CN101428171A

CN101428171A - Degradation method for polychlorinated biphenyl with staphylococcus epidermidis

Info

Publication number: CN101428171A
Application number: CNA2008100410509A
Authority: CN
Inventors: 赵晓祥; 彭瑶; 任昱宗; 庞晓倩
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2008-07-25
Filing date: 2008-07-25
Publication date: 2009-05-13

Abstract

The invention relates to a method for degradation of polychlorinated biphenyls by staphylococcus epidermidis. The method comprises the following steps: inoculating staphylococcus epidermidis in a polychlorinated biphenyl degradation culture medium and subjecting the staphylococcus epidermidis to the shaking culture at 28-32 DEG C for 24-120 hours; and analyzing the degradation effect of the bacterial strain on the PCBs after the culture is subjected to high performance liquid chromatograph determination. By the addition of extra carbon source glucose, the method can ensure that the staphylococcus epidermidis is more efficient in degrading the polychlorinated biphenyls, thereby allowing the bacteria to obtain the conditions for degrading the polychlorinated biphenyl.

Description

MRSE is to the biodegrading process of Polychlorinated biphenyls

Technical field

The invention belongs to and be used for contaminating microorganisms degraded field, particularly relate to the biodegrading process of a kind of MRSE Polychlorinated biphenyls.

Background technology

Jensen reported first in 1966 Polychlorinated biphenyls in the environment (Polychlorinated biphenyls, pollution PCBs), from then on, the hot issue that the existence of PCBs and its persistence have become people to be paid close attention to.Because the persistence of PCBs and be difficult to degradability, PCBs is one of most important pollutant in the environment, and it extensively accumulates in soil, water, deposit and atmosphere.Handling at present the method that PCBs pollutes mainly is physics or chemical technology, and the both exists or costs an arm and a leg or produce secondary pollution problem.And the microbiological treatment technology is as economy, effective method and adopted by various countries and conduct a research.

Microorganism can secrete different enzymes at different pollutants and participate in reaction, thereby changes the structure of organic pollution, and it is degraded into simple organic, reduces its adverse influence.In the degradation process of PCBs, exist two kinds of different binding modes: anaerobism dechlorination and aerobic biodegradation.

Because microorganism is strong to the adaptability of environment, and one section natural domestication of pollution course experience, thereby can be by taming the microbial degradation bacterial strain that from natural environment, screen a certain pollutant of degradable.

Summary of the invention

The purpose of this invention is to provide a kind of method of microbial degradation Polychlorinated biphenyls, Polychlorinated biphenyls is degraded, for its pollution control and application provide technical method by MRSE.

MRSE of the present invention comprises the biodegrading process of Polychlorinated biphenyls:

(1) MRSE is inoculated in 28～32 ℃ of shaking tables of Polychlorinated biphenyls degraded culture medium and cultivates 24～120h;

(2) culture can be analyzed the degradation effect of this bacterial strain to Polychlorinated biphenyls through high-performance liquid chromatogram determination;

(3) after having added additional carbon glucose, improved the degradation efficiency of Polychlorinated biphenyls, obtain the condition of degradation of polychlorinated biphenyl.

Described MRSE is to screen from the activated sludge of Songjiang, Shanghai sewage treatment plant, and this bacterial strain belongs to micrococcaceae (micrococcaceae), staphylococcus.The domestication culture medium of this bacterial strain is 0.5g/L KH ₂PO ₄, 0.5g/LK ₂HPO ₄, 0.2g/L MgSO ₄, 0.1g/LCaCl ₂, 0.2g/LNaCl, 1.0g/L (NH ₄) ₂SO ₄, 1g/L biphenyl.This bacterium colony is circular, neat in edge, and milky, smooth, moistening, projection; It is spherical that thalline is, and Gram-negative has the rete malpighii parcel around the thalline.

Described Polychlorinated biphenyls degraded culture medium is 0.5g/L KH ₂PO ₄, 0.5g/L K ₂HPO ₄, 0.2g/LMgSO ₄, 0.1g/LCaCl ₂, 0.2g/L NaCl, 1.0g/L (NH ₄) ₂SO ₄, 100mg/L 2,3 ', 4 ', 5-tetrachloro biphenyl-acetone soln, pH6.8-7.2.

The condition of described degradation of polychlorinated biphenyl is pH7.0,30 ℃, and the rotational frequency 150rpm of shaking table.

Beneficial effect:

MRSE is at pH7.0, and 30 ℃, the rotational frequency 150rpm of shaking table fermented 5 days in having added additional carbon degradation of glucose culture medium, and MRSE is to 2, and the degradation efficiency of 3 ', 4 ' 5-tetrachloro biphenyl reaches 93.33%.

Description of drawings

Fig. 1 is a population genealogical tree under the MRSE (Staphylococcus epidermidis);

Fig. 2 is 2,3 ', 4 ', and 5-tetrachloro biphenyl-acetone soln is as the high-efficient liquid phase chromatogram of standard sample;

Fig. 3 is degraded culture medium (DM) high-efficient liquid phase chromatogram of sample in contrast;

Fig. 4 is the high-efficient liquid phase chromatogram of degraded culture medium (DM) mesocuticle staphylococcus to the PCBs degraded;

Fig. 5 is adding the degradation efficiency of additional carbon degradation of glucose culture medium mesocuticle staphylococcus to PCBs.

The specific embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

The separation of embodiment 1 Polychlorinated biphenyls degradation bacteria is identified

1, the separation of bacterial strain

(1) activated sludge of collection Songjiang, Shanghai sewage treatment plant, place a long 60cm, wide 40cm, the glass domestication device of high 50cm, at Ensure Liquid material not, with air compressor machine aeration 3 days, change water then on the basis of the water outlet of not intaking exchange, the mud mixture of about 10L in the discharger adds nutrient solution and also carries out uninterrupted aeration.The waste liquid that begins to add PCBs after having cultivated 10 days when adding nutrient solution is tamed cultivation, cultivates domestication two months.The preparation of nutrient solution: glucose 5 grams, urea 2 grams, potassium dihydrogen phosphate 1 gram.Be mixed with about about 10 liters nutrient solution.

(2) precipitation of the water sample after will taming was got supernatant after 30 minutes, used 8 layers of filtered through gauze twice then, and filtrate and enriched medium by volume in the ratio wiring solution-forming immigration conical flask of 1:5, are placed on the shaking table and cultivate.Shaking speed is 150r/min, and temperature is 30 ℃, and regulator solution pH value is 7.0.The main component of enriched medium (Enrichment Medium) is: beef extract 5g/L, peptone 10g/L, sodium chloride 5g/L, pH7.4～7.6.

In cultivating, domestication utilize biphenyl to tame cultivation again as sole carbon source.Get domestication culture medium 100ml and move in the conical flask of 250ml, regulating the pH value is 7.0, moves into the muddy liquid 1ml of enriched medium under aseptic condition.Put into shaking table and cultivate, shaking speed is 150r/min, and temperature is 30 ℃, and incubation time is fortnight.The main component of domestication culture medium (Domestication Medium) is: 0.5g/L KH ₂PO ₄, 0.5g/L K ₂HPO ₄, 0.2g/LMgSO ₄, 0.1g/LCaCl ₂, 0.2g/LNaCl, 1.0g/L (NH ₄) ₂SO ₄, 1g/L biphenyl.

The bacterial strain line separates, and the main component of isolation medium SM (Separate Medium) is: beef extract 5g/L, peptone 10g/L, sodium chloride 5g/L, agar powder 20g/L, biphenyl 0.5g/L.Obtain single bacterial strain, enrichment culture in the purification enrichment culture medium.Purification enrichment culture medium PEM (Purification Enrichment Medium) main component is: 0.5g/L KH ₂PO ₄, 0.5g/L K ₂HPO ₄, 0.2g/L MgSO ₄, 0.1g/L CaCl ₂, 0.2g/L NaCl, 1.0g/L (NH ₄) ₂SO ₄, 2g/L biphenyl.

2, the evaluation of bacterial strain

(1) thalline and colony morphology characteristic

The bacterial strain individuality is spherical, and Gram-negative has the rete malpighii parcel around the thalline; Its bacterium colony is circular, neat in edge, and milky, smooth, moistening, projection.

(2) compare by 16S rDNA sequencing and analysis, the homology of this sequence and MRSE 16S rDNA sequence reaches 99%, determines that this bacterial strain is MRSE (Staphylococcus epidermidis), its 16S rDNA sequence such as table 1.

Embodiment 2 MRSEs are to the degradation analysis of Polychlorinated biphenyls

MRSE is inoculated into (0.5g/L KH in the domestication culture medium of sterilization ₂PO ₄, 0.5g/L K ₂HPO ₄, 0.2g/L MgSO ₄, 0.1g/LCaCl ₂, 0.2g/LNaCl, 1.0g/L (NH ₄) ₂SO ₄, 1g/L biphenyl), place in the constant temperature shaking table, 30 ℃, 150rpm cultivated 14 days.Be inoculated in PCBs degraded culture medium (Degradation Medium) (0.5g/L KH then ₂PO ₄, 0.5g/L K ₂HPO ₄, 0.2g/L MgSO ₄, 0.1g/L CaCl ₂, 0.2g/LNaCl, 1.0g/L (NH4) ₂SO ₄, 100mg/L 2,3 ', 4 ', 5-tetrachloro biphenyl-acetone soln, pH7.0), sample is analyzed the degradation effect of this bacterium to PCBs through high-performance liquid chromatogram determination.

Liquid phase chromatogram condition:

1. single-column list detection system, long 250mm, internal diameter 5mm activated-charcoal column.

2. carrier ratio: hplc grade methanol 10%, pure water 90%

3. column temperature: 150 ℃

4. pump and pump pressure scope: binary pump, 20～400Pa

5. input mode: hand sampling

6. sample size: 50 μ L

7. wave-length coverage: detect wavelength 230nm, reference wavelength 360nm

First with 2,3 ', 4 ', 5-tetrachloro biphenyl-acetone soln occurs crest as the PCBs that standard sample records in the sample at the 7.5min place, as Fig. 2.Determine the appearance time of PCBs standard sample, just can further analyze the degradation effect of PCBs.By in degraded culture medium (DN), do not inoculate MRSE in contrast with the high-efficient liquid phase chromatogram of inoculation MRSE sample relatively (as Fig. 3, Fig. 4) initial concentration that can obtain PCBs be 15.7ng/ μ L, ultimate density is 5.9ng/ μ L, and degradation efficiency is 62.4%.

Embodiment 3 additional carbons are to the influence of Polychlorinated biphenyls degradation efficiency

(1) with MRSE, insert in the enriched medium EM, place interior 30 ℃ of shaking table, 150rpm, enrichment culture 5 days.

(2) the degraded culture medium DM of three parts of 50ml of preparation, a copy of it adds 2g/L glucose and does additional carbon, places the 150ml conical flask.Do not inoculate for one bottle and give over to blank, inoculate 100ul enrichment bacterium liquid respectively for two bottles in addition.30 ℃ of shaking tables, 150rpm cultivates.

(3) under aseptic technique, take out 5ml bacterium liquid respectively at inoculation back 1d, 2d, 3d, 4d, 5d.

(4) sample pretreatment

1. bacterium liquid is placed in the separatory funnel, divide three times and add carrene.First and second time adds 2ml, adds 1ml for the third time.Each back that adds fully vibrates, and leaves standstill a moment.After treating complete up and down layering, take out lower floor's oil phase layer.

2. with the above-mentioned oil phase of getting, filter with anhydrous sodium sulfate.And the unified 1ml that is concentrated into.

Liquid phase chromatogram condition is identical with liquid phase chromatogram condition among the embodiment 2.

Measure by analysis, obtain the condition of MRSE degradation of polychlorinated biphenyl: at pH7.0,30 ℃, the rotational frequency 150rpm of shaking table, added in the additional carbon degradation of glucose culture medium and fermented 5 days, MRSE is to 2,3 ', the degradation efficiency of 4 ' 5-tetrachloro biphenyl reaches 93.33%, sees Fig. 5.

Table 1 16S rDNA sequence

caccttcgac?ggctagctcc?aaatggttac?tccaccggct?tcgggtgtta?caaactctcg?60

tggtgtgacg?ggcggtgtgt?acaagacccg?ggaacgtatt?caccgtagca?tgctgatcta?120

cgattactag?cgattccagc?ttcatatagt?cgagttgcag?actacaatcc?gaactgagaa?180

caactttatg?ggatttgctt?gacctcgcgg?tttcgctacc?ctttgtattg?tccattgtag?240

cacgtgtgta?gcccaaatca?taaggggcat?gatgatttga?cgtcatcccc?accttcctcc?300

ggtttgtcac?cggcagtcaa?cttagagtgc?ccaacttaat?gatggcaact?aagcttaagg?360

gttgcgctcg?ttgcgggact?taacccaaca?tctcacgaca?cgagctgacg?acaaccatgc?420

accacctgtc?actctgtccc?ccgaagggga?aaactctatc?tctagagggg?tcagaggatg?480

tcaagatttg?gtaaggttct?tcgcgttgct?tcgaattaaa?ccacatgctc?caccgcttgt?540

gcgggtcccc?gtcaattcct?ttgagtttca?accttgcggt?cgtactcccc?aggcggagtg?600

cttaatgcgt?tagctgcagc?acttaagggc?ggaaaccccc?taacactt 648

Claims

1. MRSE comprises the biodegrading process of Polychlorinated biphenyls:

(2) culture is analyzed the degradation effect of this bacterial strain to Polychlorinated biphenyls through high-performance liquid chromatogram determination;

(3) after having added additional carbon glucose, obtain the condition of degradation of polychlorinated biphenyl.

2. MRSE according to claim 1 is characterized in that the biodegrading process of Polychlorinated biphenyls: described MRSE is to screen from the activated sludge of Songjiang, Shanghai sewage treatment plant, and its domestication culture medium is 0.5g/LKH ₂PO ₄, 0.5g/L K ₂HPO ₄, 0.2g/L MgSO ₄, 0.1g/LCaCl ₂, 0.2g/LNaCl, 1.0g/L (NH ₄) ₂SO ₄, 1g/L biphenyl.

3. MRSE according to claim 1 is characterized in that the biodegrading process of Polychlorinated biphenyls: described Polychlorinated biphenyls degraded culture medium is 0.5g/L KH ₂PO ₄, 0.5g/L K ₂HPO ₄, 0.2g/LMgSO ₄, 0.1g/LCaCl ₂, 0.2g/LNaCl, 1.0g/L (NH ₄) ₂SO ₄, 100mg/L 2,3 ', 4 ', 5-tetrachloro biphenyl-acetone soln, pH6.8-7.2.

4. MRSE according to claim 1 is characterized in that the biodegrading process of Polychlorinated biphenyls: the condition of described degradation of polychlorinated biphenyl is pH7.0,30 ℃, and the rotational frequency 150rpm of shaking table.