CN103497918A

CN103497918A - Bromoxynil degrading bacterium and application thereof

Info

Publication number: CN103497918A
Application number: CN201310475449.9A
Authority: CN
Inventors: 蔡天明; 陈立伟; 徐静; 刘时旸; 胡江
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2013-10-12
Filing date: 2013-10-12
Publication date: 2014-01-08
Anticipated expiration: 2033-10-12
Also published as: CN103497918B

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

A kind of bromoxynil degradation bacteria and application thereof

Technical field

The present invention relates to the environmental microorganism technical field, particularly, relate to a kind of bromoxynil degradation bacteria and application thereof.

Background technology

A large amount of uses of weedicide have caused tremendous influence to environment.At first the use of chemical herbicide is in effective management of weeds, also reduce to a certain extent the activity of some enzymes in the quantity of Soil Microorganism and soil, thereby affect soil quality and feeder capability, and the larger impact of formulation rate is larger.Secondly, weedicide can pollute underground water, thereby affects the mankind's health.

Bromoxynil is with its octanoate, sodium salt, and after the form of sylvite is widely used as the selectivity bud abroad, cauline leaf is processed the contact killing type weedicide.Mainly by blade, absorbed, each process by inhibited photosynthesis, make rapidly tissue necrosis.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.The 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, can also and multiple weedicide be mixed, enlarge herbicidal spectrum.

Biological degradation is the main path that the occurring in nature bromoxynil is removed, the at present domestic research report that still there is no the bromoxynil efficient degrading bacteria, therefore, isolate the microorganism strains of efficient degradation bromoxynil from the environment that bromoxynil pollutes, utilize 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 ?, end is given birth to flagellum, rod-short, without gemma, mobility.

The feature that bromoxynil degradation bacteria BX03 cultivates two days later in the LB substratum is: bacterium colony is rounded, smooth moistening, the umbo shape, and 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.

The present invention also provides a kind of described bromoxynil degradation bacteria BX03 to produce the method for microbial inoculum, comprises the steps: inclined-plane kind-shaking flask kind-seeding tank-production tank-product, and the packing formulation is liquid bacterial agent or solid absorption microbial inoculum.

Implementation step is in detail:

1) activation culture: the test tube kind of bromoxynil degradation bacteria BX03 is inoculated in the LB substratum, and shaking culture is to logarithmic phase;

2) seed liquor preparation: above-mentioned cultured bacterial classification is inoculated into 500 liters of seeding tanks by 10% inoculum size, 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: seed liquor is produced to the tank cultivation and fermentation by 10% inoculum size access, and the rear nutrient solution that fermented is microbial inoculum.Produce the tank used medium identical with the seed tank culture base.

Described step 2) air flow of producing sterile air in the culturing process of tank in seeding tank and step 3) is 1:0.6-1.2, stirring velocity is 180-240 rev/min, culture temperature is 30-35 ℃, and whole technical process incubation time is 48-60 hour.

The invention provides a kind of degradation bacteria that bromoxynil pollutes of eliminating, can be produced by the general fermentation equipment of fermentation industry, and this bacterium can be take bromoxynil and be grown as sole carbon source and nitrogenous source, laboratory biological degradation experimental result shows, the degradation rate of 100mg/L bromoxynil is reached to 95%.

It is low, easy to use that the degradation bacterial agent that this invention is produced has a production cost, and the advantage that removal effect is good, promote the use of in the wastewater treatment that is adapted at containing bromoxynil.The present invention is for preserving the ecological environment, and to protect mankind healthy reduces cost for wastewater treatment and have great importance.

The 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 immobilized spherule and the free state microbial inoculum degradation experiment result to 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 are used for the present invention is described, 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 bromoxynil degradation bacteria BX03

Get the active sludge 5.0ml obtained the purification tank for liquid waste of certain the insecticide factory's production plant from producing bromoxynil and add in the 100ml inorganic salt liquid cultivation that bromoxynil concentration is 100mg/L, its formula is: every liter containing 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 ℃ of conditions, be transferred in fresh minimal medium by 5% inoculum size every 5 days, transfers continuously 5 times.

Get above bromoxynil enrichment bacterium liquid, carry out gradient dilution.Diluent is coated on the inorganic salt solid medium containing the 100mg/L bromoxynil, and culture medium prescription is: every 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, cultivate a couple of days for 30 ℃.Therefrom picking list bacterium colony, utilize HPLC to verify its degradation effect, and the strain bacterial strain that degradation efficiency is higher is stored in-20 ℃ of refrigerators with 30% glycerine.Fig. 2 is shown in by its bacterium colony picture, according to the 16SrDNA qualification result, by listed nucleotide sequence in itself and GenBank carry out homology relatively after, be accredited as bulkholderia cepasea (Burkholderia sp.); Called after: BX03.Main Biological is G-, and end is given birth to flagellum, rod-short, and without gemma, mobility.The bromoxynil of take is grown as sole carbon source and nitrogenous source, and by its mineralising.In the shake flat experiment of laboratory, cultivate 24h in 30 ℃, the shaking table of 160rpm rotating speed, by liquid phase, to measure, result shows that the degradation rate to the bromoxynil of 100mg/L reaches 95%.This bacterium can be produced by the general fermentation equipment of fermentation industry, and this bacterium is delivered to the CGMCC preservation, and deposit number is CGMCC NO.7438.

The method that embodiment 2 bromoxynil degradation bacteria BX03 produce microbial inoculum

By bromoxynil degradation bacteria BX03(CGMCC NO.7438 of the present invention) original seed on culture dish with in incubator, activate, cultivate 30h under 30 ℃ of conditions, and measure degradation property, after the completion of carrying disease germs is ripe, be inoculated on test tube slant standby.The 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, and peptone 10.00, NaCl10.00, pH7.0, constant-temperature shaking culture, to logarithmic phase, is prepared the inoculation seeding tank.500 liters of seeding tanks, 400 liters of charging capacitys, culture medium prescription 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%(above " % " means g/100ml), pH value 7.2-7.5.121 ℃ of high pressure moist heat sterilizations after feeding intake, after being cooled to 33 ℃, inoculate above-mentioned cultured shaking flask bacterial classification into 500 liters of seeding tanks by 10% inoculum size, is cultured to logarithmic phase, and stirring velocity is 220 rev/mins, and the sterile air intake is 1:0.8.The seed liquor that arrives logarithmic phase is produced to tank by 10% inoculum size access and cultivate, produce tank used medium composition identical with the seed tank culture base.Produce 5 tons of tankages, 4.5 tons of charging capacitys.Production tank 1.1kg/cm after feeding intake ²pressure under, 121 ℃ of high pressure moist heat sterilizations, be cooled to after sterilizing below 35 ℃, logical sterile air keeps sterile state standby.Postvaccinal production tank temperature is controlled at 30 ℃, and in the culturing process of production tank, the air flow of sterile air is 1:0.8-1.0, and stirring velocity is 240 rev/mins, and whole technical process incubation time is 48-60 hour.After fermentation ends thalline quantity reach 1,000,000,000/more than ml.The rear nutrient solution that fermented goes out tank and directly by plastic barrel or packing bottle, is distributed into liquid dosage form or adopts adsorption by peat to be distributed into the solid fungicide formulation with packing bag.

Embodiment 3 contain Burkholderia BX ?the cell fixation experiment of 03 microbial inoculum

Be taken in the LB substratum cultivate 8h BX ?03(CGMCC NO.7438) inoculum, centrifugal collection thalline, with sterilized water washing 2 times, making cell concentration is 20gL ^?1bacteria suspension.1.5mL bacteria suspension and 20mL2% sodium alginate soln are mixed, dropwise splash into 3%CaCl ₂form immobilization gelled pill of uniform size in solution, with aseptic washing, be placed on crosslinking curing 4h in 4 ℃ of refrigerators for twice, with standby after the sterilized water diafiltration.

First group: in bromoxynil concentration, be 100mgL ^?1the 100ml minimal medium in, the Bacteria suspension that the inoculum size by 1.5% adds above-mentioned LB to cultivate, 30 ℃, 160rmin ^?1shaking table is cultivated, and every the 4h sampling, gets 24h always, measures bromoxynil content, calculates the bromoxynil degradation rate.

Second group: in bromoxynil concentration, be 100mgL ^?1the 100ml minimal medium in, the inoculum size by 1.5% adds the immobilized spherule that above-mentioned bacteria containing amount is identical, 30 ℃, 160rmin ^?1shaking table is cultivated, and every the 4h sampling, gets 24h always, measures bromoxynil content, calculates the bromoxynil degradation rate.

Although found out that by Fig. 3 degradation rate degradation rate with respect to the free state bacterium of immobilized spherule is slightly slow, but after 24h, the degradation rate of bromoxynil is still more than 80%, illustrate bacterium BX ?03(CGMCC NO.7438) still there is the ability of degraded bromoxynil in immobilization later.

Claims

1. a bromoxynil degradation bacteria BX03, it is characterized in that this bromoxynil degradation bacteria BX03 Classification And Nomenclature is bulkholderia cepasea (Burkholderia sp.), 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. a degradation bacterial agent of producing by bromoxynil degradation bacteria claimed in claim 1.

3. degradation bacterial agent according to claim 2 is characterized in that described degradation bacterial agent is to produce by the following method:

1) described bromoxynil degradation bacteria BX03 test tube kind is inoculated in the LB culture media shaking vase, shaking culture is to logarithmic phase;

2) above-mentioned cultured bacterial classification is inoculated into 500 liters of seeding tanks by 10% inoculum size, 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%, NaCl0.01%, CaCO ₃0.3%, yeast extract paste 0.02%, pH value 7.2 ?7.5;

3) seed liquor is produced the tank cultivation by 10% inoculum size access, produces the tank used medium identical with the seed tank culture base; At seeding tank and in producing the culturing process of tank the air flow of sterile air be 1:(0.6 ?1.2), stirring velocity be 180 ?240 rev/mins, culture temperature be 30 ?35 ℃, the whole process incubation time be 48 ?60 hours, after fermentation ends thalline quantity reach 1,000,000,000/more than ml;

4) the rear nutrient solution that fermented goes out tank and directly by plastic barrel or packing bottle, is distributed into liquid dosage form or adopts adsorption by peat to be distributed into the solid fungicide formulation with packing bag.

4. the application of bromoxynil degradation bacteria BX03 in the degraded bromoxynil.