CN102492637A

CN102492637A - Atrazine degrading bacterium

Info

Publication number: CN102492637A
Application number: CN2011103717309A
Authority: CN
Inventors: 张颖; 王志刚; 汪洋; 郭火生; 孟东芳; 姜召; 曹博
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2011-11-21
Filing date: 2011-11-21
Publication date: 2012-06-13

Abstract

The invention which relates a degrading bacterium concretely relates to an atrazine degrading bacterium. The atrazine degrading bacterium which is Acinetobacter sp. DNS32 has been preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number of the atrazine degrading bacterium is CGMCC NO.5365, and the preservation date is 10/21/2011. The Acinetobacter sp. DNS32 has a high efficiency degrading capability on atrazine, so the degrading rate can reach 95.88% within 36h; the optimum temperature of the Acinetobacter sp. DNS32, which is 25-30DEG C, is 5-10DEG C lower than the optimum temperature of reported atrazine degrading bacteria; and the endurance capability of the Acinetobacter sp. DNS32 to salinity is strong, so degrading rates in 36h are higher than 60% under 1-4% salinity conditions, thereby the Acinetobacter sp. DNS32 which is better than reported bacteria with same functions is suitable for the restoration of high salinity environments polluted by atrazine.

Description

One strain G-30027 degradation bacteria

Technical field

The present invention relates to a strain degradation bacteria.

Background technology

Weedicide is the important production means that guarantee agricultural; When having brought huge economic benefit to the mankind also since weedicide long residual, have characteristics such as bio-toxicity; In case after getting into soil, air and water body, brought severe contamination also for each ecotope.The mikrobe recovery technique is regarded as effective for repairing means.

G-30027 is classified as one of most popular weedicide, in China North, Northeast China area, G-30027 and with the mixture of other agricultural chemicals still as the topmost weedicide of corn field.The mikrobe of the degraded G-30027 that is separated at present, mainly comprises bacterium, fungi, algae, actinomycetes etc.Bacterium is because its biochemical variety and very strong adaptive faculty are occupied critical role in the mikrobe of degraded G-30027.The bacterium of degraded G-30027 mainly contains: rhodococcus, pseudomonas, edaphic bacillus, acinetobacter calcoaceticus, root nodule bacteria etc.

People such as Singh once obtained many strains G-30027 degradation bacteria report separation from by G-30027 degree of depth Contaminated soil in 2004, belonged to acinetobacter through identifying.Separate a wherein strain bacterium A ₆, through to its physiological property discover that the righttest growth conditions of this bacterium is 37 ℃ of temperature, pH is between 7 to 8, and G-30027 is used as utilization of carbon source.Before this and afterwards, seldom about the report of acinetobacter degraded G-30027.

In repair process, actual temperature can not reach its requirement, and power consumption is bigger; Its optimum growing condition pH is at alkaline range, and under sour environment, growing is suppressed, and makes its range of application be restricted; The medium component cost of bacterial strain is high, is not suitable for the practical application reparation.

Summary of the invention

The invention provides a strain G-30027 degradation bacteria; Having solved at present can't be in the problem of efficient degradation G-30027 under the coldcondition.

One strain G-30027 degradation bacteria; It is acinetobacter calcoaceticus (Acinetobacter sp.) DNS32; In China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation; The preservation address is No. 3, No. 1 institute in Beichen Lu, Chaoyang District, Beijing City. Institute of Microorganism, Academia Sinica, and deposit number is CGMCC NO.5365, preservation date is on October 21st, 2011; It is a Gram-negative bacteria, and it is shaft-like that individual cells is, and thalline length is 2.03 μ m, and wide is 646nm; Bacterium colony is creamy white and has a little yellow, and is opaque, the edge rounding, and smooth surface, glossy, slightly swell.

A strain G-30027 degradation bacteria among the present invention; It is acinetobacter calcoaceticus (Acinetobacter sp.) DNS32, and its gelatin liquification test is positive, nitrate reduction test is positive, Citrate trianion utilization test is negative, hydrogen sulfide generation test is positive, the MR test is positive, the VP test is positive, catalase test is positive, the starch hydrolysis experiment is positive, glucose test is positive, cane sugar test is positive, glycerine test is negative; The growth temperature of this bacterium is 25～30 ℃, and optimum growth temperature is 30 ℃, and growth pH value is 6.5～7.5, and the righttest growth pH value is 7.2.

The present invention's one strain G-30027 degradation bacteria; It is acinetobacter calcoaceticus (Acinetobacter sp.) DNS32; Check order through 16S rDNA sequence; The result carries out homology relatively on NCBI, the sibship of bacterial strain DNS32 and Acinetobacter lwoffii DSM 2403 is nearest, and homology reaches more than 99%.

The present invention's one strain G-30027 degradation bacteria; It is acinetobacter calcoaceticus (Acinetobacter sp.) DNS32; In China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation; The preservation address is No. 3, No. 1 institute in Beichen Lu, Chaoyang District, Beijing City. Institute of Microorganism, Academia Sinica, and deposit number is CGMCC NO.5365, preservation date is on October 21st, 2011.

G-30027 degradation bacteria among the present invention, it is acinetobacter calcoaceticus (Acinetobacter sp.) DNS32, has the ability of efficient degradation G-30027, degradation rate can reach 95.88% in 36 hours, did not appear in the newspapers both at home and abroad; The optimum temperature range of bacterial strain DNS32 is 25-30 ℃, than reporting low 5-10 ℃ of bacterial strain; Bacterial strain DNS32 is stronger to the tolerance of salinity, and salinity is that following 36 hours degradation rates of 1-4% condition all are higher than 60%, and other congenerous bacterial strain that is superior to having reported is applicable to the atrazine-contaminated reparation of high salt time-sharing environment.

Description of drawings

Fig. 1 is the scanning electron microscope diagram of bacterial strain DNS32 in the embodiment one;

Fig. 2 is the phylogenetic tree of bacterial strain DNS32 in the embodiment one;

Fig. 3 is the growth of bacterial strain DNS32 under the salt concentration influence and the graphic representation of degraded situation in the embodiment one, wherein ▲ and the expression degradation curve, ◆ the expression growth curve;

Fig. 4 is the growth of bacterial strain DNS32 under different G-30027 concentration affects and the graphic representation of degraded situation in the embodiment one, wherein ▲ and the expression degradation curve, ◆ the expression growth curve;

Fig. 5 is the growth of bacterial strain DNS32 under differing temps and the figure of degraded situation in the embodiment one, and wherein oral thermometer shows degradation curve, ◆ the expression growth curve;

Fig. 6 adds the figure of the degraded situation under the nitrogenous source influence for bacterial strain DNS32 in the embodiment one in difference, and wherein oral thermometer shows the degraded situation;

Fig. 7 is the figure of bacterial strain DNS32 each time point G-30027 degraded situation in the actual repair contaminated soil in the embodiment one, wherein ◆ expression AT (G-30027) natural degradation, ▲ expression AT+DNS32.

Embodiment

Embodiment one: this embodiment one strain G-30027 degradation bacteria; It is acinetobacter calcoaceticus (Acinetobacter sp.) DNS32; In China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation; The preservation address is No. 3, No. 1 institute in Beichen Lu, Chaoyang District, Beijing City. Institute of Microorganism, Academia Sinica, and deposit number is CGMCC NO.5365, preservation date is on October 21st, 2011; It is a Gram-negative bacteria, and it is shaft-like that individual cells is, and thalline length is 2.03 μ m, and wide is 646nm; Bacterium colony is creamy white and has a little yellow, and is opaque, the edge rounding, and smooth surface, glossy, slightly swell.

The growth temperature of bacterial strain DNS32 is 25～30 ℃ in this embodiment, and optimum growth temperature is 30 ℃, and growth pH value is 6.5～7.5, and the righttest growth pH value is 7.2.

This embodiment one strain G-30027 degradation bacteria, its screening method is realized according to the following steps:

One, take by weighing 10g and pollute soil sample: the earth sample picks up from top layer, corn land for growing field crops (0-20cm) soil that Wuchang City, Heilongjiang Province uses G-30027 for many years, and soil is typical northern black earth; Adopt the diagonal lines method of layouting, establish 5 sampling spots, get the 0-10cm topsoil, mix in the open air, carry out repeatedly division with quartering, left in 1Kg to soil sample repeatedly, the laboratory is taken back in pack;

Two, the 10g in the step 1 is polluted soil sample and place minimal medium; Do not add G-30027 (purpose is that the G-30027 degradation bacteria can be survived) under nutritious condition; Cultivate after 15 days; To pollute soil sample difference transposition in minimal medium and beef-protein medium, under 30 ℃ of constant temperature, the 150r/min constant-temperature shaking culture; Be inoculated in the minimal medium of new preparation in 10% ratio weekly and continue to cultivate, increase the concentration of G-30027 gradually, the domestication enrichment is after 2 months, and G-30027 concentration is increased to 700mgL-1; With culture dilution coating, behind the cultivation 7d, picking has single bacterium colony of degraded circle to carry out plate streaking on a small quantity, and behind the separating for several times purifying, filtering out can be the bacterial strain of only nitrogen source or carbon nitrogen source growth with the G-30027; After bacterial classification inserted slant culture, move into preservation under 4 ℃ of conditions of refrigerator;

Minimal medium is by the K of 1.6g ₂HPO ₄, 0.4g KH ₂PO ₄, 0.2g MgSO ₄, 0.4g glucose, the G-30027 powder of 0.1g and the zero(ppm) water of 1000mL of NaCl, 3.0g form 121 ℃ of sterilization 30min;

Beef-protein medium is made up of the Carnis Bovis seu Bubali cream of 5.0g, the peptone of 10.0g, the NaCl of 5.0g and the zero(ppm) water of 1000ml, 121 ℃ of sterilization 30min;

The scanning electron microscope diagram of this embodiment screening gained bacterium is seen Fig. 1, and it is shaft-like that individual cells is, and thalline length is 2.03 μ m, and wide is 646nm.

The morphological feature of this embodiment screening gained bacterium shows as: bacterium colony is creamy white and has a little yellow, and is opaque, the edge rounding, and smooth surface, glossy, slightly swell.

This embodiment screening gained bacterium is carried out physiological and biochemical test:

Biochemical reactions identifies that experimental result is: gelatin liquification test is positive, nitrate reduction test is positive, Citrate trianion utilization test is negative, hydrogen sulfide generation test is positive, the MR test is positive, the VP test is positive, catalase test is positive, the starch hydrolysis experiment is positive, glucose test is positive, cane sugar test is positive, glycerine test is negative.

Molecular biology identification:

Check order through 16S rDNA sequence; The result carries out homology relatively on NCBI; The sibship of bacterial strain DNS32 and Acinetobacterlwoffii DSM 2403 is nearest, and homology reaches (as shown in Figure 2) more than 99%, bacterial strain DNS32 morphological observation, physiological and biochemical test qualification result; Confirm that bacterial strain DNS32 is a new bacterial strain of acinetobacter, called after acinetobacter calcoaceticus (Acinetobacter sp.) DNS32.

Degradation characteristic: bacterial strain DNS32 degradation rate in the liquid minimal medium that with the G-30027 is only nitrogen source is 95.88% (36 hours).As shown in Figure 3, bacterial strain DNS32 is respectively in minimal medium (being nutrient solution) salinity under 0.1%, 0.5%, 1%, 2%, 3%, 4% the condition, all can keep certain energy for growth and degradation capability, and the optimal salinity of strain growth and degraded is 1%.But when salinity was 2%-4%, the growth of bacterial strain and degraded were affected not quite, and degradation rate all was higher than 60% in 36 hours.Show (as shown in Figure 4) for the G-30027 tolerance studies: along with the increase of G-30027 concentration, the growth of bacterial strain DNS32 and degraded situation all can be affected, when G-30027 concentration surpasses 500mgL ^-1, the growth of bacterial strain receives comparatively obvious suppression, and degradation capability is not high yet.When G-30027 concentration at 1000mgL ^-1The time, bacterial strain almost can not be grown, and degradation rate is extremely low, has reached the maximum tolerated concentration of bacterial strain.

G-30027 degradation bacteria growing state and degraded under the differing temps: (as shown in Figure 5): the growth that bacterial strain DNS32 cultivates under the 36h in differing temps is different with degradation capability.Bacterial strain has preferably growth and degradation capability under 25-30 ℃ of condition, increment and the degradation rate of G-30027 reached maximum in the time of 30 ℃, and 30 ℃ is the righttest culture temperature of bacterial strain.High temperature or low temperature condition all can influence the growth and the degradation capability of bacterial strain.Though bacterial strain DNS32 degradation rate at low temperatures is relatively low, but still having certain degradation capability, also can remain on closely about 30% under 10-20 ℃ of condition to the degradation rate of G-30027, even in the time of 5 ℃, is 18.12% to the degradation rate of G-30027.

Difference adds the influence of nitrogenous source to the G-30027 degradation bacteria: (as shown in Figure 6): add the growth that nitrogenous source can effectively promote bacterial strain, this explanation bacterial strain DNS32 can utilize the nitrogenous substances more simple in structure than G-30027 to grow.Can know, add the degradation capability almost not influence of nitrogen source bacterial strain DNS32.

Actual repair effect: through the effect of experimental study G-30027 degradation bacteria actual repair contaminated soil; The result shows (as shown in Figure 7): the process of G-30027 natural degradation is very slowly in 30 days; The residual quantity of G-30027 is relatively large in the soil, and the degradation rate of G-30027 is less than 20%.But utilizing bacterial strain DNS32 to carry out in the contaminated soil of biological prosthetic, the residual quantity of G-30027 can be successively decreased along with the increase of sample time gradually.In the time of 30 days, the residual quantity of G-30027 is merely 1.85mgkg in the soil ^-1, the degradation rate of the G-30027 under this repair mode is 88.97%, bacterial strain DNS32 has good degradation characteristic.The related conclusions that combines nitrogenous source test in early stage again, bacterial strain DNS32 is the suitable G-30027 degradation bacteria strains that is applied to soil remediation of a strain.

Claims

1. a strain G-30027 degradation bacteria; It is characterized in that it is acinetobacter calcoaceticus (Acinetobacter sp.) DNS32; In China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation; The preservation address is No. 3, No. 1 institute in Beichen Lu, Chaoyang District, Beijing City. Institute of Microorganism, Academia Sinica, and deposit number is CGMCC NO.5365, preservation date is on October 21st, 2011.