CN101891305B - Application of facultative anaerobic degrading bacteria for benzene compounds - Google Patents
Application of facultative anaerobic degrading bacteria for benzene compounds Download PDFInfo
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Abstract
The invention belongs to the technical field of degrading microorganism and relates to application of facultative anaerobic degrading bacteria for benzene compounds, wherein the facultative anaerobic degrading bacteria of benzene compounds are used for degrading benzene compounds in the underground water. The application comprises the following steps of: (a) picking up a HBSD-C bacterial lawn of microbacterium schleiferi, which is kept in the way of slant refrigeration, by utilizing an inoculating loop, inoculating to a 30-ml sterilized test tube containing 10 microliters of benzene, 10 microliters of toluene, 10 microliters of xylene, 10 microliters of sym-trimethylbenzene and 10 ml of organic salt isolation medium, activating and culturing the bacteria for 36h to obtain the activated bacterial suspension; (b) mixing the benzene compounds polluted underground water and the activated bacterial suspension in the ratio of 50 ml: 50 microliters; (c) and culturing the bacteria for 72h at 15 to 40 DEG C to obtain the degraded underground water. In the invention, the benzene, the toluene, the xylene and the sym-trimethylbenzene can be degraded under facultative condition, and the degradation rate for the benzene compounds with concentration of 175.8 mg/L in three days ranges from 25.9% to 41.2%.
Description
Technical field
The invention belongs to the degraded microbial technology field, also belong to environmental pollutant biologic treating technique field, be specifically related to the application of a facultative anaerobic benzene compounds-degrading bacterium.
Background technology
Benzene compounds is one type of volatile compound fragrant hydrocarbon; Comprise benzene, toluene, ethylbenzene, neighbour,, p-Xylol etc.; Except being present in the Fuel Petroleum; Benzene compounds also is widely used in (Romy Chakraborty in the manufacturing of rubber, lubricant, dyestuff, washing composition, medicine and agricultural chemicals as starting material; John D.Coates.2005.Hydroxylationand Carboxylation-Two Crucial Steps of Anaerobic Benzene Degradation by DechloromonasStrain RCB.Applied and environmental microbiology, Sept.2005, p.5427-5432).But not paid attention to very much yet benzene compounds content in petroleum hydrocarbon is high, is water-soluble best component in the petroleum hydrocarbon, the settling-relatively low (Coleman of water dispersion coefficient; W.E., J.W.Munch, R.P.Streicher; H.P.Ringhand; And F.C.Kopfler.1984.Identification and measurement of components in gasoline, kerosene, and no.2 fuel oil thatpartition into the aqueous phase after mixing.Arch.Environ.Contam.Toxicol.13:171-178.); A little less than be adsorbed in the waterbearing stratum material; And be reversible adsorption, in underground water, be easy to migration, constituted organic pollutant common in the underground water.It has serious carcinogenic, teratogenesis, mutagenicity, is classified as priority pollutants by many countries.
Biological degradation is the important mechanisms that benzene compounds is decayed naturally; Also be major measure (the Silvia A.Mancini of dissolved benzene compounds migration in the control underground water; Ania C.Ulrich, Georges Lacrampe-Couloume, et al.2003.Carbonand Hydrogen Isotopic Fractionation during Anaerobic Biodegradation of Benzene.Applied andenvironmental microbiology; Jan.2003, p.191-198).Have under the condition of molecular oxygen existence, benzene compounds can be by biological degradation.Yet the quick consumption of underground oxygen in water can produce a large amount of anaerobic zones to mikrobe, and therefore, many researchs all concentrate on the anaerobic biodegradation of benzene compounds in the past 20 years, but anaerobic degradation mechanism is still clear fully.Simultaneously, it is lower that the biological degradation of anaerobism benzene compounds is faced with the DeR production capacity, and degraded is difficult for carrying out problems such as thorough.By contrast, amphimicrobe degrading benzene compounds technology has more practical significance.The existing benzene compounds degradation bacteria of discovering mainly contains pseudomonas (Pseudomonas), acinetobacter calcoaceticus (Acinetobacter), genus bacillus (Bacillus), rhodococcus (Rhodococcus), Nocardia bacteria (Nocardia), the logical Salmonella (Ralstonia) in Rolls, Alcaligenes (Alcaligenes) etc.
Summary of the invention
The purpose of this invention is to provide the application of a facultative anaerobic benzene compounds-degrading bacterium, it can be at the oxygen condition of holding concurrently degrade down benzene,toluene,xylene, sym-trimethylbenzene.
A facultative anaerobic benzene compounds-degrading bacterium provided by the present invention; Be Shi Shi microbacterium (Microbacteriumschleiferi) HBSD-C CCTCC NO:M209284; Chinese typical culture collection center (being called for short CCTCC), deposit number: CCTCC NO:M209284 have been preserved in on November 27th, 2009.
The colonial morphology of described Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C: light yellow, circular, translucent, neat in edge, depression, smooth surface; Cell is a direct rod shape, and Gram-positive is not moved; Main biochemical character: oxidase positive, catalase is positive, utilizes glucose, fructose, sucrose, does not utilize SANMALT-S, and denitrification is positive, and gelatin hydrolysis is negative, and amphimicrobian is 4 ℃～41 ℃ growths (cannot 60 ℃ of growths).
The application of benzene series compound facultative anaerobic degrading bacterium is characterized in that: its benzene compounds in the underground water that is applied to degrade.Concrete steps are following:
A) with the cryopreserved Shi Shi microbacterium in transfering loop picking inclined-plane (Microbacterium schleiferi) HBSD-C lawn; Be inoculated in and contain benzene,toluene,xylene and each 10 μ L of sym-trimethylbenzene; In the 30mL of the sterilization test tube of inorganic salt isolation medium 10mL; Behind constant-temperature shaking activation culture 36h under 15 ℃～40 ℃ conditions, obtain the activatory bacteria suspension;
B) by receiving benzene series compounds contaminated underground water: the proportioning=50mL of activatory bacteria suspension: 50 μ L, choose and receive benzene series compounds contaminated underground water and activatory bacteria suspension; Mixed with the activatory bacteria suspension by benzene series compounds contaminated underground water;
C) then in 15 ℃～40 ℃ cultivations, behind the cultivation 72h, the underground water after obtaining degrading.
Described benzene compounds comprises any one or any mixing more than two kinds in benzene,toluene,xylene, the sym-trimethylbenzene, and any is any proportioning when mixing more than two kinds.
The present invention receives from the Daqing oil field somewhere that screening and separating obtains the new Shi Shi microbacterium of a strain (Microbacterium schleiferi) HBSD-C the soil of crude oil pollution; It can be at the oxygen condition of holding concurrently degrade down benzene,toluene,xylene, sym-trimethylbenzene; Strong to environmental compatibility, can be applied to receive benzene series compounds contaminated phreatic improvement to repair.For the research of the oxygen benzene compounds microbiological deterioration mechanism of holding concurrently provides important foundation, will repair in environmental pollution improvement, administered in the practice by benzene series compounds contaminated underground water and play a significant role.
Description of drawings
Fig. 1 is the colonial morphology figure of Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C.
Fig. 2 be Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C pcr amplification agarose electrophoresis figure (C: amplified production, M:Marker).
Fig. 3 is the benzene compounds 72h degradation property histogram of Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C.
Fig. 4 a is the influence figure of pH to Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C growth.
Fig. 4 b is the influence figure of temperature to Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C growth.
Fig. 4 c is the influence figure of saltiness to Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C growth.
Embodiment
The screening method of 1 one facultative anaerobic benzene compounds-degrading bacteriums
1.1 material is prepared
1.1.1 bacterium source
Gather Daqing oil field greasy dirt district, the pedotheque of the following 5-10cm in top layer.
1.1.2 substratum
Inorganic salt isolation medium: 0.42gL
-1NaNO
3, 0.27gL
-1NH
4Cl, 7.9gL
-1Na
2HPO
412H
2O, 1.5gL
-1KH
2PO
4, 1.0gL
-1MgSO
47H
2O, 1.0mLL
-1(proportioning of trace element solution is trace element solution: 2.14gL
-1ZnCl
2, 2.5gL
-1MnCl
24H
2O, 0.3gL
-1CoCl
26H
2O, 0.2gL
-1CuCl
22H
2O, 0.4gL
-1NaMoO
42H
2O, 4.5gL
-1CaCl
22H
2O, 2.9gL
-1FeCl
36H
2O, 1.0gL
-1H
3BO
3, 0.1gL
-1KI, the 1L deionized water), 0.472gL
-1Soduxin, 1L zero(ppm) water, pH=7.0.
Nutrient agar: 1L inorganic salt isolation medium+20gL
-1Agar.
LB substratum: 1.0gL
-1Yeast powder (YEAST EXTRACT), 2gL
-1Peptone (TRYPTONE), 2gL
-1NaCl, 1L zero(ppm) water, pH=7.0.
Slant medium: 1.0gL
-1Yeast powder (YEASTEXTRACT), 2gL
-1Peptone (TRYPTONE), 2gL
-1NaCl, 15gL
-1Agar, 1L zero(ppm) water, pH=7.0.
Above substratum is subsequent use after respectively at steam sterilizing 30mins under 121.5 ℃ of conditions.
1.1.3 laboratory apparatus and equipment
Gas chromatograph: HP 6890N gc (tool fid detector);
Chromatographic column: HP-5 capillary column 30m * 0.32mm * 0.25 μ m;
The SW-CJ-2FD Bechtop;
TGL-16A desk type high speed refrigerated centrifuge;
The quiet oil-free air pump of CA-1 type;
SHZ-82A gas bath constant temperature oscillator;
The vertical automatic electric heating pressuresteam sterilization of LDZX-40SAI type pot;
The biochemical incubator of PHX type intelligence;
DHG-9423A type electric heating constant temperature air dry oven;
SHA-C type water bath with thermostatic control vibrator;
HH-4 digital display thermostat water bath;
The 78-1 magnetic stirring apparatus;
The BCD-301W H of Haier refrigerator;
The silver grads PCR appearance of Mastercycler ep gradient S;
UVP EC3600 gel imaging system etc.
1.2 the separation of Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C
1.2.1 the acclimation and screening of bacterial strain
Get the 5g pedotheque in the 50mL centrifuge tube, with tap water washing, centrifugal 5mins under the 8000r/min condition, totally 3 times to remove humic acid.Add 50mL inorganic salt isolation medium in the bottle shaking of 100mL, analytically pure benzene,toluene,xylene, each 6.25 μ L of sym-trimethylbenzene, the soil sample of Soduxin 23.6mg and centrifuge washing shakes up, and seals film phonograph seal with silica gel plug and Parafilm.Under 30 ℃, 200r/min condition shaking culture 2-4 days, whether the muddy degree decision of looking bacteria suspension finished the acclimation shaking culture in first cycle.After this, get bacteria suspension 200 μ L, Soduxin content constant gradient successively decreases (by 4 gradients, constant gradient successively decreases), and benzene compounds content constant gradient increases progressively (by 4 gradients, constant gradient successively decreases), the constant acclimation shaking culture of other conditions.During the 4th cycle, Soduxin content is zero in the 50mL substratum, and benzene,toluene,xylene, sym-trimethylbenzene be 50 μ L respectively, obtains the bacteria suspension of acclimation shaking culture.After acclimation period finished, the degradation flora in the bacteria suspension can utilize benzene compounds as the sole carbon source and the energy.The domestication condition is as shown in table 1.
Table 1 degradation bacteria domestication experiment condition
1.2.2 the separation and purification of bacterial strain
In the centrifuge tube of 1.5mL sterilization, the bacteria suspension 50 μ L after the adding acclimation shaking culture, sterilized water 1.0mL, 6 gradients of constant gradient dilution.When the nutrient agar sterilization is cooled to 40 ℃ of left and right sides, add analytically pure benzene,toluene,xylene, each 200 μ L of sym-trimethylbenzene, shake up, dull and stereotyped fast, cool off to such an extent that separate bacterium colony with dull and stereotyped.On the bacteria suspension 20 μ L spread plates with the 5th, the 6th concentration gradient dilution.With flat board under 30 ℃ of conditions, constant temperature culture 3-5 days.The bacterium colony that the picking growing way is good carries out pure culture, obtains the bacterium colony after the pure culture.
In the test tube of 18mm * 180mm, add 10mL inorganic salt isolation medium, add benzene,toluene,xylene, each 10 μ L of sym-trimethylbenzene, in wherein, silica gel plug and Parafilm seal film phonograph seal with the colony inoculation after the pure culture of transfering loop picking.Shaking culture is 36 hours under 30 ℃, 200r/min condition, gets the bacteria suspension coated plate, and method is with the above.Pure culture 2-3 time until obtaining single bacterial strain, is designated as bacterial strain HBSD-C [Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C], strain growth situation, colony characteristics on the observed and recorded flat board.
With bacterial strain HBSD-A (being the bacterial classification of purifying) streak inoculation on slant medium; In 4 ℃ of refrigerator storage; All use every month inorganic salt isolation medium and benzene compounds activation culture (promptly go into 10mL inorganic salt isolation medium, add benzene,toluene,xylene, each 10 μ L of sym-trimethylbenzene), spread plate to be inoculated on the inclined-plane and to use in order to follow-up test.For long-term preservation strain HBSD-C, send Chinese typical culture collection center (CCTCC) preservation on November 27th, 2009 with bacterial strain HBSD-C.
The evaluation of 2 Shi Shi microbacteriums (Microbacterium schleiferi) HBSD-C
2.116S rDNA complete sequence determination
2.1.1 the extraction of bacterial genomes DNA (gram-positive microorganism)
A) the bacterial classification HBSD-C that in the super clean bench separation is obtained is inoculated in and contains 10gL
-1The LB substratum of glucose (dip in get with aseptic toothpick), 5mL/tube * 2 are in 37 ℃ of centrifugal spending the night of 220rpm.
B) in the super clean bench 10mL bacterium liquid is transferred to the 50mL round bottom centrifuge tube.4 ℃ of centrifugal 20min of 5000rpm.
C) abandon supernatant, add 1.2mL 10mM Tris-Cl (tris buffer) (PH8.0)/25%Sucrose, vortex suspends, and divides to install to 2 1.5mL EP pipes (0.6mL/EP).
D) add 10mg/mL N,O-Diacetylmuramidase (fresh), making final concentration is 1mg/mL, mixing, 37 ℃ of water-bath 10min.
E) add 200gL
-1SDS (sodium dodecyl sulfate solution), making its final concentration is 5gL
-1Add Proteinase K, making final concentration is 100 μ g/mL, soft mixing.50 ℃ of water-bath 3h, during put upside down mixing frequently.
F) be chilled to room temperature, add equal-volume phenol/chloroform, fully put upside down mixing 10min.
G) the centrifugal 15min of room temperature 5000rpm gets supernatant.
H) repeating step f), step g) once.
I) add the equal-volume chloroform, fully put upside down mixing 10min.The centrifugal 15min of room temperature 5000rpm gets supernatant.
J) two EP merge, and divide to 3 EP pipes (about 0.4mL/EP).
K) every EP pipe adds the absolute ethyl alcohol of 2 volume precoolings and the 3MNaOAC (sodium-acetate) (making its final concentration is 0.3M) of 1/10 volume ,-20 ℃ of deposition 2h or spend the night.
L) 0 ℃ centrifugal, maximum speed of revolution (or 5000rpm) is abandoned supernatant.
M) add semicanal (about 700 μ l) 70% washing with alcohol, 4 ℃ centrifugal, and maximum speed of revolution (or 5000rpm) is abandoned supernatant.
N) drying at room temperature.
O) be dissolved in an amount of TE (PH7.4) (20～50 μ L), add 10mg/mL RNase (RNA lytic enzyme), making final concentration is 20 μ g/mL, 37 ℃ of water-bath 2h.
P) repeat phenol/chloroform extracting, absolute ethyl alcohol/3M NaOAC deposition, 70% washing with alcohol.(step and method are the same).
Q) use an amount of 3dH
2O (sterilization tri-distilled water) dissolving DNA deposition.
R)-20 ℃ preservation.
2.1.216S the pcr amplification of rDNA gene
A) pcr amplification primer:
P1: forward primer 5 ' AGAGTTTGATCCTGGCTCAG3 '
P2: reverse primer 5 ' GGTTACCTTGTTACGACTT3 '
B) reaction system:
In 50 μ L reaction volumes, add 1 μ L template DNA (0.1 μ g), 0.5 μ L P1 and P2 (final concentration is 0.5 μ M), 1 μ LdNTP (every kind of NTP0.2mM), 0.5 μ LTaq polysaccharase (2U) and 5 μ L10 * PCR damping fluid.The pcr amplification condition is: 94 ℃ of preparatory sex change 5mins; At 94 ℃ of sex change 30s, 61-65 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate 30 times; Last 72 ℃ are extended 10mins eventually.The PCR product is carried out electrophoresis detection with 1% sepharose.
2.1.316S rDNA complete sequence determination
The purifying of PCR product is accomplished by Shanghai Ying Jun Bioisystech Co., Ltd with order-checking, checks order with Applied Biosystem 373ADNA sequenator.
The present invention adopts the method for 16S rDNA complete sequence determination and analysis is carried out the evaluation of molecular level to bacterium.Nuclear DNA with bacterium is a template, is primer with the universal primer of the pcr amplification of 16S rDNA gene, carries out pcr amplification, obtains the amplified band that length is 1420bp (detecting with 1% agarose gel electrophoresis), after the PCR product is purified, measures its complete sequence.Sequence is following:
< 110>China Geological Univ. Wuhan
< 120>application of benzene series compound facultative anaerobic degrading bacterium
<160>1420
<210>1
<211>1420bp
<212>DNA
< 213>Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C
<400>1
1 TGGCCGCGTGCTTTACACATGCGAGTCGGACGGTGAAGCAGAGCTTGCTCTGTGGATCAG
61 TGGCGAACGGGTGAGTAACACGTGAGCAATCTGCCCCTGACTCTGGGATAAGCGCTGGAA
121?ACGGCGTCTAATACCGGATACGAGCTGCGAAGGCATCTTCAGCAGCTGGAAAGAATTTCG
181?GTCAGGGATGAGCTCGCGGCCTATCAGCTTGTTGGTGAGGTAACGGCTCACCAAGGCGTC
241?GACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACT
301?CCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCAACGC
361?CGCGTGAGGGACGACGGCCTTCGGGTTGTAAACCTCTTTTAGCAGGGAAGAAGCGAAAGT
421?GACGGTACCTGCAGAAAAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG
481?GGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCT
541?GCTGTGAAAACCCGAGGCTCAACCTCGGGCCTGCAGTGGGTACGGGCAGACTAGAGTGCG
601?GTAGGGGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACC
661?GATGGCGAAGGCAGATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGGGTGGGGAGCA
721?AACAGGCTTAGATACCCTGGTAGTCCACCCCGTAAACGTTGGGAACTAGTTGTGGGGACC
781?ATTCCACGGTTTCCGTGACGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCG
841?CAAGGCTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGCGGAGCATGCGGATTA
901?ATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATATACGAGAACGGGCCAGAAAT
961?GGTCAACTCTTTGGACACTCGTAAACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTG
1021?AGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCACGTAAT
1081?GGTGGGAACTCATGGGATACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAA
1141?TCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGCTG
1201?CAATACCGTAAGGTGGAGCGAATCCCAAAAAGCCGGTCCCAGTTCGGATTGAGGTCTGCA
1261?ACTCGACCTCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATA
1321?CGTTCCGGGGTCTTGTACACACCGCCCGTCAAGTCATGTAAGTCGGTAACACCCGAAGCC
1381?GGTGGCCCAACCCTTCTGGAGGGAGCCGTCGAAGGTTGGA
According to the above-mentioned characteristic of degradation bacteria HBSD-C, it is accredited as Shi Shi microbacterium (Microbacterium schleiferi).
2.2 colony morphology characteristic and physio-biochemical characteristics
2.2.1 colony morphology characteristic
See Fig. 1.That the bacterium colony of benzene compounds degradation bacteria HBSD-C is is light yellow, circular, translucent, neat in edge, depression, smooth surface.
2.2.2 physiological and biochemical property
The mensuration of Physiology and biochemistry character is with reference to " the method that uncle Jie Shi Bacteria Identification handbook is the 8th edition.See table 2.
The physiological and biochemical property that table 2 degradation bacteria HBSD-C is main
Nomenclature in the table: "+": the bacterial strain more than 90% is positive, "-": the bacterial strain more than 90% is negative.
Table 2 explanation: cell is a direct rod shape, and Gram-positive is not moved; Main biochemical character: oxidase positive, catalase is positive, utilizes glucose, fructose, sucrose, does not utilize SANMALT-S, and denitrification is positive, and gelatin hydrolysis is negative, and amphimicrobian is 4 ℃～41 ℃ growths (cannot 60 ℃ of growths).
2.3 Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C is new bacterial strain
The 16S rDNA sequence that records adopts BLAST software and international GenBank DB to carry out sequence homology relatively, and the result finds that the homology of bacterial strain and Microbacterium schleiferi (Shi Shi microbacterium) is up to more than 99%.
The Physiology and biochemistry and the Molecular Identification result of comprehensive bacterial strain, Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C is new bacterial strain (i.e. a facultative anaerobic benzene compounds-degrading bacterium).Chinese typical culture collection center (being called for short CCTCC), deposit number: CCTCC NO:M209284 have been preserved in on November 27th, 2009.
The application of 3 one facultative anaerobic benzene compounds-degrading bacteriums
3.1 Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C is to the degradation property of benzene compounds
A) with the cryopreserved a small amount of Shi Shi microbacterium in transfering loop picking inclined-plane (Microbacterium schleiferi) HBSD-C lawn; Be inoculated in and contain benzene,toluene,xylene and each 10 μ L of sym-trimethylbenzene; In the 30mL of sterilization (Φ 18*180mm) test tube of inorganic salt isolation medium 10mL; Behind constant-temperature shaking activation culture 36h under 30 ℃ of conditions, obtain the bacteria suspension of activation culture;
B) 100mL is shaken bottle after sterilization is cooled to normal temperature; In super clean bench, add 50mL inorganic salt isolation medium, analytically pure benzene,toluene,xylene, each 10 μ L of sym-trimethylbenzene; The bacteria suspension 50 μ L of activation culture, silica gel plug and Parafilm seal film phonograph seal.Single benzene compounds starting point concentration is about 175.8mg/L, establishes the blank of not inoculating simultaneously;
C) will test appearance and blank in 30 ℃ of water bath with thermostatic control shaking culture;
D) behind the 72h, the content of four kinds of benzene compounds in experiment appearance after cultivating and the blank after the cultivation is used head space---vapor-phase chromatography is measured.Before the test, with sample balance 30mins under 60 ℃, 200r/min condition.Test condition is the HP6890N gas chromatograph, band FID, and chromatographic column is HP-530m * 0.32mm * 0.25 μ m capillary column, high pure nitrogen is done carrier gas.Column temperature: 40 ℃ of initial temperatures, keep 1min, be warming up to 80 ℃ with the speed of 6 ℃/min, keep 4min; Sensing chamber's temperature: 250 ℃; Temperature of vaporization chamber: 220 ℃.Get 100 μ L head space gas samplings.
Benzene compounds degradation experiment result such as table 3 are with shown in Figure 3.
The double oxygen degradation bacteria HBSD-C of table 3 is to the degradation rate of benzene compounds 72h
Table 3 explanation: a facultative anaerobic benzene compounds-degrading bacterium can it can be at the oxygen condition of holding concurrently degrade down benzene,toluene,xylene and sym-trimethylbenzene, strong to environmental compatibility.
3.2 with Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C to receiving benzene series compounds contaminated phreatic processing
Gather Daqing oil field greasy dirt district and receive benzene series compounds contaminated underground water 500mL (being water sample), water sample is preserved in 4 ℃ of condition lower seals, 2 all inner analysis tests.Receive benzene in the benzene series compounds contaminated underground water, toluene, o-Xylol and sym-trimethylbenzene content to be respectively 12.546mg/L, 4.152mg/L, 7.213mg/L, 5.976mg/L through measuring this.
A) with the cryopreserved a small amount of Shi Shi microbacterium in transfering loop picking inclined-plane (Microbacterium schleiferi) HBSD-C lawn; Be inoculated in and contain benzene,toluene,xylene and each 10 μ L of sym-trimethylbenzene; In the 30mL of sterilization (Φ 18*180mm) test tube of inorganic salt isolation medium 10mL; Behind constant-temperature shaking activation culture 36h under 30 ℃ of conditions, obtain the activatory bacteria suspension;
B) 100mL is shaken bottle after sterilization is cooled to normal temperature, add 50mL and receive benzene series compounds contaminated underground water, activatory bacteria suspension 50 μ L, silica gel plug and Parafilm seal film phonograph seal, obtain testing water sample; Establish the blank water sample of not inoculating simultaneously;
C) will test water sample in 30 ℃ of water bath with thermostatic control shaking culture; With blank in 30 ℃ of water bath with thermostatic control shaking culture.
D) behind the 72h, the content of four kinds of benzene compounds in experiment water sample (underground water after obtaining degrading) after cultivating and the blank after the cultivation is used head space---vapor-phase chromatography is measured.Before the test, with sample balance 30mins under 60 ℃, 200r/min condition.Test condition is with the above.
The benzene compounds degradation effect is as shown in table 4.
The double oxygen degradation bacteria HBSD-C of table 4 is to the degradation rate of benzene compounds 72h
3.3 Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C is the pacing items experiment of right growth
A) in order to investigate under differing temps, different pH, different saltiness condition, the growing state of bacterial strain HBSD-C, under the identical situation of other experiment conditions, the temperature of regulating the LB substratum respectively is 4 ℃, 15 ℃, 25 ℃, 40 ℃; PH is 4,5,7,9; Saltiness was 1%, 2%, 3%, 4%, whenever measured the OD value OD of nutrient solution at a distance from 24 hours
600Experimental result such as Fig. 4 a to Fig. 4 c.
B) can find out from Fig. 4 a that bacterial strain HBSD-C is poor growth in 4 ℃ of environment, and the OD under 15 ℃, 25 ℃, 40 ℃ conditions
600Be worth all higherly, optimum growth temperature is about 25 ℃; Can find out that from Fig. 4 b bacterial strain growing state in sour environment is faint, and in the neutrality of pH7～9 and weakly alkaline environment, all can grow preferably; Can find out that from Fig. 4 c degradation bacteria all can be grown in 1%, 2%, 3%, 4% salinity, various growing state differences are little, suit in 1%～2% relatively low salinity ambient growth.
The complete sequence of benzene series compound facultative anaerobic degrading bacterium
< 110>China Geological Univ. Wuhan
< 120>application of benzene series compound facultative anaerobic degrading bacterium
<160>1420
<210>1
<211>1420bp
<212>DNA
< 213>Shi Shi microbacterium (Microbacterium schleiferi) HBSD-C
<400>1
1 TGGCCGCGTGCTTTACACATGCGAGTCGGACGGTGAAGCAGAGCTTGCTCTGTGGATCAG
61 TGGCGAACGGGTGAGTAACACGTGAGCAATCTGCCCCTGACTCTGGGATAAGCGCTGGAA
121 ACGGCGTCTAATACCGGATACGAGCTGCGAAGGCATCTTCAGCAGCTGGAAAGAATTTCG
181 GTCAGGGATGAGCTCGCGGCCTATCAGCTTGTTGGTGAGGTAACGGCTCACCAAGGCGTC
241 GACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACT
301 CCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCAACGC
361 CGCGTGAGGGACGACGGCCTTCGGGTTGTAAACCTCTTTTAGCAGGGAAGAAGCGAAAGT
421 GACGGTACCTGCAGAAAAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG
481 GGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCT
541 GCTGTGAAAACCCGAGGCTCAACCTCGGGCCTGCAGTGGGTACGGGCAGACTAGAGTGCG
601 GTAGGGGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACC
661 GATGGCGAAGGCAGATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGGGTGGGGAGCA
721 AACAGGCTTAGATACCCTGGTAGTCCACCCCGTAAACGTTGGGAACTAGTTGTGGGGACC
781 ATTCCACGGTTTCCGTGACGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCG
841 CAAGGCTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGCGGAGCATGCGGATTA
901 ATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATATACGAGAACGGGCCAGAAAT
961 GGTCAACTCTTTGGACACTCGTAAACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTG
1021 AGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCACGTAAT
1081 GGTGGGAACTCATGGGATACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAA
1141 TCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGCTG
1201 CAATACCGTAAGGTGGAGCGAATCCCAAAAAGCCGGTCCCAGTTCGGATTGAGGTCTGCA
1261 ACTCGACCTCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATA
1321 CGTTCCGGGGTCTTGTACACACCGCCCGTCAAGTCATGTAAGTCGGTAACACCCGAAGCC
1381 GGTGGCCCAACCCTTCTGGAGGGAGCCGTCGAAGGTTGGA
Claims (2)
1. the application of benzene series compound facultative anaerobic degrading bacterium is characterized in that: the benzene series compound facultative anaerobic degrading bacterium concrete steps of benzene compounds that are applied to degrade in the underground water are following:
A) with the cryopreserved Shi Shi microbacterium in transfering loop picking inclined-plane (Microbacterium schleiferi) HBSD-C lawn; Be inoculated in and contain benzene,toluene,xylene and each 10 μ L of sym-trimethylbenzene; In the 30mL of the sterilization test tube of inorganic salt isolation medium 10mL; Behind constant-temperature shaking activation culture 36h under 15 ℃～40 ℃ conditions, obtain the activatory bacteria suspension;
B) by receiving benzene series compounds contaminated underground water: the proportioning=50mL of activatory bacteria suspension: 50 μ L, choose and receive benzene series compounds contaminated underground water and activatory bacteria suspension; Mixed with the activatory bacteria suspension by benzene series compounds contaminated underground water;
C) then in 15 ℃～40 ℃ cultivations, behind the cultivation 72h, the underground water after obtaining degrading.
2. the application of benzene series compound facultative anaerobic degrading bacterium according to claim 1; It is characterized in that: described benzene compounds comprises any one or any mixing more than two kinds in benzene,toluene,xylene, the sym-trimethylbenzene, and any is any proportioning when mixing more than two kinds.
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