralising.In laboratory shake flask experiment, 30 DEG C, cultivate 24h in the shaking table of 160rpm rotating speed, measured by liquid phase, result display reaches 95% to the degradation rate of the bromoxynil of 100mg/L.This bacterium can produce by the general fermentation equipment of fermentation industry, and this bacterium is delivered CGMCC preservation, and deposit number is CGMCC NO.7438.
Embodiment 2 bromoxynil degradation bacteria BX03 produces the method for microbial inoculum
By bromoxynil degradation bacteria BX03(CGMCC NO.7438 of the present invention) original seed activate on culture dish with incubator, cultivate 30h under 30 DEG C of conditions, and measure degradation property, carry disease germs completion ripe after be inoculated on test tube slant for subsequent use.Test tube kind is inoculated in the 1000ml shaking flask containing 200ml LB substratum, LB culture medium prescription (g/L): yeast extract paste 5.00, peptone 10.00, NaCl10.00, pH7.0, and constant-temperature shaking culture, to logarithmic phase, prepares inoculation seeding tank.Seeding tank 500 liters, charging capacity 400 liters, culture medium prescription is: glucose 0.8%, (NH
4)
2sO
41%, K
2hPO
40.2%, MgSO
40.05%, NaCl0.01%, CaCO
30.3%, more than yeast extract paste 0.02%(" % " represents g/100ml), pH value 7.2-7.5.121 DEG C of high pressure moist heat sterilizations after feeding intake, after being cooled to 33 DEG C, by above-mentioned cultured shaking flask bacterial classification by 10% inoculum size inoculate into 500 liters of seeding tanks, be cultured to logarithmic phase, stirring velocity is 220 revs/min, and sterile air intake is 1:0.8.By arriving the seed liquor of logarithmic phase by the inoculum size access production tank cultivation of 10%, produce tank used medium composition identical with seed tank culture base.Produce tankage 5 tons, charging capacity 4.5 tons.Production tank 1.1kg/cm after feeding intake
2pressure under, 121 DEG C of high pressure moist heat sterilizations, are cooled to less than 35 DEG C after sterilizing, logical sterile air keeps sterile state for subsequent use.Postvaccinal production tank temperature controls at 30 DEG C, and the air flow producing sterile air in the culturing process of tank is 1:0.8-1.0, and stirring velocity is 240 revs/min, and whole technical process incubation time is 48-60 hour.After fermentation ends, thalline quantity reaches 1,000,000,000/more than ml.The rear nutrient solution that fermented goes out tank and is directly distributed into liquid dosage form by plastic barrel or packing bottle or adopts adsorption by peat packing bag to be distributed into solid fungicide formulation.
Embodiment 3 containing Burkholderia BX ?the cell fixation experiment of 03 microbial inoculum
Be taken at cultivate 8h in LB substratum BX ?03(CGMCC NO.7438) inoculum, collected by centrifugation thalline, washs 2 times with sterilized water, and making cell concentration is 20gL
?1bacteria suspension.1.5mL bacteria suspension and 20mL2% sodium alginate soln are mixed, dropwise instills 3%CaCl
2form immobilization gelled pill of uniform size in solution, be placed on crosslinking curing 4h in 4 DEG C of refrigerators for twice with aseptic washing, with for subsequent use after sterilized water diafiltration.
First group: be 100mgL in bromoxynil concentration
?1100ml minimal medium in, the inoculum size by 1.5% add above-mentioned LB cultivate Bacteria suspension, 30 DEG C, 160rmin
?1shaking table is cultivated, and every 4h sampling, gets 24h always, measures bromoxynil content, calculate bromoxynil degradation rate.
Second group: be 100mgL in bromoxynil concentration
?1100ml minimal medium in, the inoculum size by 1.5% adds the identical immobilized spherule of above-mentioned bacteria containing amount, 30 DEG C, 160rmin
?1shaking table is cultivated, and every 4h sampling, gets 24h always, measures bromoxynil content, calculate bromoxynil degradation rate.
Although find out that the degradation rate of immobilized spherule is slightly slow relative to degradation rate free state bacterium by Fig. 3, but after 24h, the degradation rate of bromoxynil still more than 80%, illustrate bacterium BX ?03(CGMCC NO.7438) still have after immobilization degraded bromoxynil ability.
Claims (1)
1. a bromoxynil degradation bacteria BX03, it is characterized in that this bromoxynil degradation bacteria BX03 Classification And Nomenclature be bulkholderia cepasea (
burkholderiasp.), be preserved in Chinese microorganism strain on April 9th, 2013 and preserve management committee's common micro-organisms center, deposit number CGMCC NO.7438.
2. the degradation bacterial agent produced with bromoxynil degradation bacteria BX03 according to claim 1.
3. degradation bacterial agent according to claim 2, is characterized in that described degradation bacterial agent produces by the following method:
1) described bromoxynil degradation bacteria BX03 test tube kind be inoculated in LB culture media shaking vase, shaking culture is to logarithmic phase;
2) by above-mentioned cultured bacterial classification by 10% inoculum size inoculate into 500 liters of seeding tanks, be cultured to logarithmic phase, seeding tank culture medium prescription mass volume ratio used is: glucose 0.8%, (NH
4)
2sO
41%, K
2hPO
40.2%, MgSO
40.05%, NaCl 0.01%, CaCO
30.3%, yeast extract paste 0.02%, pH value 7.2-7.5;
3) seed liquor is by the inoculum size access production tank cultivation of 10%, produces tank used medium identical with seed tank culture base; In the culturing process of seeding tank with production tank, the ventilation ratio of sterile air is 1:0.6-1.2, stirring velocity is 180-240 rev/min, culture temperature is 30-35 DEG C, and whole process incubation time is 48-60 hour, and after fermentation ends, thalline quantity reaches 1,000,000,000/more than ml;
4) the rear nutrient solution that fermented goes out tank and is directly distributed into liquid dosage form by plastic barrel or packing bottle or adopts adsorption by peat packing bag to be distributed into solid fungicide formulation.
4. the application of bromoxynil degradation bacteria BX03 according to claim 1 in degraded bromoxynil.
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