CN102618457B - Rhodococcussp WB-1, culture method thereof and method for degrading polychlorinated biphenyl - Google Patents
Rhodococcussp WB-1, culture method thereof and method for degrading polychlorinated biphenyl Download PDFInfo
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Abstract
The invention belongs to the technical field of pollutant biological repair, in particular relates to rhodococcussp WB-1, a separation and culture method of the rhodococcussp and a method for degrading polychlorinated biphenyl. A bacterial strain is a gram-positive bacterium and is verified to be the rhodococcussp, and a CGMCC preservation number is 5500. The bacterial strain can grow with biphenyl as the only carbon source and energy, can degrades more than 90% 2,3-dichloro diphenyl of 1mg/L, 2,4-dichloro diphenyl, 3,4-dichloro diphenyl and 2,4,4-trichlorine diphenyl in 24 hours, further can degrade more than 90% of 2,4,4-trichlorine diphenyl of 10mg/L and more than 50% of 2,2',3,3'-tetrachloro diphenyl in 72 hours. The application range of the bacterial strain comprises treatment of sewage containing the polychlorinated biphenyl and repair of polychlorinated biphenyl polluted soil.
Description
Technical field
The invention belongs to pollutent bioremediation technology field, be specifically related to a strain called after WB-1 Rhod (
RhodococcusSp. bacterial strain WB-1), this bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 29th, 2011, register on the books and be numbered: CGMCC No.5500, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.This bacterial strain is the bacterium of a high-efficiency degradation polychlorobiphenyl, also relates to separation, the cultural method of this bacterial strain, also relates to the application method of this bacterial strain in the degradation of polychlorinated biphenyl pollutant.
Background technology
Polychlorobiphenyl (Polychlorinatedbiphenyls, be called for short PCBs) be important organochlorine compound, persistence organic pollutant (the Persistent Organic Pollutants of the great ecological impact of having of synthetic and long-range circumstances harm, be called for short POPs), be to forbid all over the world in " Convention of Stockholm " or limit one of 12 kinds of persistence organic pollutants of use.The industrial goods of PCBs are widely used in various production fields, transformer oil such as transformer, capacitor equipment, the transmission medium of hydraulic efficiency system, the additive of the thermal barrier of thermal conductivity and lubricating oil, coating, tackiness agent, printing-ink, resin, rubber, paraffin etc.PCBs can discharge by industrial waste, seal and deposit a seepage, place where the garage is piled up leaching vat seepage, contain the city garbage burning of PCBs and industry is burned and the approach such as dried wet deposition of atmosphere, enter the soil deposit substance environment, account for 97 % of environment PCBs total amount.Slowly bio-transformation (biological degradation, absorption, metabolism, drainage), abiotic conversion (oxidation, photodissociation) and migration (volatilization, diffusion, absorption and sorption, desorb etc.) can occur in the PCBs homologue in the entered environment and the composition of isomer.Be present in lastingly the PCBs in soil, the Sediment environment, absorbed by plant and hydrobiont, by food chain transmission and enrichment (coefficient of concentration even up to millions of times).Have and report and detect PCB132 and the PCB149 of high density in human milk and the blue whale tissue, and have significant chiral selectivity.The toxicity toxicological study shows: entering the polychlorobiphenyl of the high density of human body or animal body, is typical environmental hormone, and strong intoxicating, carcinogenic performance are arranged, and can cause that liver injury and white corpuscle increase disease, also can pass to daughter by parent, makes the daughter deformity.Very harmful to ecotope (particularly human health).According to the WHO statistics, the whole world has produced 2 * 10 altogether
6T Industrial PC Bs.China produces 10000 t Industrial PC Bs, wherein PCB altogether between 1965～1974 years
39000 t, PCB
51000 t.PCB
3For the production of YL, YIW, CL, RLS, RLSI series power capacitor; PCB
5Main as oil paint additive.The 70's of China also import partly contain the power capacitor of polychlorobiphenyl, power transformer etc., these electrical appliance parts are retired, on a small quantity also in operation, in case it is very serious that consequence occurs to leak.
Bioremediation technology has lot of advantages, and is low such as cost, and efficient is high, do not produce secondary pollution, especially is fit to the original position reparation of POPs in the soil.The biological degradation of relevant PCBs is carried out morely in the laboratory, and it also is in recent years study hotspot (Sun Hongbin, 2006; Sun Yan, 2004).At laboratory condition, the PCBs of Cl atomicity＜5 is verified can be become inorganics by several microbiological oxidations, and the PCBs that high Cl replaces (Cl〉4) is generally considered to be persistent aerobic conditions is next.It is more that Cl replaces number, and chlorobenzene compound is more difficult by aerobic degradation.But exception is also arranged,
AlcaligenesY42,
PseudomonadSp. LB400 and
AlcaligenesEutrophus H850,
AlcaligenesSp. JB1 is verified can degrade 4-6 Cl substituent.(Zheng Hailong, 2004)
Microorganism has 2 approach usually to the degraded of PCBs: the one, take PCBs as sole carbon source with the energy, it is degraded, so that mineralising; Another approach is to obtain carbon source and the energy from other organism, and then PCBs is degraded, and also is usually said common metabolism (Borjia, 2005).But up to the present, only find
AchromobacterXylosoxidians A8 can utilize the 2-CB(2-chlordiphenyl) and 2,5-DCB(2, the 5-DCBP) as sole carbon source and the energy (Jencova, 2004), other microorganisms also are not found to utilize PCBs to grow as unique growth matrix.
Although at present there are many progress the biological degradation research of PCBs, but still have a lot of problems: 1, high chlorine is replaced the PCBs degrading microorganism research of (Cl〉4) less; 2, the microorganism cometabolism of PCBs is studied seldom; 3, lack for high chlorine and replace the degrading enzyme of PCBs of (Cl〉4) and the research of degrading genes; 4, China lags behind to the biological degradation of PCBs research, lacks degradation bacteria with independent intellectual property right and the resource of degrading genes; Although 5 have found the degrading genes segment of PCBs from the bacterium of many degraded PCBs, make up at present superpower degradation bacteria and yet there are no report for the PCBs reparation.
Summary of the invention
The objective of the invention is in the present microbial technology existing lack growth fast, cultivate present situation simple, can efficient degradation polychlorobiphenyl bacterial classification, provide a kind of fast growth, cultural method simple, can be efficient, the rhodococcus WB-1 that stablizes degradation of polychlorinated biphenyl.
Another object of the present invention provides the cultural method of rhodococcus WB-1.
The present invention also aims to provide and utilize rhodococcus WB-1 to be used for the method that degraded contains polychlorobiphenyl sewage or soil pollution polychlorobiphenyl.
Rhodococcus of the present invention (
RhodococcusSp.) WB-1, CGMCC No.5500 has following character:
The Main Morphology of this bacterial strain is characterized as: be rod-short in the Gluconic Acid Ammonium salt salt culture medium; In the LB substratum, be shaft-like; Cheng Dan, Cheng Shuan, chaining; Size (0.6 μ m-1.2 μ m) * (1.5 μ m-6.0 μ m); Colony characteristics: circle, be orange at LB, slightly do, protuberance, old culture edge is irregular, optimum growth temperature is 28-30 ℃, and the most suitable growth pH is 7.0-7.2, can be the sole carbon source growth by biphenyl (500-1000mg/L) in minimal medium, 2 of 1mg/L more than 90% can degrade in 24h, 3-DCBP, 2,4-DCBP, 3,4-DCBP and 2,4, the 4-trichloro biphenyl, 2,4 of the 10mg/L more than 90% of can also in 72h, degrading, 2 of 1mg/L more than the 4-trichloro biphenyl and 50%, 2 ', 3,3 '-tetrachloro biphenyl.
Rhodococcus WB-1 of the present invention obtains by step cultivation, separation, the screening of following order:
1) get the pedotheque 2g that is subjected to pollution by polychlorinated biphenyles 10-20, add in the 100ml enrichment medium, the prescription of this substratum is: NaCl 0.5-1.5g/L, NH
4NO
30.5-1.5g/L, K
2HPO
41.0-2.0g/L, KH
2PO
40.3-0.8g/L, MgSO
47H
2O 0.1-0.3g/L, yeast extract 0.05-0.2g/L behind the accent pH7.0-7.2, presses 500-1000mg/L and adds biphenyl, and in 28-37 ℃, the 120-180rpm shaking table is cultivated;
2) the every 1-2 of above-mentioned nutrient solution week switching culture transferring once, in the fresh described enrichment medium of the inoculum size access of 5-10%, so through the culture transferring of repeatedly transferring, domestication 3-6 month; Final cultivation bacterium liquid is carried out bacterium with plate dilution method separate, 7 of single bacterium colonies of therefrom obtain to grow soon, bacterium colony is regular;
3) detect the performance of each strains for degrading polychlorobiphenyl, method is: with single bacterium access respectively in the LB substratum cultivate 18-24h after, centrifugal collection thalline, the aseptic phosphoric acid buffer of pH7.0-7.2 cleans bacterial sediment, repeated centrifugation, cleaning are once again, use at last the aseptic phosphoric acid buffer suspension thalline of pH7.0-7.2, with bacterium liquid OD
600nmBe adjusted to 1.0-2.0,4-8 ℃ saves backup.
4) inoculum size with 5-10% contains 1-10mg/L2 with the bacteria suspension adding, 4, in the aseptic phosphate buffer solution of the pH7.0-7.2 of 4-trichloro biphenyl, detect polychlorobiphenyl content behind the 3-5d, screening obtains the highest bacterial strain of degradation rate (being numbered F-1), identify through Physiology and biochemistry, determine this bacterial strain belong to Rhod (
RhodococcusSp.), called after WB-1.Be stored in LB inclined-plane or the glycerine cryopreservation tube, activate at the substratum that contains biphenyl during use.
Rhodococcus WB-1 of the present invention is used for the method for degradation of polychlorinated biphenyl, comprises the step of following order:
A. the preparation of various substratum:
LB substratum: 2.5-7.5g/LNaCl, the 2.5-7.5g/L yeast extract paste, the 5-15g/L peptone is transferred pH to 6.0-8.0;
Minimal medium: NaCl 0.5-1.5g/L, NH
4NO
30.5-1.5g/L, K
2HPO
41.0-2.0g/L, KH
2PO
40.3-0.8g/L, MgSO
47H
2O 0.1-0.3g/L, yeast extract 0.05-0.2g/L transfers pH7.0-7.2;
Fermention medium: glucose 5-15g/ L, yeast extract paste 1-2g/ L, (NH4)
2SO
41.0-2.0g/L, NaCl0.5-1g/ L transfers pH to 6.0-8.0.
B. rhodococcus WB-1 bacterial classification is rule to solid LB flat board 30-35 ℃ of activation culture 18-24h from LB inclined-plane or glycerine cryopreservation tube.
C. the single colony inoculation after will activating is in the LB liquid nutrient medium, 30-35 ℃, the 120-180rpm shaking table is cultured to logarithmic phase, with above-mentioned medium centrifugal, after the phosphoric acid buffer cleaning with pH7.0-7.2, contain in the basic medium of 500mg/L biphenyl with the adding of 5-10% inoculum size, 30-35 ℃, the 120-180rpm shaking table is cultivated, and cultivates 3-5d and after the disappearance of biphenyl particle, use quantity and the purity of bacterium in the microscopic examination nutrient solution in nutrient solution.
D. at 4-8 ℃, 8,000-10, the centrifugal 10-15min of 000r/min collects the thalline in the nutrient solution, and with the aseptic phosphoric acid buffer cleaning bacterial sediment of pH7.0-7.2, repeated centrifugation, cleaning are once again, use at last the aseptic phosphoric acid buffer suspension thalline of pH7.0-7.2, with bacterium liquid OD
600nmBe adjusted to 1.0-2.0,4-8 ℃ saves backup.
E. the inoculum size with 5-10% contains the bacteria suspension adding in the solution or sewage of 1-10mg/L polychlorobiphenyl, 30-35 ℃, the 120-180rpm shaking table is cultivated, detect the content of polychlorobiphenyl in the solution behind the cultivation 3-5d with GC, take the solution that contains polychlorobiphenyl that do not add bacterium and sewage as contrast, the polychlorobiphenyl degradation rate is 80-90%.
F. bacteria suspension is added and contain in the contaminated soil of polychlorobiphenyl, 30-35 ℃, keep soil humidity 60-80%, extract the polychlorobiphenyl that detects in the soil after processing 15-30d, with the soil that do not add bacterium in contrast, the degradation rate of polychlorobiphenyl reaches 50-80%.
Separate the rhodococcus WB-1 strains for degrading stable performance that obtains among the present invention; not only can be in solution degradation of polychlorinated biphenyl; can also degrade residual polychlorinated biphenyl in the soil; degradation efficiency is up to 80-90%; cultural method is simple, and fast growth only needs growth 18-24h just can gather in the crops in LB substratum and fermention medium; this bacterium is expected to be applied to the reparation of pollution by polychlorinated biphenyles soil, plays a role aspect healthy in environmental pollution improvement and protection people.
Description of drawings
Fig. 1 be rhodococcus WB-1 to 2,4, the degradation curve figure of 4 '-trichloro biphenyl (PCB28);
Degradation condition: rhodococcus WB-1 inoculum size is 10%, 30 ℃ of temperature, and pH7.0, shaking speed is 180rpm.
Fig. 2 is rhodococcus WB-1 degraded 2,4, the gas chromatogram of 4 '-trichloro biphenyl (PCB28);
A is not for adding the contrast of rhodococcus WB-1, and B is the processing that adds rhodococcus WB-1, and PCB28 concentration is 10mg/L,
Analytical equipment: Agilent 7890 gas chromatographs, electron capture detector (ECD),
Chromatographic column: capillary chromatographic column, model HP-5, internal diameter 0.32mm, coat-thickness 0.25 μ m, length 30m,
Analysis condition: 28 minutes analysis times, standard substance are qualitative, and PCB138 is as internal standard substance, sample introduction 1 μ L, Splitless injecting samples.Temperature programming, 120 ℃ of initial temperatures are warming up to 180 ℃ with the speed of 15 ℃/min, speed with 10 ℃/min is warming up to 260 ℃ again, and the speed with 5 ℃/min is raised to 280 ℃ again, keeps 10min, rear operation 2min, the temperature of injection port and detector is respectively 260 ℃ and 300 ℃.
Fig. 3 is that rhodococcus WB-1 is to the gas chromatogram of 11 kinds of polychlorobiphenyl degradeds;
Polychlorobiphenyl corresponding to numbering 1-11 is respectively PCB5, PCB7, PCB12, PCB28, PCB36, PCB52, PCB47, PCB40, PCB77, PCB153, PCB138 among the figure, wherein PCB138 concentration is 0.5mg/L, all the other concentration are respectively 1mg/L, the same Fig. 2 of testing conditions and method.
Preservation information
The biomaterial Classification And Nomenclature: rhodococcus (
RhodococcusSp.) WB-1;
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center;
Deposit number: CGMCC No.5500;
Preservation date: on November 29th, 2011;
Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Embodiment
Below in conjunction with Fig. 1 to 3 and embodiment the present invention is described in further detail.
To be subjected to the pedotheque 2g of pollution by polychlorinated biphenyles, add in the 100ml enrichment medium, the prescription of this enrichment medium is: NaCl 0.5g/L, NH
4NO
30.5g/L, K
2HPO
41.0g/L, KH
2PO
40.3g/L, MgSO
47H
2O 0.1g/L, yeast extract 0.05g/L behind the accent pH7.2, presses 500mg/L and adds biphenyl, and in 28 ℃, the 120rpm shaking table is cultivated;
Per 1 week of above-mentioned nutrient solution is transferred culture transferring once, and the inoculum size with 5% accesses in the fresh described enrichment medium, so through the culture transferring of repeatedly transferring, tames 3 months; Final cultivation bacterium liquid is carried out bacterium with plate dilution method separate, 7 of single bacterium colonies of therefrom obtain to grow soon, bacterium colony is regular;
With above 7 strain bacteriums access respectively in the LB substratum cultivate 18-24h after, centrifugal collection thalline, the aseptic phosphoric acid buffer of pH7.2 cleans bacterial sediment, again repeated centrifugation, clean once, use at last the aseptic phosphoric acid buffer suspension thalline of pH7.2, with bacterium liquid OD
600nmBe adjusted to 1.0.
Inoculum size with 5% adds above-mentioned bacteria suspension and contains 2 of 1mg/L, 4, in the aseptic phosphate buffer solution of the pH7.2 of 4 '-trichloro biphenyl, at 30 ℃, detect polychlorobiphenyl content behind the degraded 3d in the shaking table of 180rpm, screening obtains the highest bacterium (being numbered F-1) of 1 strain degradation rate.
The evaluation of rhodococcus WB-1 and growth characteristics:
Through the comparison of Physiology and biochemistry and 16SrRNA gene order, the F-1 bacterial strain belong to Rhod (
RhodococcusSp.), called after WB-1.This bacterium is stored in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 29th, 2011, and deposit number is CGMCC No.5500.Growth characteristics to rhodococcus WB-1 are studied, and comprise carbon source, nitrogenous source utilization test, humid test, pH test etc.Inoculum size by 2% accesses rhodococcus WB-1 bacteria suspension in the different Carbon and nitrogen sources minimal mediums, and 30 ℃, 180rpm, shaking culture is cultivated sampling after 24 hours, measures OD
600nmValue.Inoculum size by 2% accesses the Gluconic Acid Ammonium salt salt culture medium with rhodococcus WB-1 bacteria suspension, respectively at 18 ℃, 25 ℃, 30 ℃, 37 ℃, 42 ℃, and 180rpm, shaking culture, OD is measured in sampling after 24 hours
600nmValue.The initial pH of Gluconic Acid Ammonium salt salt culture medium is transferred to respectively 5.0,6.0,7.0,8.0,9.0, the inoculum size inoculation by 2%, 30 ℃, 160rpm, shaking culture.OD is measured in sampling after 25 hours
600nmValue.
Result's demonstration, rhodococcus WB-1 can utilize the several kinds of carbon source such as wood sugar, sucrose, semi-lactosi, glucose and fructose, can not utilize lactose, and that grows in glucose is best, and fructose takes second place.Rhodococcus WB-1 can utilize multiple nitrogenous source, and growth is significantly better than inorganic nitrogen in organonitrogen, and the suitableeest organic nitrogen source is yeast extract paste, and inorganic nitrogen-sourced is ammonium sulfate.Rhodococcus WB-1 is at 30 ℃ of well-growns, the growth that all is unfavorable for it too high or too low for temperature.Rhodococcus WB-1 is well-grown under the meta-alkali condition, and pH8.0 is the optimal pH of its growth, and the pH value is lower than growth in 7.0 o'clock and obviously is suppressed.
Rhodococcus WB-1 is to the degraded of polychlorobiphenyl in the aqueous solution:
The single colony inoculation of rhodococcus WB-1 after the activation in the LB liquid nutrient medium, is added 100mg/L biphenyl, and 30 ℃, the 180rpm shaking table is cultivated 24h.With above-mentioned medium centrifugal, with the phosphoric acid buffer of pH7.0-7.2 clean, centrifugal 2 times, again with phosphoric acid buffer with thalline OD
600nmTransfer to 1.0-2.0, inoculum size with 5-10% contains the bacteria suspension adding in the solution or sewage of 1mg/L polychlorobiphenyl, and 30 ℃, the 180rpm shaking table is cultivated, cultivate the content that detects polychlorobiphenyl in the solution behind the 3-5d with GC, take the solution that contains polychlorobiphenyl that do not add bacterium and sewage as contrast.As shown in Figures 1 to 3, rhodococcus WB-1 2 of the 1mg/L more than 95% that can in 24h, degrade, 3-DCBP, 2,4-DCBP, 3,4-DCBP and 2,4,4-trichloro biphenyl, 2 of the 1mg/L more than 50% of can also in 72h, degrading, 2 ', 3,3 '-tetrachloro biphenyl.
Embodiment 4
Rhodococcus WB-1 is to the degraded of polychlorobiphenyl in the aqueous solution:
The single colony inoculation of rhodococcus WB-1 after the activation in the LB liquid nutrient medium, is added 100mg/L biphenyl, and 30 ℃, the 180rpm shaking table is cultivated 24h.With above-mentioned medium centrifugal, with the phosphoric acid buffer of pH7.0-7.2 clean, centrifugal 2 times, again with phosphoric acid buffer with thalline OD
600nmTransfer to 1.0-2.0, inoculum size with 5-10% contains the bacteria suspension adding in the solution or sewage of 5mg/L polychlorobiphenyl, and 30 ℃, the 180rpm shaking table is cultivated, cultivate the content that detects polychlorobiphenyl in the solution behind the 3-5d with GC, take the solution that contains polychlorobiphenyl that do not add bacterium and sewage as contrast.As shown in Figures 1 to 3, rhodococcus WB-1 2 of the 5mg/L more than 90% that can in 24h, degrade, 3-DCBP, 2,4-DCBP and 3,4-DCBP, 2,4 of the 5mg/L more than 90% of can also in 72h, degrading, 4-trichloro biphenyl.
Rhodococcus WB-1 is to the degraded of polychlorobiphenyl in the aqueous solution:
The single colony inoculation of rhodococcus WB-1 after the activation in the LB liquid nutrient medium, is added 100mg/L biphenyl, and 30 ℃, the 180rpm shaking table is cultivated 24h.With above-mentioned medium centrifugal, with the phosphoric acid buffer of pH7.0-7.2 clean, centrifugal 2 times, again with phosphoric acid buffer with thalline OD
600nmTransfer to 1.0-2.0, inoculum size with 5-10% contains the bacteria suspension adding in the solution or sewage of 10mg/L polychlorobiphenyl, and 30 ℃, the 180rpm shaking table is cultivated, cultivate the content that detects polychlorobiphenyl in the solution behind the 3-5d with GC, take the solution that contains polychlorobiphenyl that do not add bacterium and sewage as contrast.As shown in Figures 1 to 3, rhodococcus WB-1 2,4 of the 10mg/L more than 80% that can in 72h, degrade, 4-trichloro biphenyl.
Rhodococcus WB-1 is to the degraded of PCBs in Soil:
Soil is taken from experiment field, academy of agricultural sciences, Zhejiang Province, and soil type is yellowish soil, organic content 14.5g/kg, nitrogenous 1.2g/kg, pH value 7.2, soil moisture content 60-70%.Select in the soil veneer of soil without residual polychlorinated biphenyl, each sample 1kg soil artificially in the soil adds 2,4, and 4 '-trichloro biphenyl makes that content is 5mg/kg in the soil, and the usage quantity of rhodococcus WB-1 bacterium liquid is 10
8/ g dry ground.After executing bacterium, measure the content of PCBs in Soil respectively at the 3rd, 7,14,21d.The result shows, inoculation rhodococcus WB-1 not by the soil of pollution by polychlorinated biphenyles in, compare with control soil, the 3rd, 7,14, behind the 21d, the PCBs in Soil of inoculation rhodococcus WB-1 has degraded respectively 23.43%, 45.81%, 71.26%, 89.78%.Obviously, the rhodococcus WB-1 polychlorobiphenyl in the soil of can effectively degrading, for the reparation of pollution by polychlorinated biphenyles soil provides may.
In a word, the above only is preferred embodiment of the present invention, and all equalizations of doing according to the present patent application claim change and modify, and all should belong to the covering scope of patent of the present invention.
Claims (2)
1. a rhodococcus (Rhodococcus sp.) WB-1 is characterized in that: CGMCC No.5500, and Main Morphology is characterized as: be rod-short in the Gluconic Acid Ammonium salt salt culture medium; In the LB substratum, be shaft-like; Cheng Dan, Cheng Shuan, chaining; Size (0.6 μ m-1.2 μ m) * (1.5 μ m-6.0 μ m); Colony characteristics: circle, be orange at LB, slightly do, protuberance, old culture edge is irregular, optimum growth temperature is 28-30 ℃, and the most suitable growth pH is 7.0-7.2, the growth take the biphenyl of 500-1000mg/L as sole carbon source in minimal medium, 2 of 1mg/L more than 90% can degrade in 24h, 3-DCBP, 2,4-DCBP, 3,4-DCBP and 2,4, the 4-trichloro biphenyl, 2,4 of the 10mg/L more than 90% of can also in 72h, degrading, 2 of 1mg/L more than the 4-trichloro biphenyl and 50%, 2 ', 3,3 '-tetrachloro biphenyl.
2. rhodococcus WB-1 claimed in claim 1 is used for the method for degradation of polychlorinated biphenyl, and it is characterized in that: the method comprises the step of following order:
A. the preparation of various substratum:
LB substratum: 2.5-7.5g/LNaCl, the 2.5-7.5g/L yeast extract paste, the 5-15g/L peptone is transferred pH to 6.0-8.0;
Minimal medium: NaCl 0.5-1.5g/L, NH
4NO
30.5-1.5g/L, K
2HPO
41.0-2.0g/L, KH
2PO
40.3-0.8g/L, MgSO
47H
2O 0.1-0.3g/L, yeast extract 0.05-0.2g/L transfers pH7.0-7.2; Fermention medium: glucose 5-15g/L, yeast extract paste 1-2g/L, (NH
4)
2SO
41.0-2.0g/L NaCl0.5-1g/L transfers pH to 6.0-8.0;
B. rhodococcus WB-1 bacterial classification is rule to solid LB flat board 30-35 ℃ of activation culture 18-24h from LB inclined-plane or glycerine cryopreservation tube;
C. the single colony inoculation after will activating is in the LB liquid nutrient medium, 30-35 ℃, the 120-180rpm shaking table is cultured to logarithmic phase, with above-mentioned medium centrifugal, after the phosphoric acid buffer cleaning with pH7.0-7.2, contain in the minimal medium of 500mg/L biphenyl with the adding of 5-10% inoculum size, 30-35 ℃, the 120-180rpm shaking table is cultivated, and cultivates 3-5d and after the disappearance of biphenyl particle, use quantity and the purity of bacterium in the microscopic examination nutrient solution in nutrient solution;
D. at 4-8 ℃, 8,000-10, the centrifugal 10-15min of 000r/min collects the thalline in the nutrient solution, and with the aseptic phosphoric acid buffer cleaning bacterial sediment of pH7.0-7.2, repeated centrifugation, cleaning are once again, use at last the aseptic phosphoric acid buffer suspension thalline of pH7.0-7.2, with bacterium liquid OD
600nmBe adjusted to 1.0-2.0,4-8 ℃ saves backup;
E. the inoculum size with 5-10% contains the bacteria suspension adding in the solution or sewage of 1-10mg/L polychlorobiphenyl, 30-35 ℃, the 120-180rpm shaking table is cultivated, detect the content of polychlorobiphenyl in the solution behind the cultivation 3-5d with GC, take the solution that contains polychlorobiphenyl that do not add bacterium and sewage as contrast, obtain the polychlorobiphenyl degradation rate;
F. bacteria suspension is added and contain in the contaminated soil of polychlorobiphenyl, 30-35 ℃, keep soil humidity 60-80%, extract the polychlorobiphenyl that detects in the soil after processing 15-30d, with the soil that do not add bacterium in contrast, obtain the degradation rate of polychlorobiphenyl.
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