CN101845401A - Rhodotorula mucilaginose strain and application thereof in degradation of butachlor - Google Patents

Abstract

The invention discloses a butachlor degradation strain and application thereof. The butachlor degradation strain is rhodotorula mucilaginose Y-LCY-1 and is preserved in China General Microbiological Culture Collection Center on September 14, 2009 with a preservation number of GMCCNo.3270. The strain can maximally tolerate 1,000 mg.L<-1> butachlor; and after being cultured in an inorganic salt basal medium containing 100 mg.L<-1> butachlor for 7 days at 25 DEG C with the inoculum concentration of 5 percent and pH of 6.5, the strain can degrade 97.6 percent of the butachlor and has good degradation effect on the butachlor of 50 to 500 mg.L<-1>. The strain provided by the invention can be applied to the biological purification of water bodies and soil polluted by the butachlor.

Description

One Rhodotorula mucilaginose strain and the application in degradation of butachlor thereof

Technical field

The invention belongs to the bioremediation technology field, relate to a Rhodotorula mucilaginose strain and the application in the degrading herbicide Butachlor technical 92 thereof.

Background technology

Butachlor technical 92 (C ₁₇H ₂₆ClNO ₂) be a kind of efficient inner sucting conduction type amides preemergence herbicide, be mainly used in the gramineous weeds of preventing and kill off in rice terrace barnyard grass grass and the dry land, it is few, active high to have consumption, characteristics such as selectivity is strong, widely produce and use in China, its residual caused problem of environmental pollution in water body and soil also will be on the rise simultaneously.On the one hand, Butachlor technical 92 particularly runs off with row irrigation and precipitation with water vertical, horizontal leaching and diffusion, can cause underground water and river pollution; On the other hand, Butachlor technical 92 has restraining effect to many microbial growths such as algae, photosynthetic bacterium, nitrogen-fixing bacterias, thereby has destroyed soil micro-ecosystem balance (Zager, 1990; Lee, 1991); Moreover Butachlor technical 92 is to the toxicity of freshwater fish and Amphibians very big (Bushra et al, 2002; Bu Ning etc., 2005; Geng Baorong etc., 2005); In addition, Butachlor technical 92 has mutagenicity and genetoxic, by food chain and biomagnification etc. toxicity is transmitted step by step, finally influences human health (Ou et al, 2000; Wang et al, 1987).

Alleviate even remove approach that Butachlor technical 92 pollutes at present mainly by photodissociation and microbiological deterioration (Wang Yiru etc., 1996) in soil.Compare with photodissociation, microbial metabolism in herbicide degradation pointed strong, the cycle short, the advantage of instant effect.Therefore seek the efficient degrading bacterial strain of Butachlor technical 92, have important practical significance for the biological restoration of Butachlor technical 92 environmental pollution.Microbial resources that at present can degradation of butachlor are in the majority with bacterium, and representative have pseudomonas, genus bacillus, and filamentous fungus is less, and yeast is then more rare.

Summary of the invention

The object of the present invention is to provide effectively degradation of butachlor and can be used for Butachlor technical 92 and pollute the Rhodotorula mucilaginose that water and soil is administered of a strain.

Another object of the present invention provides a kind of by the prepared microbial inoculum of above-mentioned Rhodotorula mucilaginose.

The objective of the invention is to be achieved through the following technical solutions:

Butachlor degradation bacterial strain provided by the present invention is Rhodotorula mucilaginose (Rhodotorula mucilaginosa) Y-LCY-1, be preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) " on 09 14th, 2009, its preserving number is CGMCCNo.3270.Address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101.

The microbial inoculum of Rhodotorula mucilaginose provided by the present invention (Rhodotorula mucilaginosa) Y-LCY-1 is that bacterial classification and necessary added ingredients are mixed thus.

The application of above-mentioned Rhodotorula mucilaginose (Rhodotorula mucilaginosa) Y-LCY-1 in degradation of butachlor is characterised in that: but this bacterial strain resisting high-concentration Butachlor technical 92 growth, and the highest tolerance concentration reaches 1000mgL ^-1, when inoculum size is 5%, during 6.5,25 ℃ of pH, containing 100mgL ^-1Butachlor technical 92 inorganic salt basic culture solution in cultivate after 7 days, to the Butachlor degradation rate up to 97.6%, and to 50～500mgL ^-1Butachlor technical 92 all have good degradation effect.

Bacterial strain provided by the invention has enriched the diversity of Butachlor degradation bacterial strain, for the residual contamination problem that solves Butachlor technical 92 provides the critical strain resource, can be applicable to be subjected to the water body that Butachlor technical 92 pollutes and the purification of soil.

Described Rhodotorula mucilaginose (Rhodotorula mucilaginose) Y-LCY-1 has been preserved in China Microbial Culture Preservation Commission common micro-organisms center (being called for short CGMCC) on 09 14th, 2009, preserving number is CGMCCNo.3270.

Description of drawings

Fig. 1 Rhodotorula mucilaginose (Rhodotorula mucilaginose) Y-LCY-1 is being the sole carbon source and the energy with the Butachlor technical 92, is under 6.5 the condition, to 100mgL in 25 ℃, pH ^-1The degradation curve figure that Butachlor technical 92 is continuous 7 days.

Fig. 2 Rhodotorula mucilaginose (Rhodotorula mucilaginose) Y-LCY-1 is being the sole carbon source and the energy with the Butachlor technical 92, is under 6.5 the condition, to the continuous 7 days degradation curve figure of different concns Butachlor technical 92 in 25 ℃, pH.

Embodiment

Separation, purifying and the evaluation of example 1, Rhodotorula mucilaginose (Rhodotorula mucilaginose) Y-LCY-1 CGMCCNo.3270

Rhodotorula mucilaginose provided by the invention (Rhodotorula mucilaginose) Y-LCY-1 CGMCCNo.3270 is a separation screening from the paddy soil of suburb, Harbin long-term application Butachlor technical 92, it is concrete separate and purge process as follows:

Soil sampling 10g, placing 50mL Butachlor technical 92 concentration is 100mgL ^-1In the inorganic salt nutrient solution of basis, in 28 ℃, cultivate on the 160rpm constant temperature shaking table, insert in the fresh Butachlor technical 92 inorganic salt nutrient solution with 10% inoculum size week about then, and improve the Butachlor technical 92 consumption gradually, Butachlor technical 92 concentration reaches 1000mgL to the nutrient solution ^-1, so tamed about three months.On the inorganic salt agar plate of Butachlor technical 92 basis, separate then with the dilution coating method, place 28 ℃ to cultivate 3～5 days, obtain pure bacterial strain behind the stable bacterium colony that goes down to posterity, purifying 3～4 times, with the purifying degradation bacteria strains that obtains respectively tieback in the Butachlor technical 92 basic culture solution, verify its degradation capability, by the residual quantity of gas chromatographic detection Butachlor technical 92, finishing screen is selected a strain Butachlor technical 92 efficient degrading bacteria, transfers in Butachlor technical 92 MacConkey agar slant medium, after cultivating 48h, place 4 ℃ of refrigerators to preserve.

Described inorganic salt nutrient solution is prepared in following ratio: K ₂HPO ₄0.1g, MgSO ₄7H ₂O 0.2g, FeSO ₄7H ₂O 0.001g, (NH ₄) ₂SO ₄0.1g, CaSO ₄0.04g add distilled water to 1000mL, pH transfers to 6.5.High pressure steam sterilization (121 ℃, 20min).

Described Butachlor technical 92 basis inorganic salt nutrient agar: Butachlor technical 92 concentration is 500mgL ^-1Inorganic salt trainings liquid add the agar of 1.5～2% (W/V).

Described MacConkey agar substratum is prepared in following ratio: glucose 1.0g, KCl 1.8g, yeast extract paste 2.5g, sodium-acetate 8.2g, distilled water 1000mL, pH transfer to 6.5,115 ℃ of sterilization 20min.

Used gas chromatographic detection condition is: GC-14C type gas chromatograph, fid detector; Chromatographic column is coated with 14%OV-1701 wide bore capillary column (30m * 0.53mm i.d.) in being; Temperature condition: 250 ℃ of injection ports, 225 ℃ of column temperatures, 250 ℃ of detectors; Gas flow: nitrogen 100kpa, hydrogen 50kpa, air 50kpa, tail blows 100kpa; Split stream sampling not, retention time is 3.9min.Sample size 1 μ L.

The peak area of constant volume and extension rate and sample chromatogram peak and standard specimen chromatographic peak utilizes external standard method to calculate the concentration of Butachlor technical 92 in the nutrient solution per sample.Calculation formula:

$C_x=N\&times;\frac{V_o}{V_x}\&times;\frac{S_x}{S_o}\&times;C_o$

In the formula: C _x: the concentration (mgL of Butachlor technical 92 in the nutrient solution ^-1) N: diluted sample multiple V _o: standard specimen sample size (μ L) V _x: sample feeding amount (μ L)

S _x: sample chromatogram peak-to-peak area S _o: standard specimen chromatographic peak peak area C _o: standard specimen concentration mgL ^-1)

Calculate the degradation rate of Butachlor technical 92 according to concentration that does not connect Butachlor technical 92 in the bacterium processing and the concentration that connects Butachlor technical 92 in the bacterium processing.Calculation formula:

$X\%=\frac{(C_{\mathrm{ck}}-C_x)}{C_{\mathrm{ck}}}\&times;100\%$

In the formula: X%: the biological degradation rate C of Butachlor technical 92 _x: meet the concentration (mgL that bacterium is handled Butachlor technical 92 in the nutrient solution ^-1) C _Ck: the concentration (mgL that does not connect Butachlor technical 92 in the bacterium contrast culture liquid ^-1)

The morphological specificity and the growth characteristic of separating the Butachlor degradation bacterial strain that obtains: on the MacConkey agar substratum, cultivated 3 days under 28 ℃ of conditions, colony characteristics is the bacterium colony circle, rat, and neat in edge, opaque, moistening, thickness, orange red.Bacterial strain is individual avette or spherical, and size is 2.0～5.0 μ m, and is single, paired, do not form thecaspore or sporidium, produces pink group carotene on wort.Optimum growth temperature is 28 ℃, and optimal pH is 6.5, can grow under 4 ℃ of conditions.

It is as shown in table 1 to separate the Butachlor degradation bacterial strain part physiological and biochemical property that obtains:

Table 1 Butachlor degradation bacterial strain Y-LCY-1 physiological and biochemical property

Test subject	The result	Test subject	The result
				Nitrate reduction test	????-	Lactose fermentation	????-
Starch	????-	The lactose assimilation	????-
				Glucose fermentation	????-	The D-wood sugar	????+
The cellobiose fermentation	????-	The L-rhamnosyl	????v
				The maltose fermentation	????-	Erythritol	????-
Glycerine	????+	D-N.F,USP MANNITOL	????+
				Gelatine liquefication	????-	Citric acid	????+
The maltose assimilation	????+	The L-sorbose	????+
				The cellobiose assimilation	????+	Melibiose	????-
Inositol	????-	Melizitose	????+
				The semi-lactosi assimilation,	????+	Synanthrin	????-
The sucrose assimilation	????+	The D-sorbyl alcohol	????+

Annotate :+: positive-: negative v: variable

By the 26Sr DNA D1/D2 of this bacterial strain that increases, obtain the 26SrDNA D1/D2 sequence of length 573bp.Submit this sequence to, received by Genebank, accession number is FJ874772.By Blast software other sequences on the Genebank are carried out the homology comparison, the homology of finding this sequence and Rhodotorula mucilaginose (Rhodotorula mucilaginose) partial mode bacterial strain is up to 100%.

Comprehensive morphological is learned observation of characteristics, physiological and biochemical test and 26Sr DNA D1/D2 The sequencing results can determine that this bacterial strain is Rhodotorula mucilaginose (Rhodotorula mucilaginose), with its called after Y-LCY-1.

The preparation of example 2 Rhodotorula mucilaginoses (Rhodotorula mucilaginose) Y-LCY-1 CGMCCNo.3270 microbial inoculum

The concrete manufacture craft of microbial inoculum is:

With the bacterial classification inoculation of preservation in the slant medium in containing 100mgL ^-1Activate 12h in the minimal medium of Butachlor technical 92, then with bacterium liquid (OD ₆₀₀=1.0) inoculum size by 10% (V/V) changes in the triangular flask of the YEPD liquid nutrient medium that 150mL is housed, and the Butachlor technical 92 starting point concentration is 50mgL in the substratum ^-1, seal film and seal, place 28 ℃ of constant-temperature shaking culture casees to cultivate 3 days with the 160rpm hunting of frequency, then take out 1/4 nutrient solution, add the nutrient solution of same amount again, make that Butachlor technical 92 amine starting point concentration reaches 50mgL in the substratum ^-1, continue in the thermostat container and cultivated 3 days, be microbial inoculum.The liquid bacterial agent that obtains can be deposited in the sealed, sterile bag, is easy to promote the use of.

Described YEPD liquid culture based component is: yeast powder 10g, and peptone 20g, glucose 20g adds double distilled water to 1000mL, and pH transfers to 6.5,115 ℃ of moist heat sterilization 20min.

Example 3 Rhodotorula mucilaginoses (Rhodotorula mucilaginose) Y-LCY-1 CGMCCNo.3270 is to the biological degradation of Butachlor technical 92

(1) Rhodotorula mucilaginose (Rhodotorula mucilaginose) Y-LCY-1 CGMCCNo.3270 is to 100mgL ^-1The mensuration of Butachlor degradation ability

The bacteria suspension preparation: picking one ring Y-LCY-1 slant preservation bacterial classification under the aseptic condition, be transferred in the MacConkey medium, place 28 ℃, 160rpm constant temperature shaking table shaking culture is got a certain amount of nutrient solution to the suitableeest cell age, and the centrifugal 10min of 4000rpm abandons supernatant, uses 0.2molL ^-1The nutritive substance (totally 3 times) that thalline carries is removed in phosphoric acid buffer (pH=7) washing, collects thalline, transfers cell concentration with damping fluid, with the light absorption value OD under the spectrophotometric determination 600nm _600nm, make OD _600nmAbout 2.0.

Inoculum size by 5%, in the minimal medium of Butachlor technical 92 basis, under 6.5,25 ℃ of conditions of pH, in 160rpm constant temperature shaking table shaking culture, Butachlor technical 92 is initial dense to be 100mgL with above-mentioned bacterial suspension inoculation ^-1, cultivate 1,2,3,4,5,6,7 days, difference quantitative sampling 21mL, add a little anhydrous sodium sulphate, with heavily steaming sherwood oil 10mL, 5mL, 5mL oscillation extraction 3 times, petroleum ether layer filters through the anhydrous sodium sulphate post, merge organic phase, be concentrated near doing in 40 ℃ of rotary evaporations, to be cooled to room temperature, be settled to 7mL with acetone.With the residual concentration of gas-chromatography (GC-14C) mensuration Butachlor technical 92, draw degradation curve, compare with the substratum that does not add bacterium liquid.Above-mentioned each processing repeats for 3 times, and the result as shown in Figure 1.Fig. 1 shows, with the prolongation rhodotorula mucilaginosa Y-LCY-1 of incubation time the degradation capability of Butachlor technical 92 strengthened gradually, containing 100mgL ^-1Butachlor technical 92 inorganic salt basic culture solution in cultivate after 7 days, the Butachlor degradation rate is up to 97.6%, and in the contrast after 7 days Butachlor technical 92 natural degradation rate be lower than 15%, illustrate that this bacterial strain has great degradation capability to Butachlor technical 92.

(2) Rhodotorula mucilaginose (Rhodotorula mucilaginose) Y-LCY-1 CGMCCNo.3270 is to the tolerance and the degradation capability examination of different concns Butachlor technical 92

With Y-LCY-1 bacteria suspension (OD _600nm=2.0) be inoculated in the inorganic salt liquid nutrient solution of the Butachlor technical 92 that contains different concns by 5% inoculum size, the concentration of Butachlor technical 92 is respectively 50mgL ^-1, 100mgL ^-1, 200mgL ^-1, 300mgL ^-1, 500mgL ^-1, 800mgL ^-1, 1000mgL ^-1, 1200mgL ^-1, 25 ℃, 160rpm, one week of shaking culture.Found that, remove 1200mgL ^-1Bacterial strain is outside the growth, waits to try in the substratum all breeding in various degree at other, and it is muddy that nutrient solution all becomes.When bacterial strain is 1000mgL in Butachlor technical 92 concentration ^-1Situation under grow 3～7 days the time, find that the nutrient solution color gradient be pink.And OD under this concentration ₆₀₀Reach 0.7302.The result shows that this bacterial strain can Butachlor technical 92 be the sole carbon source growth, and can be good at tolerating the high density Butachlor technical 92.

With Y-LCY-1 bacteria suspension (OD _600nm=2.0) be inoculated in the inorganic salt liquid nutrient solution of the Butachlor technical 92 that contains different concns by 5% inoculum size, the concentration of Butachlor technical 92 is respectively 50mgL ^-1, 100mgL ^-1, 200mgL ^-1, 300mgL ^-1, 400mgL ^-1, 500mgL ^-1, be contrast with the substratum that does not connect bacterium liquid, at 25 ℃, pH is 6.5 o'clock, 160rpm cultured continuously one all bacterial strains are to different concns Butachlor degradation situation as shown in Figure 2.As seen from Figure 2, Rhodotorula mucilaginose (Rhodotorula mucilaginose) Y-LCY-1 (≤200mgL when low Butachlor technical 92 concentration ^-1), degradation effect is remarkable, and degradation rate is all above 50%; (〉=300mgL when high Butachlor technical 92 concentration ^-1), degradation capability descends.Hence one can see that, and the metabolism to bacterial strain when Butachlor technical 92 concentration surpasses certain limit has certain restraining effect.

The The above results demonstration, respond well in Rhodotorula mucilaginose (Rhodotorula mucilaginose) the Y-LCY-1 CGMCCNo.3270 liquid medium within to Butachlor degradation, have the great potential of repairing Butachlor technical 92 polluted-water and soil.

Claims (3)

1. a Rhodotorula mucilaginose strain (Rhodotorula mucilaginosa) Y-LCY-1, this bacterial strain have been preserved on 09 14th, 2009 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number are CGMCCNo.3270.

2. microbial inoculum that contains claims 1 described Rhodotorula mucilaginose (Rhodotorula mucilaginosa) Y-LCY-1.

3. the application of a Rhodotorula mucilaginose strain as claimed in claim 1 (Rhodotorula mucilaginosa) Y-LCY-1 in degradation of butachlor.

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