CN103436460A - Method for enriching efficient biphenyl degradation flora by using accelerator SRpf - Google Patents

Method for enriching efficient biphenyl degradation flora by using accelerator SRpf Download PDF

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CN103436460A
CN103436460A CN2013102542895A CN201310254289A CN103436460A CN 103436460 A CN103436460 A CN 103436460A CN 2013102542895 A CN2013102542895 A CN 2013102542895A CN 201310254289 A CN201310254289 A CN 201310254289A CN 103436460 A CN103436460 A CN 103436460A
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biphenyl
srpf
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CN103436460B (en
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苏晓梅
沈超峰
胡金星
秦智慧
黄荣浪
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Zhejiang University ZJU
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Abstract

The present invention discloses a method for enriching efficient biphenyl degradation flora by using an accelerator SRpf, wherein the accelerator SRpf is added to an enrichment culture process of biphenyl degradation bacterial, continuous acclimation is performed to obtain an enrichment liquid with an efficient biphenyl degradation performance, and bacterial growth, flora diversity and a biphenyl degradation rate can be significantly increased with addition of the accelerator SRpf, wherein specifically bacterial growth is increased by 30-50%, a biphenyl degradation rate is increased by 20-70%, and a Shannon's diversity index is increased to 2.10-2.34 from 1.56-1.70. The accelerator SRpf has functions of VBNC flora recovery and bacterial growth promotion, such that the accelerator SRpf can be provided for enriching efficient biphenyl degradation flora and promoting growth of other organic pollutant degradation flora so as to provide a safe, cheap and efficient method for exploration of potential degradation flora in PCB, PAH and other pollution environments.

Description

A kind of method of utilizing the efficient biphenyl degradation flora of promotor SRpf enrichment
Technical field
The invention belongs to pollutent microbiological deterioration field, relate in particular to a kind of method of utilizing the efficient biphenyl degradation flora of promotor SRpf enrichment.
Background technology
Biphenyl (Biphenyl) is natural to be present in coal tar, Sweet natural gas, crude oil, in the burning entered environment by fossil oil.In industrial production, biphenyl, through iodine and Ferric Chloride, can produce 209 kinds of homologues, and this compounds is called polychlorobiphenyl (Polychlorinated biphenyls is called for short PCBs).PCBs, because having flame retardant resistance, high-insulativity and thermostability preferably, once was widely used in transformer, capacitor insulation oil, fuel dispersants, lubricant, softening agent and coating etc.PCBs is as one of 12 kinds of persistence organic pollutants (POPs), because environmental persistence reaches a kind of typical heteroplasia type organic pollutant that " three cause " effect becomes current extensive concern.Because PCBs belongs to the heteroplasia type organism of difficult degradation, but have many decades in physical environment more than, therefore, although PCBs forbid for many years, but the problem of environmental pollution that it causes deeply becoming increasingly conspicuous along with research.Chemical stability due to the PCBs uniqueness, traditional physics and chemistry method is because existing poor efficiency, secondary pollution problems to become the application bottleneck, microorganism is as the natural resources of a large amount of existence, and microbiological deterioration improvement technology has safety, efficient, economic dispatch characteristics, therefore, the microorganism recovery technique of PCBs contaminate environment has application prospect preferably.
The microorganism of PCBs contaminate environment is repaired with mineralising or is total to two kinds of modes of metabolism and carries out.Some low chlorine PCBs can be used as the required carbon source of microorganism self Growth and reproduction and the energy and carry out aerobic degradation.And, for high chlorine (chlorine replaces number >=5) PCBs, need be converted into low chlorine PCBs through anaerobic dechlorination, just can carry out aerobic degradation.Its degradation pathway is: by dioxygenase, prosposition is carried out to oxidation, dehydrogenation obtains pyrocatechol, then by the phenylformic acid that obtains of ring-opening reaction.This pathways metabolism is similar to other aromatics, as toluene, and biphenyl.And Biphenyl Degrading plays a significant role in metabolism altogether at PCBs, most of Biphenyl Degradings have been proved the effect that has equally degraded PCB homologue.In addition, when screening PCBs degradation bacteria, generally take biphenyl as sole carbon source, first screening can utilize the bacterial strain of biphenyl, and then checks its degradation property to the PCB homologue.
There are very large potentiality although microorganism is repaired PCBs, and carried out a large amount of research work, mostly still be limited at present the laboratory study stage.PCBs efficient degradation function bacterial classification scarcity of resources becomes the maximum bottleneck that it is applied.In addition, the efficient degrading bacteria that laboratory obtains often presents low degraded in the actual repair place active, even the phenomenon of inactivation.Now, although structure PCBs Degrading genetical engineering of microorganism and immobilization degrading enzyme are the approach that builds High Efficiency PC Bs degradation bacteria, there are a lot of problems, as selection, gene level transfer and the immobilization high in cost of production of host cell.
Therefore, how from the work as the overwhelming majority (90%～99%) Microbial resources physical environment but non-educable state (" viable but non-culturable ", abbreviation VBNC) in flora, find potential PCBs and biphenyl degradation flora, the degradation efficiency that improves bacterial strain becomes to be broken the microorganism recovery technique apply the key point of bottleneck in the PCBs contaminate environment.The discovery of gamboge coccus Resuscitation-promoting Factor (resuscitation-promoting factor, Rpf) is the important breakthrough of VBNC state bacterium recovery.Not only can the recover high GC gram-positive microorganism of multiple nearly edge of gamboge coccus Rpf, as: mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium bovis (Mycobacterium bovis), mycobacterium kansasii (Mycobacterium kansasii), M. smegmatics (Mycobacterium smegmatis) and mycobacterium avium (Mycobacterium avium) etc.Simultaneously, low GC gram-positive microorganism and part Gram-negative bacteria (Curvibacter fontana sp.) are also had to recovery promotion functions preferably.At present, the concern for Rpf recovery and promoter action, mainly concentrate on medical science, epidemiology basin, and from Microbial resources and environmental functional angle, do not launch research.
In sum, utilize the promotor SRpf(Supernant from Micrococcus luteus containing Rpf containing Rpf) not only can be for the efficient biphenyl degradation flora of enrichment, improve the degradation efficiency of flora, become the key breakthrough that obtains efficient biphenyl degradation flora.In addition, promotor SRpf also can be used for promoting the growth of other organic pollutant degradation flora, for exploring potential degradation flora in the contaminate environment such as PCBs, PAHs, provides a kind of safe, cheap, efficient method.
Summary of the invention
The objective of the invention is that the strain degradation usefulness that exists in the microorganism recovery technique for existing PCBs contaminate environment is low, the speed of growth is slow, the low deficiency of tolerance concentration, provide a kind of method of utilizing the efficient biphenyl degradation flora of promotor SRpf enrichment.The method is easy and simple to handle, efficient, non-secondary pollution.
The objective of the invention is to be achieved through the following technical solutions: a kind of method of utilizing the efficient biphenyl degradation flora of promotor SRpf enrichment comprises the following steps:
(1) gamboge coccus (Micrococcus Luteus IAM14879) is inoculated in to the LMM liquid nutrient medium, makes its nutrient solution OD 600nmfor 0.05-0.2; The LMM liquid nutrient medium forms: the NH of 2.5-4.5g/L 4cl, the KH of 1.0-2.0g/L 2pO 4, the vitamin H of 0.005g/L, the L-Methionine of 0.02g/L, the VITMAIN B1 of 0.04g/L, the inosine of 0.5-1.5g/L, the MgSO of 0.03g/L 4, the Pfansteihl lithium salts of 8.0-10.0g/L, mineral salts solution (the 0.375g/L CuSO of 1.5-3.0ml/L 45H 2o, 0.785g/L MnCl 24H 2o, 0.18g/LFeSO 47H 2o, 0.029g/L Na 2moO 42H 2o, 0.089g/L ZnSO 47H 2o), pH is 6.5-8.0, cultivates 24-36h, obtains seed culture fluid;
(2) seed culture fluid step 1 obtained is by 3-5%(v/v) inoculum size be seeded in the LMM liquid nutrient medium, 160r/min, cultivate 48-60h, fermented liquid centrifugal (8000-10000r/min) 10-15min is removed to thalline, carry out sterile filtration through 0.22 μ m filtering membrane, the supernatant liquor obtained is promotor SRpf, and its protein content is 23.96-25.34mg/L, is stored under-20 ℃ of conditions standby;
(3) promotor SRpf step 2 obtained by volume mark is 15% to add to and take in the minimal medium that biphenyl is sole carbon source, after mixing enrichment medium, wherein the composition of minimal medium is: the KH of 1-2g/L 2pO 4, the K of 2.5-3.5g/L 2hPO 43H 2o, the MgSO of 0.2g/L 4, the FeSO of 0.02g/L 47H 2o, the NaCl of the 1g/L, (NH of 2-4g/L 4) 2sO 4, the CaCl of 0.01g/L 2, the trace salt solution (MoO of 4mg/L of 2mL/L 3, the ZnSO of 28mg/L 45H 2o, the CuSO of 0.02mg/L 45H 2o, the H of 4mg/L 3bO 3, the MnSO of 4mg/L 45H 2o, the CoCl of 4mg/L 26H 2o), pH is 7.3-7.5, and biphenyl is 500-3000mg/L;
(4) get the PCBs contaminated soil and be added in the enrichment medium that step 3 obtains, the mass volume ratio of PCBs contaminated soil and enrichment medium is 35-65g/L; 30 ℃, the cultivation of 180-200r/min shaking table, every culture 3-5d, go down to posterity and cultivate 5-6 time, and initial biphenyl concentration is 500mg/L, with 500mg/L, increases progressively, and in enrichment medium, biphenyl concentration reaches 2500-3000mg/L;
(5) nutrient solution step 4 obtained is by 4-6%(v/v) inoculum size be seeded in the minimal medium containing 500-4500mg/L biphenyl, 30 ℃, 180-200r/min, cultivation 24-64h.
The fermented liquid that step 5 is obtained carries out biphenyl degradation rate and thalli growth flow measurement, the effect that result shows to add promotor SRpf is remarkable, is specially: the thalli growth amount improves that 30-50%, biphenyl degradation rate improve 20-70%, Shannon diversity index is increased to 2.10-2.34 by 1.56-1.70.
The present invention compares with existing Biphenyl Degrading enriching method, and its beneficial effect is:
1, the present invention promotor SRpf used is by gamboge coccus secretion, have with low cost, raw material is easy to get, safety non-toxic, the characteristics such as easy and simple to handle.
2, the present invention utilizes the recovery promoter action of promotor SRpf, the VBNC flora in not only can recovery PCBs contaminated soil, and can promote the growth of low degraded usefulness flora.Thereby the efficient biphenyl degradation flora of enrichment, give full play to the effect of indigenous microorganism in the PCBs contaminated soil.
3, in the present invention, promotor SRpf is added in the enrichment medium of biphenyl degradation flora, by the SRpf(promotor SRpf with adding inactivation through 121 ℃, autoclaving 20min) contrasted, the interpolation that shows promotor SRpf can significantly improve thalli growth amount, flora diversity, and the biphenyl degradation rate.
4, the present invention by volume mark be 15% interpolation promotor SRpf, 30 ℃, 180-200r/min, cultivate 24h, to 1500mg/L biphenyl, degradation rate is 95.7-98.2%, and known promotor SRpf can effectively improve the biphenyl degradation rate, has fast, the characteristics such as non-secondary pollution simultaneously, fully demonstrate the recycling economy feature of practicality, economy, the feature of environmental protection, have application value.
5, the promotor SRpf in the present invention not only can be for the efficient biphenyl degradation flora of enrichment, also can be used for promoting the growth of other organic pollutant degradation flora, for exploring potential degradation flora in the contaminate environment such as PCBs, PAHs, provide a kind of safe, cheap, efficient method.
In sum, the present invention meets the environmental protection concept of green safety, realize Microbial resources, can effectively bring into play the degradation characteristic of indigenous microorganism in the PCBs contaminate environment, for promoting applying of microorganism recovery technique, provide a kind of safety non-toxic, method with low cost.
The accompanying drawing explanation
Fig. 1 adds the affect figure of promotor SRpf on thalli growth amount and biphenyl degradation rate.
Embodiment
The present invention utilizes the method for the efficient biphenyl degradation flora of promotor SRpf enrichment, comprises the following steps:
(1) gamboge coccus (Micrococcus Luteus IAM14879) is inoculated in to the LMM liquid nutrient medium, makes its nutrient solution OD 600nmfor 0.05-0.2; The LMM liquid nutrient medium forms: the NH of 2.5-4.5g/L 4cl, the KH of 1.0-2.0g/L 2pO 4, the vitamin H of 0.005g/L, the L-Methionine of 0.02g/L, the VITMAIN B1 of 0.04g/L, the inosine of 0.5-1.5g/L, the MgSO of 0.03g/L 4, the Pfansteihl lithium salts of 8.0-10.0g/L, mineral salts solution (the 0.375g/L CuSO of 1.5-3.0ml/L 45H 2o, 0.785g/L MnCl 24H 2o, 0.18g/LFeSO 47H 2o, 0.029g/L Na 2moO 42H 2o, 0.089g/L ZnSO 47H 2o, solvent is water), pH is 6.5-8.0, cultivates 24-36h, obtains seed culture fluid;
(2) seed culture fluid step 1 obtained is by 3-5%(v/v) inoculum size be seeded in the LMM liquid nutrient medium, 160r/min, cultivate 48-60h, fermented liquid centrifugal (8000-10000r/min) 10-15min is removed to thalline, carry out sterile filtration through 0.22 μ m filtering membrane, the supernatant liquor obtained is promotor SRpf, and its protein content is 23.96-25.34mg/L, is stored under-20 ℃ of conditions standby;
(3) promotor SRpf step 2 obtained by volume mark is 15% to add to and take in the minimal medium that biphenyl is sole carbon source, after mixing enrichment medium, wherein the composition of minimal medium is: the KH of 1-2g/L 2pO 4, the K of 2.5-3.5g/L 2hPO 43H 2o, the MgSO of 0.2g/L 4, the FeSO of 0.02g/L 47H 2o, the NaCl of the 1g/L, (NH of 2-4g/L 4) 2sO 4, the CaCl of 0.01g/L 2, the trace salt solution (MoO of 4mg/L of 2mL/L 3, the ZnSO of 28mg/L 45H 2o, the CuSO of 0.02mg/L 45H 2o, the H of 4mg/L 3bO 3, the MnSO of 4mg/L 45H 2o, the CoCl of 4mg/L 26H 2o, solvent is water), pH is 7.3-7.5, biphenyl is 500-3000mg/L;
(4) get the PCBs contaminated soil and be added in the enrichment medium that step 3 obtains, the mass volume ratio of PCBs contaminated soil and enrichment medium is 35-65g/L; 30 ℃, the cultivation of 180-200r/min shaking table, every culture 3-5d, go down to posterity and cultivate 5-6 time, and initial biphenyl concentration is 500mg/L, with 500mg/L, increases progressively, and in enrichment medium, biphenyl concentration reaches 2500-3000mg/L;
(5) nutrient solution step 4 obtained is by 4-6%(v/v) inoculum size be seeded in the minimal medium containing 500-4500mg/L biphenyl, 30 ℃, 180-200r/min, cultivation 24-64h.
The fermented liquid that step 5 is obtained carries out biphenyl degradation rate and thalli growth flow measurement, the effect that result shows to add promotor SRpf is remarkable, is specially: the thalli growth amount improves that 30-50%, biphenyl degradation rate improve 20-70%, Shannon diversity index is increased to 2.10-2.34 by 1.56-1.70.
Below according to embodiment, describe the present invention in detail, it is more obvious that purpose of the present invention and effect will become:
The present invention utilizes the method for the efficient biphenyl degradation flora of promotor SRpf enrichment, comprises the following steps:
1, the preparation of promotor SRpf
Gamboge coccus (Micrococcus Luteus IAM14879) can be purchased from Japanese RIKEN's microbial strains preservation center.
The LMM substratum consists of: 4.0g/L NH 4cl, 1.4g/L KH 2pO 4, 0.005g/L vitamin H, 0.02g/LL-methionine(Met), 0.04g/L VITMAIN B1,1.0g/L inosine, 0.03g/L MgSO 4, 8.01g/L Pfansteihl lithium salts, 2.0ml/L mineral salts solution (0.375g/L CuSO 45H 2o, 0.785g/L MnCl 24H 2o, 0.18g/LFeSO 47H 2o, 0.029g/L Na 2moO 42H 2o, 0.089g/L ZnSO 47H 2o), pH is 7.5.
The cultivation of actication of culture and seed liquor: from slant tube, M.luteus is inoculated in culture dish, cultivates 3d for 30 ℃.Picking 3 ring M.luteus lawns are to the 250mL triangular flask that 50mL LMM liquid nutrient medium is housed, and 30 ℃, 160r/min, cultivate 36h.
M.luteus fermentation culture: get seed culture fluid by 4%(v/v) inoculum size is equipped with access in the 50mL triangular flask of 15mLLMM nutrient solution, and 30 ℃, 160r/min cultivates 48h.
2, the enrichment culture of Biphenyl Degrading
Consisting of of minimal medium: 1g/L KH 2pO 4, 3g/L K 2hPO 43H 2o, 0.2g/L MgSO 4, 0.02g/L FeSO 47H 2o, 1g/L NaCl, 3g/L (NH 4) 2sO 4, 0.01g/L CaCl 2, trace salt solution 2mL/L(4mg/L MoO 3, 28mg/L ZnSO 45H 2o, 0.02mg/L CuSO 45H 2o, 4mg/L H 3bO 3, 4mg/L MnSO 45H 2o, 4mg/L CoCl 26H 2o), pH is 7.3.
Enrichment medium: biphenyl content is: 500-2500mg/L, treatment group: press the 15%(volume fraction in minimal medium) add promotor SRpf, control group: press the 15%(volume fraction in minimal medium) SRpf of interpolation inactivation.
Enrichment culture: get 5g soil and be added to respectively and be equipped with in the enrichment medium of 100mL containing the treatment group of 500mg/L biphenyl and control group, 30 ℃, after 180r/min cultivates 6d, the pregnant solution of the first-generation is seeded to and is equipped with in the 150mL Erlenmeyer flask of 50mL containing the enrichment medium of 1000mg/L biphenyl by 6% inoculum size, 30 ℃, after the 180r/min shaking table is cultivated 6d, the pregnant solution of the s-generation is seeded to and is equipped with in the 150mL Erlenmeyer flask of 50mL containing the enrichment medium of 1500mg/L biphenyl by 4% inoculum size, 30 ℃, after the 180r/min shaking table is cultivated 4d, the pregnant solution of the third generation is seeded to and is equipped with in the 150mL Erlenmeyer flask of 50mL containing the enrichment medium of 2000mg/L biphenyl by 4% inoculum size, 30 ℃, after the 180r/min shaking table is cultivated 3d, the pregnant solution in the 4th generation is seeded to and is equipped with in the 150mL Erlenmeyer flask of 50mL containing the enrichment medium of 2000mg/L biphenyl by 4% inoculum size, 30 ℃, the 180r/min shaking table is cultivated, after 5 generation enrichment culture, the final biphenyl concentration of pregnant solution reaches 2500mg/L, obtain respectively control group and treatment group enrichment culture liquid.
3, promotor SRpf adds the impact on thalli growth and biphenyl degradation rate
The pregnant solution of control group and treatment group is seeded to and 20mL is housed respectively containing 500 by 5% inoculum size, 1000, 1500, 2000, 2500, 3000, 3500, 4000, in the inorganic salt nutrient solution of 4500mg/L biphenyl, each concentration is all done three groups of parallel laboratory tests, 30 ℃, 200r/min, after cultivating 64h, add isopyknic ethyl acetate extraction, after vibration 25min, pour in the 50ml colorimetric cylinder, standing 20min, after getting the upper organic phase stepwise dilution, adopt gas chromatograph-mass spectrometer (GC-MS) to measure the residual quantity of biphenyl, take off layer inorganic phase and carry out the mensuration of thalli growth amount.
The mensuration of biphenyl content: get upper organic phase and measure residual biphenyl concentration.The condition that GC-MS measures: GC condition: GC(Agilent7890) measured DB-5 quartz capillary column (30m * 0.25mm * 0.25 μ m); The detector radioactive source is 63Ni; Injector temperature: 270 ℃; Detector temperature: 280 ℃; Carrier gas: high pure nitrogen, constant current 1.0mL/min; Without split stream sampling 1 μ L.Heating schedule is: 70 ℃ of the temperature that begins, keep 1min, and 30 ℃/min rises to 280 ℃, keeps 7min, total run time 8min.The MS condition: MS(5975C inter MSD) the EI ion source temperature is 200 ℃, 150 ℃ of level Four bar temperature, and detecting pattern is for selecting ion scan, solvent delay 3min.Biphenyl has three kinds to select ion, and m/z is respectively 152,153,154.TMX has four kinds to select ion, and m/z is respectively 207,209,242,244.
Thalli growth amount (OD 600) mensuration: thalli growth is by measuring the optical density value (OD under 600nm 600) meaned.Get the inorganic phase fermented liquid, the centrifugal 10min of 10000r/min, collect thalline, after distilled water wash 3 times, is diluted to original volume, with UV-5500 (PC) type spectrophotometer in wavelength 600nm place mensuration optical density value, with OD 600mean the thalli growth amount.
Measure by analysis, result shows that the interpolation of promotor SRpf can significantly improve thalli growth amount and biphenyl degradation rate, is specially: the thalli growth amount improves 30-50%, and the degradation rate of biphenyl improves 20-70%, sees Fig. 1.4, promotor SRpf adds the multifarious impact of flora
Treatment group by enrichment culture after 5 generations and control group pregnant solution, extract respectively total DNA, by bacterial 16 S rDNA primers F, 357-GC/R518 carries out pcr amplification to extracted DNA, PCR reaction system 50 μ L comprise: EasyTaq Mix:25 μ L, primer 338F-GC (5 '-CGCCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCAGCA G-3 ') and 518R (5 '-ATTACCGCGGCTGCTGG-3 ') each 1 μ L, template DNA 2 μ L, deionized water: 21 μ L.The PCR reaction conditions is: 94 ℃ of denaturation 4min, and 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 20s, carry out 30 circulations, and 72 ℃ are extended 5min, 16 ℃ of maintenances eventually.Amplified fragments is about 200bp, product detects and carries out deformation gradient gel electrophoresis (DGGE) with 1% agarose gel electrophoresis, DGGE adopts 8% polyacrylamide gel, sex change concentration is 30%～60%, PCR product applied sample amount is 45 μ L, 6 * loading the buffer that adds 20 μ L, 1 * TAE, electrophoresis 6h under the 160V voltage conditions.Electrophoresis poststaining 30min, take pictures.Structural changes and the dominant microflora of analyzing flora according to the band number presented and brightness.By Quantity One4.0 software processes DGGE collection of illustrative plates, analyze the peak density of each band in each swimming lane, calculate Shannon diversity index (Shannon-Weaver diversity index, H): H=-∑ (n i/ N) log (n i/ N), n wherein ifor the peak density of each band, N is all band peak value summations, and S is DNA band number.
The outward appearance of pregnant solution and the demonstration of DGGE analytical results, the Shannon diversity index of control group and treatment group is respectively 1.76 and 2.11, shows that the interpolation of promotor SRpf makes thalli growth amount and flora diversity be improved significantly.
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Claims (3)

1. a method of utilizing the efficient biphenyl degradation flora of promotor SRpf enrichment, is characterized in that, comprises the following steps:
(1) the gamboge coccus is inoculated in to the LMM liquid nutrient medium, makes its nutrient solution OD 600nmfor 0.05-0.2; The LMM liquid nutrient medium forms: the NH of 2.5-4.5g/L 4cl, the KH of 1.0-2.0g/L 2pO 4, the vitamin H of 0.005g/L, the L-Methionine of 0.02 g/L, the VITMAIN B1 of 0.04 g/L, the inosine of 0.5-1.5g/L, the MgSO of 0.03g/L 4, the Pfansteihl lithium salts of 8.0-10.0g/L, the mineral salts solution of 1.5-3.0ml/L, pH is 6.5-8.0; Cultivate 24-36h, obtain seed culture fluid;
(2) seed culture fluid step 1 obtained is by 3-5%(v/v) inoculum size be seeded in the LMM liquid nutrient medium, 160r/min, cultivate 48-60h, fermented liquid centrifugal (8000-10000r/min) 10-15min is removed to thalline, carry out sterile filtration through 0.22 μ m filtering membrane, the supernatant liquor obtained is promotor SRpf, and its protein content is 23.96-25.34 mg/L, is stored under-20 C conditions standby;
(3) promotor SRpf step 2 obtained by volume mark is 15% to add to and take in the minimal medium that biphenyl is sole carbon source, after mixing enrichment medium, wherein the composition of minimal medium is: the KH of 1-2g/L 2pO 4, the K of 2.5-3.5g/L 2hPO 43H 2o, the MgSO of 0.2g/L 4, the FeSO of 0.02g/L 47H 2o, the NaCl of 1 the g/L, (NH of 2-4g/L 4) 2sO 4, the CaCl of 0.01g/L 2, the trace salt solution of 2mL/L, pH is 7.3-7.5, biphenyl is 500-3000mg/L;
(4) get the PCBs contaminated soil and be added in the enrichment medium that step 3 obtains, the mass volume ratio of PCBs contaminated soil and enrichment medium is 35-65g/L; 30 ℃, the cultivation of 180-200r/min shaking table, every culture 3-5d, go down to posterity and cultivate 5-6 time, and initial biphenyl concentration is 500mg/L, with 500mg/L, increases progressively, and in enrichment medium, biphenyl concentration reaches 2500-3000mg/L;
(5) nutrient solution step 4 obtained is by 4-6%(v/v) inoculum size be seeded in the minimal medium containing 500-4500mg/L biphenyl, 30 ℃, 180-200r/min, cultivation 24-64h.
2. utilize according to claim 1 the method for the efficient biphenyl degradation flora of promotor SRpf enrichment, it is characterized in that, in described step 1, the consisting of of described mineral salts solution: the CuSO of 0.375g/L 45H 2o, the MnCl of 0.785g/L 24H 2o, the FeSO of 0.18g/L 47H 2o, the Na of 0.029g/L 2moO 42H 2o, the ZnSO of 0.089g/L 47H 2o, solvent is water.
3. utilize according to claim 1 the method for the efficient biphenyl degradation flora of promotor SRpf enrichment, it is characterized in that, in described step 1, the consisting of of described trace salt solution: the MoO of 4mg/L 3, the ZnSO of 28mg/L 45H 2o, the CuSO of 0.02mg/L 45H 2o, the H of 4mg/L 3bO 3, the MnSO of 4mg/L 45H 2o, the CoCl of 4mg/L 26H 2o, solvent is water.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104531600A (en) * 2014-10-18 2015-04-22 浙江大学 VBNC biphenyl degrading bacterium isolation and screening method and application
CN105817476A (en) * 2016-03-24 2016-08-03 华南理工大学 Degradation reinforcing method for polychlorinated biphenyl in soil or sediment
CN106987517A (en) * 2017-05-02 2017-07-28 广东省微生物研究所（广东省微生物分析检测中心） A kind of VOCs degradation floras quickly tame the system and method for enrichment
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CN107929997A (en) * 2017-10-13 2018-04-20 浙江大学 A kind of application of rape straw charcoal in Polychlorinated biphenyls anaerobic reductive dechlorination is mediated
CN110484465A (en) * 2019-08-01 2019-11-22 浙江浩岚投资有限公司 A kind of recovery medium and the preparation method and application thereof of VBNC bacterium
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