CN103555606A - Efficient pyrene degradation bacterial and application thereof - Google Patents
Efficient pyrene degradation bacterial and application thereof Download PDFInfo
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- CN103555606A CN103555606A CN201310283838.1A CN201310283838A CN103555606A CN 103555606 A CN103555606 A CN 103555606A CN 201310283838 A CN201310283838 A CN 201310283838A CN 103555606 A CN103555606 A CN 103555606A
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to efficient pyrene degradation bacterial and an application thereof. The efficient pyrene degradation bacterial is Achromobactersp., and has the preservation number of CGMCC No.7741. According to the present invention, the Achromobactersp. screening separation method is simple, and pyrene can be effectively degraded so as to easily restore pyrene-polluted soil; in a shake flask, the optimal pyrene degradation conditions comprise that the temperature is 25 DEG C, the pH value is 6.0, the initial inoculation amount is 1 wt%, and pyrene degradation rate can be significantly promoted with addition of phenanthrene; and the degradation efficiency of the strain on pyrene in soil can be increased when planting pumpkin and adding glucose.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of pyrene efficient degrading bacteria and application thereof.
Background technology
Polycyclic aromatic hydrocarbons (Polycyclic Aromatic Hydrocarbons, PAHs) be that a class is extensively present in the organic pollutant in edatope, also be to be found the earliest and carcinogens that quantity is maximum, the carinogenicity PAHs finding at present and derivative thereof are over 400 kinds.Because PAHs has the features such as bio-toxicity, bioconcentration and half volatile also can exist lastingly in environment, be divided into persistence organic pollutant (Persistent Organic Pollutants, POPs), be subject to the extensive concern of national governments and scientific circles.In 129 kinds " priority pollutants " of EPA (USEPA) in 1976 proposition, PAHs compounds just has l6 kind; Nineteen ninety, in Chinese environmental priority pollutant Black List was also listed 7 kinds of carinogenicity PAHs by China State Environmental Protection Administration.
Microorganism mainly contains two kinds to the degraded mode of PAHs: (1) take PAHs as sole carbon source and the energy; (2) PAHs and other organic substances carry out common metabolism.Lower molecular weight PAHs (naphthalene, phenanthrene, anthracene, fluorenes etc.) can be used as sole carbon source and energy is utilized by microorganism and degrades, and more than its biological degradations of PAHs of 4 rings, more depends on cometabolism process.The microorganism that PAHs is sole carbon source of take being separated at present has Rhodopseudomonas, Flavobacterium, Nocardia, Vibrio, the Pseudomonas etc. of unlinking; Whiterot fungi, smoke pipe bacterium etc. can be degraded to the above PAHs of 4 ring by common metabolic way.But the bacterial strain of having reported at present still can not meet for PAHs degradation efficiency more than Fourth Ring the needs that actual pollution is controlled, therefore still need screening obligate degradation bacteria more efficiently, and study its actual soil remediation effect.
Summary of the invention
In order to overcome the deficiencies in the prior art, the object of the present invention is to provide a kind of pyrene efficient degrading bacteria and application thereof.
A kind of pyrene efficient degrading bacteria provided by the invention, this strain bacterium be achromobacter (
achromobactersp.) PY1, and with Achromobacter xylosoxidans (
achromobacter xylosoxidan) similarity is 99%, near its polluted agricultural land An Zhen service station, Wuxi City, Jiangsu Province, soil separation obtains.
Achromobacter provided by the invention (
achromobactersp) PY1 bacterial strain, its Classification And Nomenclature is: Achromobacter xylosoxidans (
achromobacter xylosoxidan), on June 17th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, referred to as: CGMCC; Address is: Datun Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences; Deposit number is: CGMCC No. 7741.
The morphological specificity of achromobacter PY1 provided by the invention is as follows: bacterial strain 25 ℃ of constant temperature culture after 3 days on LB flat board, that bacterium colony is is faint yellow, circular, dimpling, neat in edge, moistening, opaque.Gram-negative.Micro-Microscopic observation, bacterium is straight or slightly curved tyrothricin (0.4 ~ 0.6 μ m * 2.0 ~ 5.0 μ m), the single or irregular group of cell shape occurs.
The main physiological and biochemical property of achromobacter PY1 provided by the invention is as follows: non-fermentation, do not form brood cell, dynamic, oxydase and catalase is positive, oxidation wood sugar produces acid.
Beneficial effect of the present invention is: achromobacter (
achromobactersp.) method for screening and separating of PY1 is simple, isolated achromobacter (
achromobactersp.) the PY1 pyrene of effectively degrading, is conducive to the reparation of pyrene contaminated soil.
Accompanying drawing explanation
Fig. 1 be achromobacter (
achromobactersp.) growth curve of PY1.
Fig. 2 be achromobacter (
achromobactersp.) phylogenetic tree of PY1.
Fig. 3 be achromobacter (
achromobactersp.) growth tendency and the pyrene degradation efficiency figure of PY1 in pyrene-minimal medium.
Fig. 4 be different culture temperature to achromobacter (
achromobactersp.) PY1 growth and pyrene degradation rate affects figure.
Fig. 5 be the different pH of cultivation values to achromobacter (
achromobactersp.) PY1 growth and pyrene degradation rate affects figure.
Fig. 6 is the affect figures of different initial inoculums on pyrene degradation rate.
Fig. 7 is the affect figure of different luxuriant and rich with fragrance concentration on pyrene degradation rate.
Fig. 8 is the affect figure of inoculation degradation bacteria on soil pyrene degradation rate.
Embodiment
The screening of embodiment 1 pyrene efficient degrading bacteria
Gather near the contaminated soil in Wuxi City An Zhen service station, get 1 g fresh soil sample and join with 100 mg L
-1pyrene is in the inorganic salt liquid substratum (pyrene-inorganic salt liquid substratum) of sole carbon source, in temperature, is that 25 ℃, rotating speed are shaking culture in the shaking table of 120 rpm, after one week, by the inoculum size of 1wt%, is transferred to 100 new mg L
-1in pyrene inorganic salt liquid substratum, continue shaking table and cultivate 1 week.So repeat, after 5 times, to get pregnant solution and carry out gradient dilution, coat on pyrene-inorganic salt double-layer plate.Cultivate after one week, on flat board, picking can produce single bacterium colony of hydrolysis circle, in the flat lining out separation and purification of LB, until obtain pure single bacterium colony.Bacterial strain, after cultivating, then is inoculated into separately to shake-flask culture in pyrene-inorganic salt liquid substratum, choose the speed of growth faster bacterial strain as research object.
Wherein:
The composition of inorganic salt liquid substratum and content: (NH
4)
2sO
41.5 g, NaCl 0.5 g, KH
2pO
41.5 g, K
2hPO
40.5 g, MgSO
47H
2o 0.2 g, 1 000 mL H
2o, pH 7.0.
100 mg L
-1the compound method of pyrene-inorganic salt liquid substratum: pyrene is first mixed with 1 g L
-1acetone concentrated solution, therefrom get 1 mL and add in 100 mL minimal mediums, 121 ℃ of sterilizing 30 min.
The evaluation of bacterial strain:
Separated bacterial classification is extracted to total DNA, and total DNA, after pcr amplification, obtains 16S rDNA product ，You Shanghai Shenergy Biocolor BioScience & Technology Company purifying the order-checking of size approximately 1.4 kb.The 16S rDNA gene order of this bacterial strain is as shown in sequence table SEQ ID NO.1.
Isolated strains 16S rDNA sequence is compared by Blast program and GenBank amplifying nucleic acid data, set up phylogenetic tree result as shown in Figure 2.
With reference to < < common bacteria system identification handbook > >, carry out Physiology and biochemistry evaluation, finally identify that bacterial strain is to genus simultaneously.Result shows: this strain bacterium be achromobacter (
achromobactersp.), and with
achromobacter xylosoxidan(Achromobacter xylosoxidans) similarity is 99%.
In the present invention, by the bacterial strain called after achromobacter obtaining (
achromobactersp.) PY1.
The growth curve of bacterial strain in LB liquid nutrient medium as shown in Figure 1, reached stationary phase in 14 h.
The Degrading experiment of embodiment 2 pyrenes
By the speed of growth obtaining in embodiment 1 faster achromobacter (
achromobactersp.) in PY1 inoculation to 100 mL pyrene-inorganic salt liquid substratum, 25 ℃, pH7.0, inoculum size 1wt%, 250 rpm shaking tables are cultivated one week, get 10 mL nutrient solutions, use isopyknic n-hexane extraction, after 40 ℃ of water-bath rotary evaporations, add anhydrous sodium sulphate, the concentrated constant volume of rotary evaporation, finally uses Agilent 6890N gas chromatograph (FID, HP25 capillary column) to measure pyrene residual content again.
Parameter arranges: the temperature of injection port is 280 ℃, and the temperature of detector is 300 ℃, temperature programming, and 80 ℃ of the temperature that begins, constant temperature 2 min, 15 ℃/min is raised to 280 ℃, constant temperature 3 min; Carrier gas N
22 ml/min, H
240 ml/min, air 400 ml/min; Splitless injecting samples, sample size 1 μ l.
Bacterial strain to the degradation efficiency of pyrene as shown in Figure 3.Result shows: after the adaptive phase of 2 d, enter rapid degradative phase (3-5 d), along with the increase of thalline quantity, the degradation rate of pyrene also increases gradually, when being cultured to the 5th d, it is maximum that thalline quantity reaches, and the degradation rate of pyrene still slowly increases, when cultivating the 6th d, thalli growth amount is substantially steady, explanation, because substrate is utilized and the accumulation of meta-bolites gradually, causes bacterial strain mortality ratio to increase gradually, the most finally breeding potential balance.Although thalli growth amount held stationary, pyrene degradation rate is still in slow increase, and while being cultured to the 7th d, the degradation rate of pyrene is 89.20%.
The impact of embodiment 3 varying environment conditions on pyrene degraded
By the speed of growth obtaining in embodiment 1 faster achromobacter (
achromobactersp) in PY1 inoculation to 100 mL pyrene-inorganic salt liquid substratum, shaking table is cultivated one week, get 10 mL nutrient solutions, use isopyknic n-hexane extraction, after 40 ℃ of water-bath rotary evaporations, add anhydrous sodium sulphate, the more concentrated constant volume of rotary evaporation, finally use Agilent 6890N gas chromatograph (FID, HP25 capillary column) to measure pyrene residual content.In 100 ml pyrene-inorganic salt liquid substratum, test differing temps (15,20,25,30,35 ℃), pH(4,5,6,7,8,9) (adopting the dilute hydrochloric acid of 0.1 M sodium hydroxide or 5% to regulate pH), inoculum size (0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%) and add metabolism substrate (1,2,5,10 mg L altogether
-1phenanthrene) impact on its degraded.
Achromobacter (
achromobactersp) PY1 inoculation, in minimal medium, is cultivated respectively at 15,20,25,30,35 ℃, and result as shown in Figure 4.Can find out, within the scope of 15-25 ℃, the degradation rate of pyrene raises and increases with temperature, and wherein pyrene degradation rate reaches maximum in the time of 25 ℃.When culture temperature is 30 ℃, the increment of bacterial strain PY1 is the highest, but the degradation rate of pyrene slightly declines.Degradation rate by pyrene can find, also corresponding raising of pyrene degradation rate when thalli growth amount is large, illustrates that bacterial strain PY1 is in vigorous growth, and the secretion of the enzyme relevant with metabolism is also in the most vigorous stage.Result shows, 25 ~ 30 ℃ all highly beneficial for thalli growth or the degraded of pyrene.
Different pH values to achromobacter (
achromobactersp) usefulness of PY1 strains for degrading pyrene has impact in various degree, and result as shown in Figure 5.Achromobacter (
achromobactersp) PY1 bacterial strain is grown and is subject to inhibition to a certain extent in strong acidic environment.Along with weakening of acidity, bacterial strain starts raised growth breeding, and degradation efficiency also obviously improves.Bacterial strain is grown the most vigorous when pH is 6 left and right, and bacterial number rolls up, and the degradation efficiency of pyrene is also reached to the highest.Under neutral meta-alkalescence condition, bacterial strain slightly declines to the degradation effect of pyrene.
On the impact of pyrene degradation rate as shown in Figure 6, result shows different initial inoculums, and initial inoculum is in 1wt%, and one week degradation rate of pyrene reaches 84.1%, and after this strengthen inoculum size, final degradation rate is not had to too much influence again.Illustrate under shaking flask condition, 1wt% is proper inoculum size.
Add metabolism substrate (phenanthrene) altogether on pyrene degraded affect result as shown in Figure 7, result shows between pyrene and phenanthrene, there is significantly metabolism relation altogether.Along with the content increase of luxuriant and rich with fragrance concentration, achromobacter (
achromobactersp) PY1 bacterial strain can be take phenanthrene fast as carbon source for growth, the relevant degrading enzyme class of secretion simultaneously, the degradation rate of raising pyrene.Bacterial strain reaches maximum value to the degraded of pyrene when luxuriant and rich with fragrance concentration content is 20 mg/L, and along with the increase of luxuriant and rich with fragrance content, degradation efficiency sharply declines afterwards.This may be, because luxuriant and rich with fragrance concentration increases, bacterial strain is also had to certain toxicity, has suppressed the growth of thalline.
Culture presevation: in the fresh LB liquid nutrient medium of bacterial strain access sterilizing, shaking table constant temperature oscillation, to stationary phase, adds 20% sterile glycerol, in cold storage pipe ,-80 ℃ of preservations 1 year.
The reparative experiment of embodiment 4 pyrene efficient degrading bacterias to contaminated soil
(1) preparation in pyrene contaminated soil
Pyrene is dissolved in acetone, configures 5 g L
-1concentrated solution.Take 1 kg and cross 100 mesh sieve soil samples and be put in stainless steel pallet, add 100 ml pyrene-acetone concentrated solutions, with scoop, stir, be placed in stink cupboard and regularly and stir, acetone is volatilized completely.The Polluted Soil that 1 kg is prepared and 49 kg stir by the cleaning soil of 2 mm sieves, and preparing pyrene concentration is 10 mg kg
-1pollution soil sample, for pot experiment.
(2) recovery dynatron effect of pyrene efficient degrading bacteria to contaminated soil
Adopt the basin alms bowl of 2 kg to carry out pot experiment, each basin alms bowl dress pollutes soil sample 1.5 kg, but after dress basin, water irrigates and do not produce percolating water.Select golden hook pumpkin as test plant, select full seed, 10% H
2o
230 min that sterilize, distilled water flushing three times.Inoculating strain is (3 g L in 100 ml enrichment mediums
-1extractum carnis, 5 g L
-1peptone, 5 g L
-1naCl, 7.0,121 ℃ of moist heat sterilization 30 min of pH), 30 ℃ of shaking culture 48 h, it is centrifugal that (10000 turn min
-1, 5 min) and collection thalline, 0.1 mol L
-1mgSO
47H
2o washing thalline 3 times, finally uses 0.1 mol L
-1mgSO
47H
2o suspends thalline, measures the optical density(OD) (λ=600 nm) of bacterial suspension and be adjusted to density to be about 1012 ml
-1.
Test is divided into four processing: 1, plant separately pumpkin (Pu); 2, plantation pumpkin+inoculation degradation bacteria (Pu+PY1); 3, plantation pumpkin+interpolation glucose (Pu+Glu); 4, plantation pumpkin+inoculation degradation bacteria+interpolation glucose (Pu+PY1+Glu), the blank basin alms bowl that pumpkin is not planted in setting is simultaneously (CK) in contrast.Each processes 3 repetitions, and glucose adds with 0.6% amount; Bacterium pours into 2% bacteria suspension, and the processing of inoculated bacteria does not impose 0.1 mol L of equivalent
-1mgSO
4solution, mixes with soil.6 Pumpkin Seed of sterilizing of every basin sowing, thinning to 3 after germinateing.Basin alms bowl random alignment in heliogreenhouse, every day weighting method to keep soil moisture content be field capacity 60%, Routine Management, gathers in the crops after 60 d.
During 60 d, plant is divided overground part and underground part results, with tap water, rinses rear distilled water flushing well three times, and 70 ℃ of oven dry, take dry weight, pulverize standby; Each basin alms bowl takes to mix soil sample 200 g, and a part is stored in 4 ℃ of refrigerators for microbiological analysis, and another part is air-dry to be ground, and crosses 100 mesh sieves standby.
The mensuration of pyrene residual quantity in soil: get 2.0 g soil samples in centrifuge tube, add 2 g anhydrous sodium sulphate, mix; Add the organic extraction agent of 30 mL (acetone: normal hexane=1: 1), ultrasonic extraction 30 min at 40 ℃; 4000 r min
-1centrifugal, collect supernatant liquor.This step in triplicate, merges supernatant liquor.Supernatant liquor is evaporated to 2 mL with Rotary Evaporators, crosses Cleanert C18-SPE post, with 10 mL normal hexane wash-outs, N at 40 ℃
2dry up, with normal hexane constant volume, to 2 mL, GC analyzes.
As shown in Figure 8, result shows result, plants pumpkin and can significantly improve soil pyrene degradation rate, is mainly due to the root exudates of plant, to increase the carbon source of soil, has improved soil property simultaneously.In plantation pumpkin, inoculation degradation bacteria and interpolation dextrose treatment soil pyrene degradation rate have the trend of increase, but compare and there is no significant difference with plantation pumpkin.In inoculation degradation bacteria, add a small amount of glucose as additional carbon, to improving the survival rate of degradation bacteria in soil, there is very important effect, soil pyrene degradation rate is also significantly higher than the processing of only adding glucose simultaneously.
SEQUENCE LISTING
<110> Zhejiang A & F University
<120> pyrene efficient degrading bacteria and application thereof
<130> 001
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 694
<212> DNA
<213> achromobacter (Achromobacter sp) PY1
<400> 1
gccttacaca tgcaagtcga acggcagcac ggacttcggt ctggtggcga gtggcgaacg 60
ggtgagtaat gtatcggaac gtgcccagta gcgggggata actacgcgaa agcgtagcta 120
ataccgcata cgccctacgg gggaaagcag gggatcgcaa gaccttgcac tattggagcg 180
gccgatatcg gattagctag ttggtggggt aacggctcac caaggcgacg atccgtagct 240
ggtttgagag gacgaccagc cacactggga ctgagacacg gcccagactc ctacgggagg 300
cagcagtggg gaattttgga caatggggga aaccctgatc cagccatccc gcgtgtgcga 360
tgaaggcctt cgggttgtaa agcacttttg gcaggaaaga aacgtcgcgg gttaataccc 420
cgcggaactg acggtacctg cagaataagc accggctaac tacgtgccag cagccgcggt 480
aatacgtagg gtgcaagcgt taatcggaat tactgggcgt aaagcgtgcg caggcggttc 540
ggaaagaaag atgtgaaatc ccagagctta actttggaac tgcattttta actaccgggc 600
tagagtgtgt cagagggagg tggaattccg cgtgtagcag tgaaatgcgt agatatgcgg 660
aggaacaccg atggcgaagg cagcctcctg ggat 694
Claims (5)
1. a pyrene efficient degrading bacteria, its be achromobacter (
achromobactersp.) PY1, its preservation registration number is CGMCC No. 7741.
Achromobacter (
achromobactersp.) application of PY1 CGMCC No. 7741 in degraded pyrene.
3. application claimed in claim 2, is characterized in that: temperature during degraded is 25 ℃, and pH is 6.0, and initial inoculum is 1wt%.
4. application according to claim 2, is characterized in that: during degraded, add phenanthrene to carry out co-degradation.
5. application according to claim 2, is characterized in that: in the time of in degraded pyrene contaminated soil, plant pumpkin and add glucose in soil.
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| CN115415299A (en) * | 2022-08-29 | 2022-12-02 | 东华大学 | Method for repairing pyrene contaminated soil through low-temperature heat treatment-pyrene degradation microbial inoculum combination |
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Application publication date: 20140205 |