CN101845401B - Rhodotorula mucilaginose strain and application thereof in degradation of butachlor - Google Patents
Rhodotorula mucilaginose strain and application thereof in degradation of butachlor Download PDFInfo
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- CN101845401B CN101845401B CN2009100732526A CN200910073252A CN101845401B CN 101845401 B CN101845401 B CN 101845401B CN 2009100732526 A CN2009100732526 A CN 2009100732526A CN 200910073252 A CN200910073252 A CN 200910073252A CN 101845401 B CN101845401 B CN 101845401B
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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
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 are widely produced and are used in China, and 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 toxicity very big (Bushra et al, 2002 of freshwater fish and Amphibians; Bu Ning etc., 2005; Geng Baorong etc., 2005); In addition, Butachlor technical 92 has mutagenicity and genetoxic, through 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 the approach that Butachlor technical 92 pollutes at present and mainly lean on 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 prosthetic 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 realize through following technical scheme:
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 variety of Butachlor degradation bacterial strain, for the residual contamination problem that solves Butachlor technical 92 provides the critical strain resource, can be applicable to receive 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 instance 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 following:
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 with the dilution coating method then, place 28 ℃ to cultivate 3～5 days, stable bacterium colony goes down to posterity; Obtain pure bacterial strain behind the 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; Through 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 zero(ppm) 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, zero(ppm) 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, RT 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:
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:
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 that bacterium colony is circular, 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, on wort, produces pink group Serlabo.Optimum growth temperature is 28 ℃, and ph optimum is 6.5, can under 4 ℃ of conditions, grow.
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 SANMALT-S fermentation | - | Erythritol | - |
| Glycerine | + | D-N.F,USP MANNITOL | + |
| Gelatine liquefication | - | Hydrocerol A | + |
| The SANMALT-S 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
Through 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.Through 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 confirm that this bacterial strain is Rhodotorula mucilaginose (Rhodotorula mucilaginose), with its called after Y-LCY-1.
The preparation of instance 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
-1Activation 12h in the minimal medium of Butachlor technical 92 is 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.
(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 to the righttest cell age is got a certain amount of nutrient solution, 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; Respectively quantitative sampling 21mL adds a little SODIUM SULPHATE ANHYDROUS 99PCT, and with heavily steaming sherwood oil 10mL, 5mL, 5mL oscillation extraction 3 times, petroleum ether layer filters through the SODIUM SULPHATE ANHYDROUS 99PCT 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 gc (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 is 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%, explain 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.The result finds, removes 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 as shown in Figure 2 to different concns Butachlor degradation situation.Visible by Fig. 2, Rhodotorula mucilaginose (Rhodotorula mucilaginose) Y-LCY-1 is (≤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 CGMCC No.3270.
2. microbial inoculum that contains the described Rhodotorula mucilaginose of claim 1 (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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| CN105400709B (en) * | 2015-12-21 | 2018-12-11 | 湖南省植物保护研究所 | Cement rhodotorula bacterial strain, microbial inoculum and its preparation method and application |
| CN111088172A (en) * | 2020-03-06 | 2020-05-01 | 中国水稻研究所 | Method for enhancing weeding toxicity of helminthosporium peregrinum spores |
| CN112322510B (en) * | 2020-11-19 | 2022-05-17 | 江苏海洋大学 | Rhodotorula mucilaginosa SYM-1 and screening method and application thereof |
| CN113249128A (en) * | 2021-06-01 | 2021-08-13 | 南京农业大学 | Composite soil remediation agent and preparation method and application thereof |
| CN114806905B (en) * | 2022-04-21 | 2023-08-01 | 宁夏回族自治区食品检测研究院 | Rhodotorula mucilaginosa strain and application thereof |
| CN116694486B (en) * | 2023-02-24 | 2024-05-28 | 天津科技大学 | Rhodotorula mucilaginosa strain and application thereof in preparation of soy sauce |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101270346A (en) * | 2008-04-25 | 2008-09-24 | 浙江大学 | Butachlor degradation bacterium and application thereof |
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| CN101270346A (en) * | 2008-04-25 | 2008-09-24 | 浙江大学 | Butachlor degradation bacterium and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Jun Xu et al..Isolation and characterization of a Pseudomonas oleovorans degrading the chloroacetamide herbicide acetochlor.《Biodegradation》.2006,219-225. * |
| 李川等.丁草胺高效真菌的分离及性能研究.《农业环境科学学报》.2004,第23卷(第3期),611-614. * |
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