CN104152373B - Bacterial strain capable of efficiently degrading pendimethalin and application thereof - Google Patents

Abstract

The invention belongs to the field of environmental microorganisms, and particularly relates to a bacterial strain capable of efficiently degrading pendimethalin. The bacterial strain is named brevibacterium halotolerans (Brevibacterium halotolerans) Y-1-4, and is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms; the preservation date is February 27, 2014; and the preservation number is CGMCCNo.8823. The brevibacterium halotolerans Y-1-4 capable of efficiently degrading pendimethalin can be separated and obtained from the soil in which vegetables are planted for a long period of time, the brevibacterium halotolerans Y-1-4 has the ability of efficiently degrading the pendimethalin, and the residual pendimethalin in the soil and vegetables can be degraded, therefore, the bacterial strain disclosed by the invention has the advantages of good degradation characteristic, high degradation rate, high degradation efficiency and the like.

Description

A kind of can the bacterial strain of efficient degradation Pendimethalin and application thereof

Technical field

The present invention relates to a kind of degradation of pesticide microorganism, particularly relating to one can efficient degradation Pendimethalin Bacterial strain, belong to field of environment microorganism.

Background technology

Pendimethalin is dinitroaniline herbicide before a kind of selectivity Seedling, mainly suppresses separate living tissue Cell division, does not affect the sprouting of seed, and its selectivity is physiology and potential difference selection, due to its low-residual Highly active feature, and be widely used in agricultural product.But U.S. Environmental Protection Agency (EPA) (EPA) research discovery two The happy spirit of first penta belongs to three grades of toxicity class materials, and the mankind are had carcinogenesis.Extensive due in agricultural product Application, its residual in the environment is the most serious, therefore, Pendimethalin residual in research environment Removal problem is extremely the most urgent.

The minimizing technology of Pendimethalin mainly has physics, chemical and biological method.Traditional physics side Method (wrap up, burn and bury) and chemical method (include oxidation, reduce and hydrolyze) cost are high, can New pollutant can be produced, and act on relatively slow.Biology or biological product are utilized to carry out degradation of contaminant Biological renovation method has the advantages such as nontoxic, noresidue, non-secondary pollution, is to eliminate and removing toxic substances high concentration Pesticide residues a kind of safely, effectively, cheap method.Microorganism have that kind is many, variation is fast and The features such as gene easily operation, are the important living resources of environmental organism reparation.With microorganism or acellular Enzyme preparation is cut down the bioremediation technology of pollution by pesticides and is demonstrated wide application prospect.

Summary of the invention

The present invention is directed to the deficiencies in the prior art, it is provided that this bacterium is also used for degrading two by a kind of brevibacterium halotolerans The happy spirit of first penta, improves by Pendimethalin contaminated soil and the quality of agricultural product.

The technical scheme is that a kind of efficient degradation Pendimethalin agriculture The degradation bacteria of medicine, named brevibacterium halotolerans (Brevibacterium halotolerans) Y-1-4, preservation In China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is the clear sun in Beijing North Star West Road, district 1 institute 3, preservation date is on February 27th, 2014, and preserving number is CGMCC No.8823。

Degradation bacteria Y-1-4 of the present invention is through form, Analysis of The Physiological And Biochemical Properties and 16SrDNA gene order Homology analysis, is accredited as brevibacterium halotolerans (Brevibacterium halotolerans).

Y-1-4 bacterial strain is on LB solid plate, and bacterium colony is big, and edge is irregular, and white is opaque, table Face is smooth, aging after in sundown.Gram-positive, shaft-like, not there is spore.

Preferably, the condition of culture of brevibacterium halotolerans Y-1-4 is: be inoculated into by brevibacterium halotolerans Y-1-4 Basal medium, cultivates 18-24h for 25-36 DEG C, and the pH value range of cultivation is 5-8；The training of described basis Support the composition of base and proportioning is: NH₄NO₃1.0g, MgSO₄0.5g, (NH₄)₂SO₄0.5g, KH₂PO₄, 0.5g, K₂HPO₄1.5g, NaCl 0.5g, yeast extract 0.05g, 121 DEG C of sterilizing 30min. Solid medium then adds 2% agar.

It is furthermore preferred that the condition of culture of brevibacterium halotolerans Y-1-4 is: brevibacterium halotolerans Y-1-4 is inoculated To basal medium, cultivating 24h for 36 DEG C, the pH value range of cultivation is 7.0 ± 1.

Y-1-4 is at pH5-pH8, and temperature 25-36 DEG C growth, degradation characteristic well, can be with Pendimethalins Grow for sole carbon source.

Second aspect present invention discloses the degradation bacteria of aforementioned efficient degradation Pendimethalin pesticide, and (salt tolerant is short Bacillus Y-1-4) degraded Pendimethalin pesticide residues in application.

By the gas chromatography-mass spectrometry analysis Y-1-4 bacterial strain degradation rate to Pendimethalin: just When the OD650nm of beginning bacterium is 0.02, Y-1-4 bacterial strain can be by 100mg L in 72h^-lPendimethalin Spirit degraded 97.3%.

Third aspect present invention discloses a kind of Pendimethalin degradation bacterial agent, containing aforementioned brevibacterium halotolerans Y-1-4。

Preferably, in described Pendimethalin degradation bacterial agent, the thalline quantity of brevibacterium halotolerans Y-1-4 reaches To 10¹⁰CFU ml^-1Above.

Fourth aspect present invention discloses a kind of method remaining Pendimethalin in soil of degrading, for soil Earth applies aforementioned brevibacterium halotolerans Y-1-4.

Further, the mode applied described in can be to spray.

The invention has the beneficial effects as follows: separate from the soil of long-term planting vegetable and obtain a high-efficiency degradation The brevibacterium halotolerans Y-1-4 of Pendimethalin, this brevibacterium halotolerans Y-1-4 cultural method is simple, has The ability of efficient degradation Pendimethalin.Can at pH5-pH8, well-grown under conditions of 25 DEG C-36 DEG C, And pH5-pH8, keep under conditions of 25 DEG C-42 DEG C more than 65% degradation of pesticide ability.Can accelerate Degraded soil and the Pendimethalin of Residues in Vegetables, under certain condition, by Pendimethalin in soil Half-life foreshortened to 9d by 24d, there is degradation characteristic good, degradation rate is fast, and degradation efficiency is high Advantage.

Accompanying drawing explanation

Fig. 1 is Y-1-4 bacterial strain cultivation form on LB solid plate and characteristic pattern；

Fig. 2 is the initial pH impact on Y-1-4 degradation efficiency；

Fig. 3 is the cultivation temperature impact on Y-1-4 degradation efficiency；

Fig. 4 is Pendimethalin concentration curve in Y-1-4 growth curve and culture fluid；

Fig. 5 is Pendimethalin degradation dynamic variation diagram in green stem vegetable；

Fig. 6 is Pendimethalin degradation dynamic variation diagram in soil.

Detailed description of the invention

Being described principle and the feature of the present invention below in conjunction with accompanying drawing, example is served only for explaining this Invention, is not intended to limit the scope of the present invention.

Embodiment 1 can the screening of efficient degradation Pendimethalin pesticide degradation bacteria

1. soil sample source

Soil sample: pick up from Laiyang Shandong Province growing vegetables base.

2. the enrichment of degradation bacteria and separation:

The pedotheque gathered is inoculated into containing Pendimethalin 100mg L with the amount of 5%^-lPendimethalin Spirit culture fluid carries out enrichment culture, 36 DEG C of shaken cultivation 48h, takes appropriate pregnant solution and be inoculated in containing diformazan Penta happy spirit 200mg L^-lPendimethalin culture fluid in cultivate.So transfer and improve constantly diformazan penta The concentration of Pendimethalin in happy spirit culture fluid, until the concentration of Pendimethalin brings up to 1000 mg·L^-1.Take last culture fluid to be transferred to containing Pendimethalin 100mg L^-lPendimethalin solid Purification in culture medium, transferring continuously and improving constantly the concentration of Pendimethalin is finally reached 1000mg·L^-1.The strain Strain Designation choosing the speed of growth the fastest is Y-1-4；The classification of this degradation bacteria Named: brevibacterium halotolerans (Brevibacterium halotolerans), protect on February 17th, 2014 Being stored in " China Committee for Culture Collection of Microorganisms's common micro-organisms center ", its preserving number is CGMCC No.8823。

Described Pendimethalin culture fluid is by adding Pendimethalin acquisition in basal medium.Institute Composition and the proportioning of stating basal medium are every liter of culture medium: NH₄NO₃1.0g, MgSO₄0.5g, (NH₄)₂SO₄0.5g, KH₂PO₄0.5g, K₂HPO₄1.5g, NaCl 0.5g, yeast extract 0.05g, pH 7.2-7.3.Solid medium then adds 2% agar.Culture medium prepares rear 121 DEG C of sterilizing 30min.

The interpolation of Pendimethalin: take 33% and execute field benefit cream 1.0ml, add 32.0ml sterilized water and obtain 1mg·ml^-1The aqueous solution of Pendimethalin, adds in the basal medium of sterilizing according to different ratios, Obtain the Pendimethalin culture fluid (or solid medium) of different Pendimethalin concentration, and be sub-packed in Sterilizing test tubes.

3. the qualification of degradation bacteria and attribute:

1) form and physiological and biochemical property:

Y-1-4 bacterial strain is on LB solid plate, and bacterium colony is big, and edge is irregular, and white is opaque, table Face is smooth, aging after in sundown.Gram-positive, shaft-like, not there is spore, see attached Fig. 1.Indole experiment feminine gender, the casein hydrolysis positive, specific features is shown in Table 1.

The physiology of table 1 pesticide degradation bacteria Y-1-4, biochemical characteristic

Note: "+" representing reacting positive, "-" represents reaction negative

2) the 16S rDNA sequence homology analysis of bacterial strain Y-1-4

With BLAST software, the 16S rDNA sequence (shown in SEQ ID NO:1) of Y-1-4 bacterial strain is carried Send to Genbank is carried out nucleotide sequence homology comparison, it was found that Y-1-4 bacterial strain and having carried The many strains brevibacterium halotolerans sequence height handed over is similar, and homology is higher, illustrates that this bacterium developmentally belongs at molecule In brevibacterium halotolerans, after the order-checking of 16SrDNA comparing homology, according to homology in phylogeny In principle of classification and combining form, physiological and biochemical property, with reference to " Bergey ' s mannual of Determinative bacteriology " the 9th edition, bacterial strain Y-1-4 ranges salt tolerant quarter butt the most at last Bacterium (Brevibacterium halotolerans).

Table 2 bacterial strain Y-1-416SrRNA gene order BLAST comparison result

The preparation of embodiment 2 degradation bacterial agent

1) test tube kind being inoculated in basic culture solution, concussion is cultivated to logarithmic (log) phase, it is thus achieved that seed liquor；

2) above-mentioned seed liquor is inoculated by the inoculum concentration of 10% in the basal medium of 10L fermentation tank and carry out Fermentation；

3) in sweat, the ventilation of filtrated air is 1:0.8, and mixing speed is 150 revs/min, training Foster temperature is 36 DEG C, and overall process incubation time is 60h.

After fermentation ends, thalline quantity reaches 10¹⁰CFU ml^-1Above, rear culture fluid has been fermented directly with bag Bottling is distributed into liquid agent, is degradation bacterial agent.

Embodiment 3 bacterial strain Y-1-4 degradation characteristic

1. Y-1-4 degradation characteristic is affected by initial pH

1.1 test method

Preparation Pendimethalin culture fluid subpackage, regulation culture fluid pH value respectively 5.0,6.0,7.0, 8.0,9.0, the concentration of Pendimethalin is 100mg L^-1.Inoculum concentration by 10% inoculates embodiment 2 The degradation bacteria Y-1-4 seed liquor of preparation, cultivates 24h sampling, measures the degradation rate of Pendimethalin for 36 DEG C And the increment of antibacterial.The mensuration of bacterial growth amount: measure the OD value under culture fluid 650nm；Degraded The computing formula of rate is as follows:

$x\%=\frac{\mathrm{Cck}-\mathrm{Cx}}{\mathrm{Cck}}\×100\%$

In formula:

The biological degradation rate of x%: pesticide

Cx: connect bacterium and process culture fluid culture fluid Pesticides concentration (mg L^-1)

C_CK: do not meet concentration (the mg L of bacterium comparison culture fluid Pesticides^-1)

1.2 result of the test

Culture fluid original ph is essentially identical on the impact of degradation rate and increment, all reaches when pH is 7 To maximum, degradation rate is 80.7%.Y-1-4 is not strict to the requirement of pH value, and subject range is wider, Can keep higher degradation rate in the range of pH value 5～8, experimental result is shown in Fig. 2.

2. the cultivation temperature impact on Y-1-4 degradation characteristic

2.1 test method

Preparation Pendimethalin culture fluid subpackage, the initial pH7.0 of regulation culture fluid, Pendimethalin Concentration is 100mg L^-1.By 10% inoculum concentration inoculate embodiment 2 preparation Y-1-4 seed liquor after in 10 DEG C, 25 DEG C, 36 DEG C, 42 DEG C, shaken cultivation, sampling and measuring Pendimethalin after 24h at 50 DEG C Degraded and the increment of antibacterial.

2.2 result of the test

Experimental result is shown in Fig. 3, and cultivation temperature has considerable influence to the efficiency of Y-1-4 degraded Pendimethalin, Rise high degradation rate with temperature first to increase, more excellent in the range of 25 DEG C-42 DEG C, reach maximum at 36 DEG C, degraded Rate is 86.59%, and degradation rate declines subsequently.

Embodiment 4 liquid degradation is tested

Bacterial strain Y-1-4 inoculation basal medium 36 DEG C is cultivated after 24h, with the inoculum concentration of 10% be inoculated into Pendimethalin is sole carbon source and its concentration is 100mg L^-1Pendimethalin fluid medium in, Pendimethalin residual concentration and degradation efficiency in 3h sampling and measuring culture medium.

Result of the test is shown in Fig. 4, and in starting 3h, Pendimethalin lowering of concentration is quickly, and Pendimethalin is degraded Rate quickly increases, and then slows down, and to 15～24h, the lowering of concentration speed of Pendimethalin reaches maximum. Analyze the growth curve of Pendimethalin concentration curve and microorganism it can be seen that from the beginning of cultivating just Within several hours, reaching in the 24h of stable trophophase to Y-1-4, Pendimethalin degradation rate is very fast.Just In stage beginning, thalline number increases slow but pesticide concentration declines fast, and this is probably thalline and makes the absorption of pesticide Become.It follows that Y-1-4 has reached exponential phase, along with the increase of metabolic activity, diformazan penta The lowering of concentration speed of happy spirit is also accelerated.After 24h, Y-1-4 is slack-off to the degradation rate of Pendimethalin, Lowering of concentration speed reduces, and gradually tends to be steady, and the growth of microorganism reaches decline in contrast Phase, this is because after cultivating certain time, in culture fluid, the utilizable active substance of microorganism reduces, And the growth of the metabolite suppression antibacterial produced itself, the profit so thalli growth slows down, to pesticide With minimizing.

Embodiment 5 vegetable degradation experiment

With green stem vegetable as test plant, Laiyang Shandong Province Vegetable Base is experimental plot, if 3 (only process Spray Pendimethalin T1, spray Pendimethalin and degradation bacteria T2, spray clear water comparison CK1), 3 weights Multiple.Use every mu of 200mL of pendimethalin missible oil of recommended density 33%, simultaneously with every mu of 4000mL Use the degradation bacterial agent (broadcasting dispenser before rear Seedling) of embodiment 2 preparation.Green stem vegetable sample the 3d of dispenser, 5d, 7d, 9d, 12d, 15d, 18d, 21d, 24d sample, and in self-styled band ,-20 DEG C of preservations are to be measured.

Result of the test is shown in Fig. 5.Under two kinds of processing modes of T1 and T2, the residual concentration of Pendimethalin is all Prolongation in time and be gradually reduced.In the T1 only using Pendimethalin processes, through 24 days Degrading pesticide residues concentration is the highest is maintained at 4.0mgkg-1.Using Pendimethalin and bacterial strain During the T2 of Y-1-4 processes, the residual quantity decrease speed of Pendimethalin quickly, T2 process group during 12d In green stem vegetable, Pendimethalin residual quantity has fallen to 2.7mgkg-1, illustrates that the pesticide of more than 50% is residual Stay and be the most degraded.In the green stem vegetable sample of T1 process group, Pendimethalin residual quantity is apparently higher than at T2 Reason group.Obviously, bacterial strain Y-1-4 accelerates the degraded of Pendimethalin in green stem vegetable, makes in green stem vegetable two First penta happy spirit residual quantity is degraded to more low-level within a short period of time.

Embodiment 6 soil degrading is tested

With green stem vegetable as test plant, if 3 process (only spray Pendimethalin T1, spray pendimethalin Spirit and degradation bacteria T2, spray clear water comparison CK1), repeat for 3 times.Use the pendimethalin of recommended density 33% Missible oil every mu 200mL, simultaneously uses the degradation bacterial agent of embodiment 2 preparation with every mu of 4000mL and (broadcasts Dispenser before rear Seedling).Respectively at 1d, 2d, 3d, 5d, 7d, 9d, 12d, 15d, 18d, 21d of dispenser, By 5 point samplings, gather pedotheque.In self-styled band ,-20 DEG C of preservations are to be measured.

Result of the test is shown in Fig. 6.Under two kinds for the treatment of conditions of T1 and T2, in soil, Pendimethalin pesticide is residual Allowance within two days residual concentration be substantially the same, the 3rd day, prolongation in time started to be gradually reduced.Only The T1 process group decrease speed spraying Pendimethalin pesticide is relatively slow, during to 24d, and this group pendimethalin The residual quantity of spirit is the most substantially in the half of 30mgkg-1 level, substantially degraded to initial concentration, namely In the soil of Pendimethalin, the half-life is 24 days, and this numerical value is slightly below the pendimethalin of forefathers' report 31.9 days soil half-life of spirit.In the T2 process group of spray Pendimethalin and bacterial strain Y-1-4, diformazan The residual quantity of penta happy spirit is also gradually reduced after 2d, and decrease speed is very fast, when the 9th day, and soil Sample Pesticide Residues concentration has reached 28.4mgkg-1, less than the half of initial concentration.Also just say, Spray while Pendimethalin degradation bacteria Y-1-4, make the soil half-life of Pendimethalin be reduced to less than 9 day time.Pendimethalin residual quantity in the soil of T1 process group is apparently higher than T2 process group soil Sample.

Compared with Example 5, in the pedotheque and green stem vegetable sample of same process, pendimethalin Spirit persticide residue is significantly different.In same process, Pendimethalin persticide residue in pedotheque The content of Pendimethalin pesticide residues in green stem vegetable to be significantly larger than.This reason may contact with vegetable Sunlight and air are abundant, promote pesticide degradation bacteria relevant to the degraded of Pendimethalin.

The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all in the present invention Spirit and principle within, any modification, equivalent substitution and improvement etc. made, should be included in this Within bright protection domain.

Claims (6)

1. the degradation bacteria of a Pendimethalin pesticide of degrading, it is characterised in that the named brevibacterium halotolerans of this bacterium (Brevibacterium halotolerans) Y-1-4, it being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date is on February 27th, 2014, and preserving number is CGMCC No.8823.

2. the degradation bacteria of degraded Pendimethalin pesticide application in degraded Pendimethalin pesticide residues described in claim 1.

3. a Pendimethalin degradation bacterial agent, containing the degradation bacteria of Pendimethalin pesticide of degrading described in claim 1.

4. Pendimethalin degradation bacterial agent as claimed in claim 3, it is characterised in that in described Pendimethalin degradation bacterial agent, the thalline quantity of brevibacterium halotolerans Y-1-4 reaches 10¹⁰CFU ml^-1Above.

5. degrade the method remaining Pendimethalin in soil, for applying Pendimethalin degradation bacterial agent described in claim 3 or 4 any claim in soil.

6. the method remaining Pendimethalin in degraded soil as claimed in claim 5, it is characterised in that described in the mode that applies for spraying.