CN102250770A - Microbial agent and preparation method and application thereof - Google Patents
Microbial agent and preparation method and application thereof Download PDFInfo
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- CN102250770A CN102250770A CN2011101694798A CN201110169479A CN102250770A CN 102250770 A CN102250770 A CN 102250770A CN 2011101694798 A CN2011101694798 A CN 2011101694798A CN 201110169479 A CN201110169479 A CN 201110169479A CN 102250770 A CN102250770 A CN 102250770A
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- rhodococcus ruber
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- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 230000000813 microbial effect Effects 0.000 title abstract description 8
- 241000589516 Pseudomonas Species 0.000 claims abstract description 79
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 62
- 241001656677 Gordonia alkanivorans Species 0.000 claims abstract description 40
- 241000028625 Gordonia amicalis Species 0.000 claims abstract description 40
- 239000003208 petroleum Substances 0.000 claims abstract description 28
- 239000003209 petroleum derivative Substances 0.000 claims abstract description 26
- 239000002689 soil Substances 0.000 claims abstract description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 241000607142 Salmonella Species 0.000 claims description 86
- 241000187563 Rhodococcus ruber Species 0.000 claims description 82
- 238000000034 method Methods 0.000 claims description 47
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 43
- 235000013305 food Nutrition 0.000 claims description 43
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- 244000005700 microbiome Species 0.000 claims description 11
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 3
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- 238000012216 screening Methods 0.000 description 26
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a microbial agent, which comprises a culture medium and a thallus, wherein the thallus comprises psesudoxanthomonasjaponensis with the collection number CGMCC No.4797, pseudomonas with the collection number CGMCC No.4793, gordoniaamicalis with the collection number CGMCC No.4794, phodococcusruber with the collection number CGMCC No.4795 and gordoniaalkanivorans with the collection number CGMCC No.4796. The invention further provides a preparation method of the microbial agent, application of the microbial agent to degradation of petroleum hydrocarbon, application of the microbial agent to biological repair of petroleum pollution, application of the microbial agent to biological repair of soil suffering from petroleum pollution and application of the microbial agent to biological repair of water bodies suffering from petroleum pollution.
Description
Technical field
The present invention relates to a kind of microbiobacterial agent and its production and application; Or rather, relate to a kind of microbiobacterial agent and preparation method thereof, also relate to application, application in biological restoration petroleum pollution, application in the soil of biological restoration petroleum pollution and the application in the water body of biological restoration petroleum pollution of described bacteria agent in decomposing petroleum hydrocarbon.
Background technology
The world today, oil has become one of human topmost energy, and a large amount of oil and processed goods thereof enter the petroleum pollution that soil causes soil, bring harm for human mater to whole biosphere, thereby become international environmental problem.In the research to petroleum pollution, biological restoration as a kind of potential efficiently, cleaning technique come into one's own just day by day cheaply.
Biological restoration (Bioremediation) is meant by biological (particularly microorganism) catalyze and degrade organic pollutants, thus the controlled or spontaneous process of carrying out of repairing the pollutent in contaminated environment or the elimination environment.
" petroleum hydrocarbon " is the main pollutant component in oil and the processed goods thereof, discovers that microorganism can play Degradation to petroleum hydrocarbon.The microbiological deterioration petroleum hydrocarbon is to find for 19 end of the centurys.Before the 1950's, Bel is representative with U.S. C.E. assistant, and the marine microorganism decomposing petroleum hydrocarbon has been carried out extensive studies.The beginning of the fifties, gas-chromatography was come out, and the widespread usage of radio-label method has play a part positive to the microbiological deterioration mechanism of studying petroleum hydrocarbon.Since the sixties,, impelled many maritime nations,, actively developed the research work of relevant marine microorganism decomposing petroleum hydrocarbon as states such as the U.S., Canada, Japan, Britain and the Soviet Union because offshore oil pollution is on the rise.The mid-1970s, American scholar have also been cultivated " super microorganism " with engineered technology, in the hope of decomposing petroleum hydrocarbon effectively.
China is from 1975, successively Jiaozhou Bay, Qingdao, the Bohai Sea, PORT OF XIAMEN, the Huanghai Sea and East Sea oil degradation microbial numbers, distribution, kind formed and influenced degradation factors etc. and investigate.
In influencing the principal element of microorganism to the degraded of petroleum hydrocarbon, the physiological property of microorganism is one of key factor of decision decomposing petroleum hydrocarbon ability.It is also lower to the petroleum hydrocarbon degradation ability at present to have separated the microorganism strains and the microbial inoculum that obtain, therefore, presses for and develops a kind of microbiobacterial agent that can the efficient degradation petroleum hydrocarbon.
Summary of the invention
The objective of the invention is to overcome the existing microbiobacterial agent shortcoming low, microbiobacterial agent of a kind of decomposing petroleum hydrocarbon efficiently and its production and application is provided the degradation capability of petroleum hydrocarbon.
In order to realize first goal of the invention, the invention provides a kind of microbiobacterial agent, this microbiobacterial agent comprises substratum and thalline, wherein, described thalline comprise preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis), preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas), preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis), preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) and preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans).
In order to realize second goal of the invention, the present invention also provides a kind of preparation method of microbiobacterial agent, and wherein, this method comprises: with preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis), preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas), preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis), preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) and preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans) be inoculated in the substratum and cultivate.
The present invention also provides the application of described microbiobacterial agent in decomposing petroleum hydrocarbon.
The present invention also provides the application of described microbiobacterial agent in the biological restoration petroleum pollution.
The present invention also provides the application of described microbiobacterial agent in the soil of biological restoration petroleum pollution.
In addition, the present invention also provides the application of described microbiobacterial agent in the water body of biological restoration petroleum pollution.
According to the present invention, the bacterial strain that uses in the described microbiobacterial agent screens by a large amount of screening operations, and these bacterial strains have very strong degradation capability to petroleum hydrocarbon.These bacterial strains be stored on 04 28th, 2011 China Committee for Culture Collection of Microorganisms common micro-organisms center (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), wherein, Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) preserving number be CGMCC No. 4797, pseudomonas (
Pseudomonas) preserving number be CGMCC No. 4793, friendly Gordon Salmonella (
Gordonia amicalis) preserving number be CGMCC No. 4794, Rhodococcus ruber (
Rhodococcus ruber) preserving number be CGMCC No. 4795, food alkane Gordon Salmonella (
Gordonia alkanivorans) preserving number be CGMCC No. 4796.Microbiobacterial agent provided by the invention has very strong degradation capability to petroleum hydrocarbon, can be widely used in decomposing petroleum hydrocarbon and to the reparation of petroleum pollution, as to the reparation of the soil of petroleum pollution with to reparation of the water body of petroleum pollution etc.
Description of drawings
Fig. 1 shown the preserving number of the present invention's screening be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxant homonas japonensis) 24 hours form of cultivation on solid TSA substratum;
Fig. 2 shown the preserving number of the present invention's screening be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxan thomonas japonensis) cultivating 24 hours on the solid TSA substratum after the form under 100 times of oily mirrors of lawn dyeing back at opticmicroscope;
Fig. 3 shown the preserving number of the present invention's screening be CGMCC No. 4793 pseudomonas (
Pseudomonas) 24 hours form of cultivation on solid TSA substratum;
Fig. 4 shown the preserving number of the present invention's screening be CGMCC No. 4793 pseudomonas (
Pseudomonas) cultivating 24 hours the form of (10 * 100 times) under opticmicroscope oil mirror afterwards on the solid TSA substratum after lawn dyes;
Fig. 5 shown the preserving number of the present invention's screening be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) 24 hours form of cultivation on solid TSA substratum;
Fig. 6 shown the preserving number of the present invention's screening be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) cultivating 24 hours on the solid TSA substratum after the form under 100 times of oily mirrors of lawn dyeing back at opticmicroscope;
Fig. 7 for the preserving number of the present invention screening be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) after cultivating 24 hours on the solid TSA substratum stereoscan photograph (* 10K);
Fig. 8 for the preserving number of the present invention screening be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) after cultivating 48 hours on the solid TSA substratum stereoscan photograph (* 10K);
Fig. 9 has shown that the preserving number of the present invention's screening is the form that the Rhodococcus ruber (Rhodococcus ruber) of CGMCC No. 4795 was cultivated 24 hours on solid TSA substratum;
Figure 10 has shown that the preserving number of the present invention's screening is that the Rhodococcus ruber (Rhodococcus ruber) of CGMCC No. 4795 was being cultivated 24 hours on the solid TSA substratum after the form under 100 times of oily mirrors of lawn dyeing back at opticmicroscope;
Figure 11 for the preserving number of the present invention screening be stereoscan photograph after cultivating 24 hours on the solid TSA substratum of the Rhodococcus ruber (Rhodococcus ruber) of CGMCC No. 4795 (* 10K);
Figure 12 for the preserving number of the present invention screening be stereoscan photograph after cultivating 48 hours on the solid TSA substratum of the Rhodococcus ruber (Rhodococcus ruber) of CGMCC No. 4795 (* 10K);
Figure 13 shown the preserving number of the present invention's screening be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans) 24 hours form of cultivation on solid TSA substratum;
Figure 14 shown the preserving number of the present invention's screening be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans) cultivating 24 hours on the solid TSA substratum after the form under 100 times of oily mirrors of lawn dyeing back at opticmicroscope;
Figure 15 for the preserving number of the present invention screening be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans) after cultivating 24 hours on the solid TSA substratum stereoscan photograph (* 10K);
Figure 16 for the preserving number of the present invention screening be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans) after cultivating 48 hours on the solid TSA substratum stereoscan photograph (* 10K).
Embodiment
The invention provides a kind of microbiobacterial agent, this microbiobacterial agent comprises substratum and thalline, wherein, described thalline comprise preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis), preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas), preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis), preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) and preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans).
Through 16s rDNA amplification and sequencing analysis, deposit number is that the bacterial strain of CGMCC No. 4797 has the 16s rDNA sequence shown in the SEQ ID NO:1; Deposit number is that the bacterial strain of CGMCC No. 4793 has the 16s rDNA sequence shown in the SEQ ID NO:2; Deposit number is that the bacterial strain of CGMCC No. 4794 has the 16s rDNA sequence shown in the SEQ ID NO:3; Deposit number is that the bacterial strain of CGMCC No. 4795 has the 16s rDNA sequence shown in the SEQ ID NO:4; Deposit number is that the bacterial strain of CGMCC No. 4796 has the 16s rDNA sequence shown in the SEQ ID NO:5.
The amount of the thalline that contains in the described microbiobacterial agent can in very large range change, and under the preferable case, the contained total viable count of the described microbiobacterial agent of every gram is 0.5-1 * 10
10Individual.Under most preferred situation, be benchmark with the total viable count in the described microbiobacterial agent, preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) viable count be the 10-50% of total viable count, the pseudomonas that preserving number is CGMCC No. 4793 (
Pseudomonas) viable count be 10-50%, the preserving number of total viable count be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) viable count be the 10-50% of total viable count, the Rhodococcus ruber that preserving number is CGMCC No. 4795 (
Rhodococcus ruber) viable count be the 10-50% of total viable count, food alkane Gordon Salmonella that preserving number is CGMCC No. 4796 (
Gordonia alkanivorans) viable count be the 10-50% of total viable count; Preferably, preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) viable count be the 15-35% of total viable count, the pseudomonas that preserving number is CGMCC No. 4793 (
Pseudomonas) viable count be 15-35%, the preserving number of total viable count be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) viable count be the 15-35% of total viable count, the Rhodococcus ruber that preserving number is CGMCC No. 4795 (
Rhodococcus ruber) viable count be the 15-35% of total viable count, food alkane Gordon Salmonella that preserving number is CGMCC No. 4796 (
Gordonia alkanivorans) viable count be the 15-35% of total viable count.
According to the present invention, the kind of described substratum can in very large range change, can be the various substratum of cultivating Japanese false Xanthomonas campestris, pseudomonas, friendly Gordon Salmonella, Rhodococcus ruber and food alkane Gordon Salmonella that can be used in, for example, can be in beef-protein medium, oil screening culture medium and the PDA substratum one or more, be preferably beef-protein medium.Above-mentioned substratum can be commercially available or prepare according to the record of " microbiological culture media handbook " (Microbiology Culture Media Manual).For example, described beef-protein medium can contain extractum carnis, peptone, NaCl and water, wherein, extractum carnis with respect to 100 weight parts, the content of peptone can be the 150-250 weight part, the content of NaCl can be the 50-150 weight part, and the content of water can be the 15000-25000 weight part, and the pH value can be 7.2-7.4; The agar that adds 1.2 weight % in the substratum can be made into solid medium.
The present invention also provides the preparation method of described microbiobacterial agent, wherein, this method comprise with preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis), preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas), preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis), preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) and preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans) be inoculated in the substratum and cultivate.
According to the present invention, described cultured method comprises: independently cultivate respectively in the culture system separately the Japanese false Xanthomonas campestris that preserving number is CGMCC No. 4797 (
Pseudoxanthomonas japonensis), preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas), preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis), preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) and preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans), and will cultivate the microorganism that obtains respectively and proportionally mix.Under the preferable case, by separately independently the cultivation in the culture system and cultivate after mixing, the total viable count that makes the every gram microbiobacterial agent that obtains is 0.5-1 * 10
10Individual.
In a kind of preferred implementation, by separately independently the cultivation in the culture system and cultivate after proportionally mixing, described blended condition has no particular limits, as long as the microbiobacterial agent that obtains is met the following conditions: total so that the viable count in the described microbiobacterial agent is a benchmark, preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) viable count be the 10-50% of total viable count, the pseudomonas that preserving number is CGMCC No. 4793 (
Pseudomonas) viable count be 10-50%, the preserving number of total viable count be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) viable count be the 10-50% of total viable count, the Rhodococcus ruber that preserving number is CGMCC No. 4795 (
Rhodococcus ruber) viable count be the 10-50% of total viable count, food alkane Gordon Salmonella that preserving number is CGMCC No. 4796 (
Gordonia alkanivorans) viable count be the 10-50% of total viable count; Preferably, preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) viable count be the 15-35% of total viable count, the pseudomonas that preserving number is CGMCC No. 4793 (
Pseudomonas) viable count be 15-35%, the preserving number of total viable count be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) viable count be the 15-35% of total viable count, the Rhodococcus ruber that preserving number is CGMCC No. 4795 (
Rhodococcus ruber) viable count be the 15-35% of total viable count, food alkane Gordon Salmonella that preserving number is CGMCC No. 4796 (
Gordonia alkanivorans) viable count be the 15-35% of total viable count.
According to the present invention, described preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) cultural method have no particular limits, for example, can in the beef-protein medium of 100 weight parts, inoculate the 2-5 weight part Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) bacterial strain, 37 ℃ of cultivations, until the false Xanthomonas campestris of Japan (
Pseudoxanthomonas japonensis) viable count be 0.5-1 * 10
10The substratum of individual/gram.
Described preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas) cultural method have no particular limits, for example, can in the beef-protein medium of 100 weight parts, inoculate the 2-5 weight part pseudomonas (
Pseudomonas) bacterial strain, 37 ℃ of cultivations, until pseudomonas (
Pseudomonas) viable count be 0.5-1 * 10
10The substratum of individual/gram.
Described preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) cultural method have no particular limits, for example, can in the beef-protein medium of 100 weight parts, inoculate the 2-5 weight part friendly Gordon Salmonella (
Gordonia amicalis) bacterial strain, 37 ℃ of cultivations, until friendly Gordon Salmonella (
Gordonia amicalis) viable count be 0.5-1 * 10
10The substratum of individual/gram.
Described preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) cultural method have no particular limits, for example, can in the beef-protein medium of 100 weight parts, inoculate the 2-5 weight part Rhodococcus ruber (
Rhodococcus ruber) bacterial strain, 37 ℃ of cultivations, until Rhodococcus ruber (
Rhodococcus ruber) viable count be 0.5-1 * 10
10The substratum of individual/gram.
Described preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans) cultural method have no particular limits, for example, can in the beef-protein medium of 100 weight parts, inoculate the 2-5 weight part food alkane Gordon Salmonella (
Gordonia alkanivorans) bacterial strain, 37 ℃ of cultivations, until food alkane Gordon Salmonella (
Gordonia alkanivorans) viable count be 0.5-1 * 10
10The substratum of individual/gram.
The present invention also provides the application of described microbiobacterial agent in decomposing petroleum hydrocarbon.
The present invention also provides the application of described microbiobacterial agent in the biological restoration petroleum pollution.
The present invention also provides the application of described microbiobacterial agent in the soil of biological restoration petroleum pollution.
In addition, the present invention also provides the application of described microbiobacterial agent in the water body of biological restoration petroleum pollution.
Below by specific embodiment the present invention is further illustrated.
Experiment material
One, the oil sample of strain screening:
The crude oil and the commercially available 0# diesel oil that pick up from Shengli Oil Field are even by the mixed of 1:4, as the experiment oil.
Two, pedotheque is the mixture of 1:1:1 that picks up from three kinds of different oil-polluted soils samples of Shandong Shengli Oil Field, and it is respectively:
1) the ground crude oil contaminated soil around the producing well;
2) oil sediment; With
3) stack the topsoil soil of oil sediment for a long time.
Three,Substratum
1) enrichment medium: K
2HPO
43H
2O 1.0g, KH
2PO
41.0g, MgSO
47H
2O 0.5g, NH
4NO
31.0g, CaCl
20.02g distilled water 1000mL transfers to PH7.0,121 ℃ of sterilization 20min add the 5g oil after cooling.
2) isolation medium (PDA substratum): potato powder 6.0g, glucose 20g, agar 20g adds distilled water until 1000ml, and standby 115 ℃ of autoclavings 20 minutes.
3) strain store medium: beef-protein medium
4) oil screening culture medium (liquid): NaNO
31.5g, (NH4)
2SO
41.5g, K
2HPO
41g, MgSO
47H
2O 0.5 g, KCl 0.5g, FeSO
47H
2O 0.01g, CaCl
20.002g distilled water 1000mL transfers to pH8.0,121 ℃ of sterilization 20min, and the cooling back adds the 2.5g oil.The agar that adds 1.2 weight % in the substratum is made the solid screening culture medium.
5) solid TSA substratum: Tryptones 15g, soya peptone 5g, sodium-chlor 30g, agar 15g, adding distilled water is 1000ml until final volume, the pH value is 7.1-7.5.
Embodiment 1
1, the concentration and separation of petroleum hydrocarbon degradation bacterial strain
Taking by weighing the adding of 5g oil-polluted soils mixture is equipped with in the 250mL triangular flask of 100mL enrichment medium, under 28 ℃, the constant temperature shaking table condition of 180r/min, cultivate, draw the 5mL nutrient solution after 5 days and be forwarded to fresh enrichment medium, the same terms was cultivated 5 days, enrichment culture is 25 days so continuously, nutrient solution is through being coated on behind the gradient dilution on the solid PDA substratum, in 28 ℃ of cultivations; After treating that flat board grows bacterium colony, single bacterium colony of picking different shape adopts the line partition method to carry out separation and purification repeatedly, and up to the pure growth that obtains single strain, the bacterial strain behind the purifying is stored in the beef-protein medium inclined-plane.
2, the screening of high degrading activity bacterial strain
The bacterial strain of above-mentioned purifying is inoculated into respectively in the 250mL triangular flask that the 100mL screening culture medium is housed, cultivated 5 days under 28 ℃, the condition of 180r/min, be coated on the solid screening culture medium after 100 times of the nutrient solution dilutions, observe the growing state of each bacterial strain in solid medium, filter out grow very fast, the more bacterial strain of bacterium colony.
Single bacterium colony of each bacterial strain of picking is inoculated into respectively in the 250ml triangular flask that 50ml liquid beef-protein medium is housed, in 28 ℃, the constant temperature shaking table of 180r/min, cultivate 24h, measure the absorbancy of bacterium liquid at the 600nm place, regulate its mass concentration with the beef-protein medium of sterilization and make OD600nm be about 0.4, make seed inoculation screening culture medium with this bacterium liquid that dilutes.
Drawing the ready seed liquor of 5ml is inoculated in the 250mL triangular flask that the 100mL screening culture medium is housed, measure oil content after in 28 ℃, the constant temperature shaking table of 180r/min, cultivating 4 days, the processing triplicate of each seed liquor, calculate average degradation rate, filter out 5 the highest bacterial strains of oil degradation rate, and this bacterial strain carried out biological preservation, preserving number is respectively CGMCC No. 4793, CGMCC No. 4794, CGMCC No. 4795, CGMCC No. 4796 and CGMCC No. 4797.
Embodiment 2
According to experiment content and experimental technique of record in " common bacteria system identification handbook " [4] and " actinomycetes systematics principle, method and put into practice " [5], respectively to sieve preserving number be that the bacterial strain of CGMCC No. 4793, CGMCC No. 4794, CGMCC No. 4795, CGMCC No. 4796 and CGMCC No. 4797 carries out strain identification.
1, morphological analysis
Be that preserving number is that the bacterial strain of CGMCC No. 4793, CGMCC No. 4794, CGMCC No. 4795, CGMCC No. 4796 and CGMCC No. 4797 carries out morphological analysis and observes under opticmicroscope to preserving number respectively, the result is respectively shown in Fig. 1-16.
Colonial morphology is observed and is carried out on solid TSA substratum, after the bacterial strain streak inoculation, observes colonial morphology in 24 hours in 28 ℃ of constant temperature culture.
Preserving number is that the bacterium colony of the bacterial strain of CGMCC No. 4793 is colourless translucent, circle, and the edge is irregular, and quality is smooth, and the surface is flat.Picking is cultivated 24 hours lawn dyeing backs and is observed under 100 times of oily mirrors of opticmicroscope.Can see clearly that at microscopically preserving number is that the somatic cells of the bacterial strain of CGMCC No. 4793 is stock shape, and single appearance.
Preserving number is that the bacterium colony of the bacterial strain of CGMCC No. 4794 is a light orange, circle, and quality is smooth, rat.Picking is cultivated 24 hours lawn dyeing backs and is observed under 100 times of oily mirrors of opticmicroscope.Can see clearly that at microscopically preserving number is that the somatic cells of bacterial strain of CGMCC No. 4794 is less relatively, and in pairs or gather into Xiao Cong and occur.
Preserving number is that the bacterium colony of the bacterial strain of CGMCC No. 4795 is a pink colour, and circle, quality are slightly coarse, surface elevation.Picking is cultivated 24 hours lawn dyeing backs and is observed under 100 times of oily mirrors of opticmicroscope.Can see clearly that at microscopically preserving number is that the somatic cells of bacterial strain of CGMCC No. 4795 is less relatively, and in pairs or gather into Xiao Cong and occur.
Preserving number is that the bacterium colony of the bacterial strain of CGMCC No. 4796 is orange, circle, and quality is smooth, rat.Picking is cultivated 24 hours lawn dyeing backs and is observed under 100 times of oily mirrors of opticmicroscope.Can see clearly that at microscopically preserving number is that the somatic cells of bacterial strain of CGMCC No. 4796 is less relatively, and in pairs or gather into Xiao Cong and occur.
Preserving number is that the bacterium colony of the bacterial strain of CGMCC No. 4797 is light yellow, circle, and quality is smooth, surface elevation.Picking is cultivated 24 hours lawn dyeing backs and is observed under 100 times of oily mirrors of opticmicroscope.Can see clearly that at microscopically preserving number is that the somatic cells of the bacterial strain of CGMCC No. 4797 is stock shape, and single appearance.
2,16s rDNA identifies
The pcr amplification of 16s rDNA and order-checking: adopt test kit (the full Shi Jin in Beijing biotech company) to extract and respectively the purifying preserving number be total DNA of the bacterial classification of CGMCC No. 4793, CGMCC No. 4794, CGMCC No. 4795, CGMCC No. 4796 and CGMCC No. 4797, be template with total DNA then, 16s rDNA conserved sequence carried out pcr amplification obtain target gene fragment.
The pcr amplification primer is respectively:
27f(5 '-AGAGTTTGATCCTGGCTCAG-3 ') and;
1492r（5’-TACGGCTACCTTGTTACGACTT-3’）。
PCR total reaction system is 50uL, specifically comprises:
| 10×Buffer | 5μL |
| 10 mmol/ L dNTP | 4μL |
| Reverse primer | 1μL |
| Forward primer | 1μL |
| Taq enzyme (5U/ μ L) | 1μL |
| Template DNA | 2μL |
| ddH 2O | Complement to 50 μ L |
The PCR reaction conditions is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 1min, 50 ℃ of annealing 1min, 72 ℃ are extended 2min, and this step is carried out 35 circulations altogether, and last 72 ℃ are extended 10min.
Amplified production carries out the electrophoresis check with 1% sepharose.The examining order of PCR product is finished by Beijing three rich polygala root biotech companies.Sequencing result shows that deposit number is that the bacterial strain of CGMCC No. 4797 has the 16s rDNA sequence shown in the SEQ ID NO:1; Deposit number is that the bacterial strain of CGMCC No. 4793 has the 16s rDNA sequence shown in the SEQ ID NO:2; Deposit number is that the bacterial strain of CGMCC No. 4794 has the 16s rDNA sequence shown in the SEQ ID NO:3; Deposit number is that the bacterial strain of CGMCC No. 4795 has the 16s rDNA sequence shown in the SEQ ID NO:4; Deposit number is that the bacterial strain of CGMCC No. 4796 has the 16s rDNA sequence shown in the SEQ ID NO:5.
Through Phylogenetic Analysis: land http://www.ncbi.nlm.nih.gov, check order row are analyzed by the sequence in blast program and the nucleic acid database search same or analogous nucleotide sequence, utilize sequence alignment software ClustalX 2.0 and Phylogenetic Analysis software MEGA 4.0.2 constructing system evolutionary tree then.In MEGA software, the method of constructing system evolutionary tree is used the Neighbor-Joining method, the base alternative model is selected the Kimura two-parameter model, the evolutionary tree certificate authenticity is selected 1000 duplicate detection of check (bootstrap) of bootstrapping for use, and the conversion in the DNA sequence variations is given identical weighted value with transversion.Show by analysis, preserving number be CGMCC No. 4797 bacterial strain belong to Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis), preserving number be the bacterial strain of CGMCC No. 4793 belong to pseudomonas (
Pseudomonas), preserving number be the bacterial strain of CGMCC No. 4794 belong to friendly Gordon Salmonella (
Gordonia amicalis), preserving number be the bacterial strain of CGMCC No. 4795 belong to Rhodococcus ruber (
Rhodococcus ruber), preserving number be the bacterial strain of CGMCC No. 4796 belong to food alkane Gordon Salmonella (
Gordonia alkanivorans).
Embodiment 3-7
Use respectively preserving number as the Japanese false Xanthomonas campestris of CGMCC No. 4797 (
Pseudoxanthomonas japonensis), preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas), preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis), preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) and preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans) the oil-polluted soils mixture is handled, concrete steps are as follows:
Taking by weighing 10g oil-polluted soils mixture (oil content 1.25 gram) adds and the 100mL beef-protein medium is housed (inoculum size of bacterial classification is that the contained total viable count of every gram substratum is 0.3 * 10
10Individual) the 250mL triangular flask in, under 28 ℃, the constant temperature shaking table condition of 180r/min, cultivate, detect the oil content in the oil-polluted soils mixture after 3 days, detect the residual petroleum content in the oil-polluted soils mixture after handling, and the calculating microbiobacterial agent is to the degradation rate of oil, oil content before oil content/processing after oil content-processing before degradation rate=processing, the result is as shown in table 1 below.
Table 1
From the data of last table 1 as can be seen, the preserving number that the present invention filters out be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis), preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas), preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis), preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) and preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans), petroleum hydrocarbon is had good degradation capability, can be widely used in decomposing petroleum hydrocarbon and to the reparation of petroleum pollution, as to the reparation of the soil of petroleum pollution with to reparation of the water body of petroleum pollution etc.
Embodiment 8
(1) in the beef-protein medium of 100 weight parts the inoculation 3 weight parts preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis), 37 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 0.5 * 10 until the viable count of the false Xanthomonas campestris of Japan
10The substratum of individual/gram.
(2) in the beef-protein medium of 100 weight parts the inoculation 4 weight parts preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas), 37 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 0.5 * 10 until the viable count of pseudomonas
10The substratum of individual/gram.
(3) in the beef-protein medium of 100 weight parts the inoculation 2 weight parts preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis), 37 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 0.5 * 10 until the viable count of friendly Gordon Salmonella
10The substratum of individual/gram.
(4) in the beef-protein medium of 100 weight parts the inoculation 2 weight parts preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber), 37 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 0.5 * 10 until the viable count of Rhodococcus ruber
10The substratum of individual/gram.
(5) in the beef-protein medium of 100 weight parts the inoculation 4 weight parts preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans), 30 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 0.5 * 10 until the viable count of food alkane Gordon Salmonella
10The substratum of individual/gram.
(6) with the Japanese false Xanthomonas campestris that obtains respectively in the step (1) to (5) (
Pseudoxanthomonas japonensis), pseudomonas (
Pseudomonas), friendly Gordon Salmonella (
Gordonia amicalis), Rhodococcus ruber (
Rhodococcus ruber) and food alkane Gordon Salmonella (
Gordonia alkanivorans) proportionally mix, make in the microbiobacterial agent that obtains, preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) viable count be total viable count 15%, preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas) viable count be total viable count 15%, preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) viable count be total viable count 15%, preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) viable count be total viable count 20%, preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans) viable count be 20% of total viable count, obtain microbiobacterial agent A1.
Embodiment 9
(1) in the beef-protein medium of 100 weight parts the inoculation 5 weight parts preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis), 37 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 1 * 10 until the viable count of the false Xanthomonas campestris of Japan
10The substratum of individual/gram.
(2) in the beef-protein medium of 100 weight parts the inoculation 3 weight parts preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas), 37 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 1 * 10 until the viable count of pseudomonas
10The substratum of individual/gram.
(3) in the beef-protein medium of 100 weight parts the inoculation 4 weight parts preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis), 37 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 1 * 10 until the viable count of friendly Gordon Salmonella
10The substratum of individual/gram.
(4) in the beef-protein medium of 100 weight parts the inoculation 5 weight parts preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber), 37 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 1 * 10 until the viable count of Rhodococcus ruber
10The substratum of individual/gram.
(5) in the beef-protein medium of 100 weight parts the inoculation 2 weight parts preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans), 30 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 1 * 10 until the viable count of food alkane Gordon Salmonella
10The substratum of individual/gram.
(6) with the Japanese false Xanthomonas campestris that obtains respectively in the step (1) to (5) (
Pseudoxanthomonas japonensis), pseudomonas (
Pseudomonas), friendly Gordon Salmonella (
Gordonia amicalis), Rhodococcus ruber (
Rhodococcus ruber) and food alkane Gordon Salmonella (
Gordonia alkanivorans) proportionally mix, make in the microbiobacterial agent that obtains, preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) viable count be total viable count 25%, preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas) viable count be total viable count 25%, preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) viable count be total viable count 25%, preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) viable count be total viable count 15%, preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans) viable count be 15% of total viable count, obtain microbiobacterial agent A2.
Embodiment 10
(1) in the beef-protein medium of 100 weight parts the inoculation 4 weight parts preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis), 37 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 0.8 * 10 until the viable count of the false Xanthomonas campestris of Japan
10The substratum of individual/gram.
(2) in the beef-protein medium of 100 weight parts the inoculation 2 weight parts preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas), 37 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 0.8 * 10 until the viable count of pseudomonas
10The substratum of individual/gram.
(3) in the beef-protein medium of 100 weight parts the inoculation 3 weight parts preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis), 37 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 0.8 * 10 until the viable count of friendly Gordon Salmonella
10The substratum of individual/gram.
(4) in the beef-protein medium of 100 weight parts the inoculation 4 weight parts preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber), 37 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 0.8 * 10 until the viable count of Rhodococcus ruber
10The substratum of individual/gram.
(5) in the beef-protein medium of 100 weight parts the inoculation 3 weight parts preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans), 30 ℃ of cultivations are taken a sample in culturing process and are observed by the microscope direct-counting method, are 0.8 * 10 until the viable count of food alkane Gordon Salmonella
10The substratum of individual/gram.
(6) with the Japanese false Xanthomonas campestris that obtains respectively in the step (1) to (5) (
Pseudoxanthomonas japonensis), pseudomonas (
Pseudomonas), friendly Gordon Salmonella (
Gordonia amicalis), Rhodococcus ruber (
Rhodococcus ruber) and food alkane Gordon Salmonella (
Gordonia alkanivorans) proportionally mix, make in the microbiobacterial agent that obtains, preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) viable count be total viable count 10%, preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas) viable count be total viable count 10%, preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) viable count be total viable count 10%, preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) viable count be total viable count 35%, preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans) viable count be 35% of total viable count, obtain microbiobacterial agent A3.
Embodiment 11
Prepare microbiobacterial agent A4 according to the method identical with embodiment 5, difference is, with the Japanese false Xanthomonas campestris that obtains respectively in the step (1) to (5) (
Pseudoxanthomonas japonensis), pseudomonas (
Pseudomonas), friendly Gordon Salmonella (
Gordonia amicalis), Rhodococcus ruber (
Rhodococcus ruber) and food alkane Gordon Salmonella (
Gordonia alkanivorans) proportionally mix, make in the microbiobacterial agent that obtains, preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) viable count be total viable count 35%, preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas) viable count be total viable count 35%, preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) viable count be total viable count 10%, preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) viable count be total viable count 10%, preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans) viable count be 10% of total viable count, obtain microbiobacterial agent A4.
Embodiment 12-15
Use the microbiobacterial agent A1-A4 that makes among the embodiment 8-11 that the oil-polluted soils mixture is handled respectively, concrete steps are as follows:
Taking by weighing 10g oil-polluted soils mixture (oil content 1.25 grams) adds and is equipped with in the 250mL triangular flask of 100mL microbiobacterial agent, under 28 ℃, the constant temperature shaking table condition of 180r/min, cultivate, detect the oil content in the oil-polluted soils mixture after 3 days, detect the residual petroleum content in the oil-polluted soils mixture after handling, and the calculating microbiobacterial agent is to the degradation rate of oil, oil content before oil content/processing after oil content-processing before degradation rate=processing, the result is as shown in table 2 below.
Table 1
By the result in the last table 2 as can be seen, microbiobacterial agent provided by the invention has good degradation capability to petroleum hydrocarbon, can be widely used in decomposing petroleum hydrocarbon and to the reparation of petroleum pollution, as to the reparation of the soil of petroleum pollution with to reparation of the water body of petroleum pollution etc.
SEQUENCE LISTING
＜110〉Institute of Environment and Sustainable Development in Agriculture, CAAS
＜120〉a kind of false Xanthomonas campestris and microbiobacterial agent and application thereof
<160> 5
<170> PatentIn version 3.4
<210> 1
<211> 1408
<212> DNA
＜213〉Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) 16s rDNA
<400> 1
ctacacatgc aagtcgaacg gcagcacagg agagcttgct ctctgggtgg cgagtggcgg 60
acgggtgagg aatacatcgg aatctacctt ttcgtggggg ataacgtagg gaaacttacg 120
ctaataccgc atacgaccta cgggtgaaag tgggggaccg caaggcctca cgcgattaga 180
tgagccgatg tccgattagc tagttggcgg ggtaatggcc caccaaggcg acgatcggta 240
gctggtctga gaggatgatc agccacactg gaactgagac acggtccaga ctcctacggg 300
aggcagcagt ggggaatatt ggacaatggg cgcaagcctg atccagccat accgcgtggg 360
tgaagaaggc cttcgggttg taaagccctt ttgttgggaa agaaatccta tcggttaata 420
accggtgggg atgacggtac ccaaagaata agcaccggct aacttcgtgc cagcagccgc 480
ggtaatacga agggtgcaag cgttactcgg aattactggg cgtaaagcgt gcgtaggtgg 540
tggtttaagt ctgctgtgaa agccctgggc tcaacctggg aattgcagtg gatactggat 600
cactagagtg tggtagaggg atgcggaatt tctggtgtag cagtgaaatg cgtagagatc 660
agaaggaaca tccgtggcga aggcggcatc ctgggccaac actgacactg aggcacgaaa 720
gcgtggggag caaacaggat tagataccct ggtagtccac gccctaaacg atgcgaactg 780
gatgttgggt gcaacttggc acccagtatc gaagctaacg cgttaagttc gccgcctggg 840
gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900
tatgtggttt aattcgatgc aacgcgaaga accttacctg gtcttgacat ccacggaact 960
ttccagagat ggattggtgc cttcgggaac cgtgagacag gtgctgcatg gctgtcgtca 1020
gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg tccttagttg 1080
ccagcacgta atggtgggaa ctctaaggag accgccggtg acaaaccgga ggaaggtggg 1140
gatgacgtca agtcatcatg gcccttacga ccagggctac acacgtacta caatggtggg 1200
gacagagggc tgcaaacccg cgagggtgag ccaatcccag aaaccctatc tcagtccgga 1260
ttggagtctg caactcgact ccatgaagtc ggaatcgcta gtaatcgcag atcagcattg 1320
ctgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg ggagtttgtt 1380
gcaccgggag caggtagctt aacgtcgg 1408
<210> 2
<211> 1414
<212> DNA
＜213〉pseudomonas (
Pseudomonas) 16s rDNA
<400> 2
gctacacatg caagtcgagc ggatgaaggg agcttgctcc tggattcagc ggcggacggg 60
tgagtaatgc ctaggaatct gcctggtagt gggggataac gttccgaaag gaacgctaat 120
accgcgtacg tcctacggga gaaagcaggg gaccttcggg ccttgcgcta tcagatgagc 180
ctaggtcgga ttagctagtt ggtgaggtaa tggctcacca aggcgacgat ccgtaactgg 240
tctgagagga tgatcagtca cactggaact gagacacggt ccagactcct acgggaggca 300
gcagtgggga atattggaca atgggcgaaa gcctgatcca gccatgccgc gtgtgtgaag 360
aaggtcttcg gattgtaaag cactttaagt tgggaggaag ggctgtaggc taatatcctg 420
cggttttgac gttaccaaca gaataagcac cggctaactt cgtgccagca gccgcggtaa 480
tacgaagggt gcaagcgtta atcggaatta ctgggcgtaa agcgcgcgta ggtggttcgt 540
taagttggat gtgaaagccc cgggctcaac ctgggaactg catccaaaac tggcgagcta 600
gagtacggta gagggtagtg gaatttcctg tgtagcggtg aaatgcgtag atataggaag 660
gaacaccagt ggcgaaggcg actacctgga ctgatactga cactgaggtg cgaaagcgtg 720
gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgtc aactagccgt 780
tggaatcctt gagattttag tggcgcagct aacgcattaa gttgaccgcc tggggagtac 840
ggccgcaagg ttaaaactca aatgaattga cgggggcccg cacaagcggt ggagcatgtg 900
gtttaattcg aagcaacgcg aagaacctta cctggccttg acatgctgag aactttccag 960
agatggattg gtgccttcgg gaactcagac acaggtgctg catggctgtc gtcagctcgt 1020
gtcgtgagat gttgggttaa gtcccgtaac gagcgcaacc cttgtcctta gttaccagca 1080
cgttatggtg ggcactctaa ggagactgcc ggtgacaaac cggaggaagg tggggatgac 1140
gtcaagtcat catggccctt acggccaggg ctacacacgt gctacaatgg tcggtacaaa 1200
gggttgccaa gccgcgaggt ggagctaatc ccataaaacc gatcgtagtc cggatcgcag 1260
tctgcaactc gactgcgtga agtcggaatc gctagtaatc gtgaatcaga atgtcacggt 1320
gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgggagtgg gttgctccag 1380
aagtagctag tctaaccttc ggggggacgg tacc 1414
<210> 3
<211> 1393
<212> DNA
＜213〉friendly Gordon Salmonella (
Gordonia amicalis) 16s rDNA
<400> 3
ccatgcagtc gaacggaaag gcccgcttgc gggtactcga gtggcgaacg ggtgagtaac 60
acgtgggtga tctgccctgg actctgggat aagcctggga aactgggtct aataccggat 120
atgaccttac atcgcatggt gtttggtgga aagcttttgc ggttcaggat gggcccgcgg 180
cctatcagct tgttggtggg gtaatggcct accaaggcga cgacgggtag ccgacctgag 240
agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg 300
gggaatattg cacaatgggc gcaagcctga tgcagcgacg ccgcgtgagg gatgacggcc 360
ttcgggttgt aaacctcttt caccagggac gaagcgcaag tgacggtacc tggagaagaa 420
gcaccggcca actacgtgcc agcagccgcg gtaatacgta gggtgcgagc gttgtccgga 480
attactgggc gtaaagagct cgtaggcggt ttgtcgcgtc gtctgtgaaa ttctgcaact 540
caattgtagg cgtgcaggcg atacgggcag acttgagtac tacaggggag actggaattc 600
ctggtgtagc ggtgaaatgc gcagatatca ggaggaacac cggtggcgaa ggcgggtctc 660
tgggtagtaa ctgacgctga ggagcgaaag cgtgggtagc gaacaggatt agataccctg 720
gtagtccacg ccgtaaacgg tgggtactag gtgtggggct catttcacga gttccgtgcc 780
gtagctaacg cattaagtac cccgcctggg gagtacggcc gcaaggctaa aactcaaagg 840
aattgacggg ggcccgcaca agcggcggag catgtggatt aattcgatgc aacgcgaaga 900
accttacctg ggtttgacat acaccagaaa gctgtagaga tatagccccc cttgtggttg 960
gtgtacaggt ggtgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1020
caacgagcgc aacccttgtc ctgtattgcc agcgggttat gccggggact tgcaggagac 1080
tgccggggtc aactcggagg aaggtgggga tgacgtcaag tcatcatgcc ccttatgtcc 1140
agggcttcac acatgctaca atggctggta cagagggctg cgataccgtg aggtggagcg 1200
aatcccttaa agccagtctc agttcggatt ggggtctgca actcgacccc atgaagtcgg 1260
agtcgctagt aatcgcagat cagcaacgct gcggtgaata cgttcccggg ccttgtacac 1320
accgcccgtc acgtcatgaa agtcggtaac acccgaagcc ggtggcctaa cccttgtgga 1380
gggagcttcg aag 1393
<210> 4
<211> 1410
<212> DNA
＜213〉Rhodococcus ruber (Rhodococcus ruber) 16s rDNA
<400> 4
cttacacatg caagtcgaac gatgaagccc agcttgctgg gtggattagt ggcgaacggg 60
tgagtaacac gtgggtgatc tgccctgcac ttcgggataa gcctgggaaa ctgggtctaa 120
taccggatag gacctcggga tgcatgttcc ggggtggaaa ggttttccgg tgcaggatgg 180
gcccgcggcc tatcagcttg ttggtggggt aacggcccac caaggcgacg acgggtagcc 240
ggcctgagag ggcgaccggc cacactggga ctgagacacg gcccagactc ctacgggagg 300
cagcagtggg gaatattgca caatgggcgc aagcctgatg cagcgacgcc gcgtgaggga 360
tgacggcctt cgggttgtaa acctctttca gtaccgacga agcgcaagtg acggtaggta 420
cagaagaagc accggcccaa ctacgtgcca gcagccgcgg taatacgtag ggtgcgagcg 480
ttgtccggga attactgggc gtaaagagct cgtaggcggt ttgtcgcgtc gtctgtgaaa 540
acccgcagct caactgcggg cttgcaggcg atacgggcag acttgagtac tgcaggggag 600
actggaattc ctggtgtagc ggtgaaatgc gcagatatca ggaggaacac cggtggcgaa 660
ggcgggtctc tgggcagtaa ctgacgctga ggagcgaaag cgtgggtagc gaacaggatt 720
agataccctg gtagtccacg ccgtaaacgg tgggcgctag gtgtgggttt ccttccacgg 780
gatccgtgcc gtagctaacg cattaagcgc cccgcctggg gagtacggcc gcaaggctaa 840
aactcaaagg aattgacggg ggcccgcaca agcggcggag catgtggatt aattcgatgc 900
aacgcgaaga accttacctg ggtttgacat acaccggacc gccccagaga tggggtttcc 960
cttgtggtcg gtgtacaggt ggtgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg 1020
ttaagtcccg caacgagcgc aacccttgtc ctgtgttgcc agcacgtaat ggtggggact 1080
cgcaggagac tgccggggtc aactcggagg aaggtgggga cgacgtcaag tcatcatgcc 1140
ccttatgtcc agggcttcac acatgctaca atggccggta cagagggctg cgataccgcg 1200
aggtggagcg aatcccttaa agccggtctc agttcggatc ggggtctgca actcgacccc 1260
gtgaagtcgg agtcgctagt aatcgcagat cagcaacgct gcggtgaata cgttcccggg 1320
ccttgtacac accgcccgtc acgtcatgaa agtcggtaac acccgaagcc ggtggcctaa 1380
cccctcgtgg gagggagccg tcgaaggtgg 1410
<210> 5
<211> 1408
<212> DNA
＜213〉food alkane Gordon Salmonella (
Gordonia alkanivorans) 16s rDNA
<400> 5
gcttacacat gcaagtcgaa cggaaaggcc cagcttgctg ggtactcgag tggcgaacgg 60
gtgagtaaca cgtgggtgat ctgccctgaa ctttgggata agcctgggaa actgggtcta 120
ataccggata tgaccttgga gtgcatgctc tggggtggaa agcttttgcg gttcaggatg 180
ggcccgcggc ctatcagctt gttggtgggg taatggccta ccaaggcgac gacgggtagc 240
cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag 300
gcagcagtgg ggaatattgc acaatgggcg caagcctgat gcagcgacgc cgcgtgaggg 360
atgacggcct tcgggttgta aacctctttc accagggacg aagcgcaagt gacggtacct 420
ggagaagaag caccggccaa ctacgtgcca gcagccgcgg taatacgtag ggtgcgagcg 480
ttgtccggaa ttactgggcg taaagagctc gtaggcggtt tgtcgcgtcg tctgtgaaat 540
tctgcaactc aattgtaggc gtgcaggcga tacgggcaga cttgagtact acaggggaga 600
ctggaattcc tggtgtagcg gtgaaatgcg cagatatcag gaggaacacc ggtggcgaag 660
gcgggtctct gggtagtaac tgacgctgag gagcgaaagc gtgggtagcg aacaggatta 720
gataccctgg tagtccacgc cgtaaacggt gggtactagg tgtggggctc atttcacgag 780
ttccgtgccg tagctaacgc attaagtacc ccgcctgggg agtacggccg caaggctaaa 840
actcaaagga attgacgggg gcccgcacaa gcggcggagc atgtggatta attcgatgca 900
acgcgaagaa ccttacctgg gtttgacata caccagacgc atgtagagat acatgttccc 960
ttgtggttgg tgtacaggtg gtgcatggct gtcgtcagct cgtgtcgtga gatgttgggt 1020
taagtcccgc aacgagcgca acccttgtcc tgtattgcca gcgggttatg ccggggactt 1080
gcaggagact gccggggtca actcggagga aggtggggat gacgtcaagt catcatgccc 1140
cttatgtcca gggcttcaca catgctacaa tggctggtac agagggctgc gataccgtga 1200
ggtggagcga atcccttaaa gccagtctca gttcggattg gggtctgcaa ctcgacccca 1260
tgaagtcgga gtcgctagta atcgcagatc agcaacgctg cggtgaatac gttcccgggc 1320
cttgtacaca ccgcccgtca cgtcatgaaa gtcggtaaca cccgaagccg gtggcctaac 1380
cccttgtggg agggagctgt cgaaggtg 1408
Claims (10)
1. microbiobacterial agent, this microbiobacterial agent comprises substratum and thalline, it is characterized in that, described thalline comprise preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis), preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas), preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis), preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) and preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans).
2. microbiobacterial agent according to claim 1, wherein, the contained total viable count of the described microbiobacterial agent of every gram is 0.5-1 * 10
10Individual.
3. microbiobacterial agent according to claim 2 wherein, is a benchmark with the total viable count in the described microbiobacterial agent, preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) viable count be the 10-50% of total viable count, the pseudomonas that preserving number is CGMCC No. 4793 (
Pseudomonas) viable count be 10-50%, the preserving number of total viable count be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) viable count be the 10-50% of total viable count, the Rhodococcus ruber that preserving number is CGMCC No. 4795 (
Rhodococcus ruber) viable count be the 10-50% of total viable count, food alkane Gordon Salmonella that preserving number is CGMCC No. 4796 (
Gordonia alkanivorans) viable count be the 10-50% of total viable count; Preferably, preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) viable count be the 15-35% of total viable count, the pseudomonas that preserving number is CGMCC No. 4793 (
Pseudomonas) viable count be 15-35%, the preserving number of total viable count be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) viable count be the 15-35% of total viable count, the Rhodococcus ruber that preserving number is CGMCC No. 4795 (
Rhodococcus ruber) viable count be the 15-35% of total viable count, food alkane Gordon Salmonella that preserving number is CGMCC No. 4796 (
Gordonia alkanivorans) viable count be the 15-35% of total viable count.
4. the preparation method of the described microbiobacterial agent of claim 1 is characterized in that, this method comprises: with preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis), preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas), preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis), preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) and preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans) be inoculated in the substratum and cultivate.
5. preparation method according to claim 4, wherein, described being inoculated in carried out cultured method and comprised in the substratum: independently cultivate respectively in the culture system separately the Japanese false Xanthomonas campestris that preserving number is CGMCC No. 4797 (
Pseudoxanthomonas japonensis), preserving number be CGMCC No. 4793 pseudomonas (
Pseudomonas), preserving number be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis), preserving number be CGMCC No. 4795 Rhodococcus ruber (
Rhodococcus ruber) and preserving number be CGMCC No. 4796 food alkane Gordon Salmonella (
Gordonia alkanivorans), and will cultivate the microorganism that obtains respectively and proportionally mix.
6. preparation method according to claim 5, wherein, by separately independently the cultivation in the culture system and cultivate after mixing, the total viable count that makes the every gram microbiobacterial agent that obtains is 0.5-1 * 10
10Individual, wherein, preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) viable count be the 10-50% of total viable count, the pseudomonas that preserving number is CGMCC No. 4793 (
Pseudomonas) viable count be 10-50%, the preserving number of total viable count be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) viable count be the 10-50% of total viable count, the Rhodococcus ruber that preserving number is CGMCC No. 4795 (
Rhodococcus ruber) viable count be the 10-50% of total viable count, food alkane Gordon Salmonella that preserving number is CGMCC No. 4796 (
Gordonia alkanivorans) viable count be the 10-50% of total viable count; Preferably, preserving number be CGMCC No. 4797 Japanese false Xanthomonas campestris (
Pseudoxanthomonas japonensis) viable count be the 15-35% of total viable count, the pseudomonas that preserving number is CGMCC No. 4793 (
Pseudomonas) viable count be 15-35%, the preserving number of total viable count be CGMCC No. 4794 friendly Gordon Salmonella (
Gordonia amicalis) viable count be the 15-35% of total viable count, the Rhodococcus ruber that preserving number is CGMCC No. 4795 (
Rhodococcus ruber) viable count be the 15-35% of total viable count, food alkane Gordon Salmonella that preserving number is CGMCC No. 4796 (
Gordonia alkanivorans) viable count be the 15-35% of total viable count.
7. any application of described microbiobacterial agent in decomposing petroleum hydrocarbon among the claim 1-3.
8. any application of described microbiobacterial agent in the biological restoration petroleum pollution among the claim 1-3.
9. any application of described microbiobacterial agent in the soil of biological restoration petroleum pollution among the claim 1-3.
10. any application of described microbiobacterial agent in the water body of biological restoration petroleum pollution among the claim 1-3.
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| CN103695528A (en) * | 2013-07-19 | 2014-04-02 | 中国海洋石油总公司 | Specific primer for quantitative determination of gordonia alkanivorans |
| CN104830723A (en) * | 2015-04-30 | 2015-08-12 | 大连民族学院 | Bacterium AW25 with petroleum degradation function, application thereof and bacterial vector |
| CN106957809A (en) * | 2017-05-15 | 2017-07-18 | 中国农业科学院农业环境与可持续发展研究所 | The biological synergetic restorative procedure of soil organic contamination |
| CN108101235A (en) * | 2018-01-22 | 2018-06-01 | 中国人民解放军陆军勤务学院 | A kind of method of biodegradation mineral oil in water |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695528A (en) * | 2013-07-19 | 2014-04-02 | 中国海洋石油总公司 | Specific primer for quantitative determination of gordonia alkanivorans |
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| CN106957809B (en) * | 2017-05-15 | 2020-05-22 | 中国农业科学院农业环境与可持续发展研究所 | Biological synergistic remediation method for soil organic pollution |
| CN108101235A (en) * | 2018-01-22 | 2018-06-01 | 中国人民解放军陆军勤务学院 | A kind of method of biodegradation mineral oil in water |
| CN108101235B (en) * | 2018-01-22 | 2021-02-02 | 中国人民解放军陆军勤务学院 | Method for biodegrading mineral oil in water |
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