CN109913563A - The appraisal procedure of Natural Attenuation can occur by anaerobe degradation for chlorobenzene class organic contamination place - Google Patents
The appraisal procedure of Natural Attenuation can occur by anaerobe degradation for chlorobenzene class organic contamination place Download PDFInfo
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- 150000008422 chlorobenzenes Chemical class 0.000 title claims abstract description 17
- 241000894006 Bacteria Species 0.000 claims abstract description 20
- 239000003673 groundwater Substances 0.000 claims abstract description 13
- 238000011529 RT qPCR Methods 0.000 claims abstract description 5
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- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention discloses the appraisal procedures that Natural Attenuation can occur by anaerobe degradation for a kind of chlorobenzene class organic contamination place, this method is by carrying out real-time quantitative PCR (qPCR) detection to groundwater sample in contaminated site, obtain all Chlorobenzens reductive dehalogenation bacterium (including the Dehalobacter being currently known, Dehalogenimonas and Dehalococcoides) absolute quantity, fast, economical determine place in chloro- benzenes contamination object whether can pass through anaerobe degradation occur Natural Attenuation.
Description
Technical field
The present invention relates to underground water pollutions to repair field, and especially a kind of chlorobenzene class organic contamination place can be micro- by anaerobism
The appraisal procedure of biodegradation generation Natural Attenuation.
Background technique
Underground water is the important strategic resource in China, it is played not in terms of safeguarding urban economy Community health
Alternative effect.The domestic water of the northern area of China 65%, 50% industrial water and 33% agricultural irrigation water come
From underground water, in national 655 cities, using underground water as drinking water source, (Chinese Ministry of Environmental Protection, national underground water are dirty in a city about more than 400
Water Pollution Control Planning, 2011).Recently as the high speed development of economic society, groundwater environment pressure is gradually increased, and China is main
Urban groundwater, especially phreatic water are by different degrees of pollution (69,2011. China Gao Cunrong, Wang Juntao city
Organic Contamination of Groundwater properties study earth journal [J], 32:581-591), and by extending partially into region, recessive change
Organic contamination is developed to for dominant, inorganic pollution.Underground water pollution has persistence, concealment, complexity and is difficult to administer
Feature produces ecology and human health and greatly threatens (QIU J 2010.China faces up to
Groundwater crisis.Nature [J], 466:308), the highest attention by domestic and international scientific research personnel and government organs.
But China's underground water pollution is repaired still in its infancy, and there is an urgent need to develop various economic, effective, environmentally protective reparation skills
Art.
Chlorobenzens refer to the chemical substance formed after hydrogen atom is substituted by a chlorine atom both on phenyl ring, according to the number for replacing chlorine
Amount and on phenyl ring position difference, one shares 12 kinds of Chlorobenzens.The type organic is as a kind of industrial chemicals and centre
Body, be widely used in dyestuff, medicine, pesticide, rubber, electric equipment products production industry (Wang Fang, DRFLER U, SCHMID M,
The identification and functional analysis environmental science [J] of et al.2007.1,2,4- trichloro-benzenes mineralized bacterium, 28:1082-108).Due to it
A large amount of use detects in the surrounding mediums such as soil, underground water, deposit, vegetables with unreasonable processing, chloro- benzenes contamination object
(2011. microbial degradation 1,2,4- trichloro-benzenes progress soil [J] of Song Yang, Wang Fang, Jiang Xin, 43:343-349).In addition,
Organo-chlorine pesticide six six six can also generate Chlorobenzens by the reaction of the reduction dechlorination of biology or abiologic process
(DOESBURG W,EEKERT M H,MIDDELDORP P J,et al.2005.Reductive dechlorination of
β-hexachlorocyclohexane(β-HCH)by a Dehalobacter species in coculture with a
Sedimentibacter sp.FEMS Microbiol.Ecol.[J],54:87-95；LIU X,PENG P A,FU J,et
al.2003.Effects of FeS on the Transformation Kinetics ofγ-
Hexachlorocyclohexane.Environ Sci Technol[J],37:1822-1828).The pollutant is also China
Common pollutant (the 2015. chlorinatedorganics pollution such as Chen Ranran, Zhu Xin, Lin Yu lock in the soil and underground water of industrial sites
The monitoring Natural Attenuation in place repairs pre-test Journal of Chemical Industry and Engineering [J], 66:2361-2369).Chlorobenzens chemical property is stablized,
Can prolonged stay in the environment, and be easy to biological concentration, people's hepatorenal damage can be caused, there is potential carcinogenicity, to ecological environment and
Human health constitutes a threat to.
Therefore, carrying out chloro- benzenes contamination site remediation has important practical usage.And in existing recovery technique, it declines naturally
Subtraction has that operating cost is low, high efficiency, disturbs to place small, and one for can be used as some low concentration pollution places is long
The recovery technique of phase and the advantages that use, be increasingly taken seriously.And microbiological deterioration be it is most important, destructive from
Right attenuation.It is generally acknowledged that microbiological deterioration is the key that Natural Attenuation method success or not.But aerobic microbiological reparation
Usually limited by the following aspects factor: 1) underground water oxygen content is lower；2) a small amount of oxygen dissolved in underground water holds
Easily rapidly depleted by the simple molecule of aeration bacteria degrading texture；3) oxygen of sufficient amount is pumped into underground water, engineering
Complicated and higher cost；4) Chlorobenzens belong to weight nonaqueous phase organic matter, easily migrate downward into deep layer under the effect of gravity and detest
Oxygen artesian aquifer.Therefore, anaerobe degradation is most important in the Natural Attenuation in chloro- benzenes contamination place.That
, implementing to pass through the Natural Attenuation method under anaerobe degradation, needing to carry out anaerobe to contaminated site first
The assessment for feasibility of degrading.Before anaerobe in situ is repaired, microorganism remediation feasibility assessment need to be carried out to contaminated site.
Method (the Technical mostly announced at present with reference to Environmental Protection Agency in 1998 for the assessment of place Natural Attenuation
Protocol for Evaluating Natural Attenuation of Chlorinated Solvents in Ground
Water).Evidence of this method suggestion in terms of following three confirms the generation of place Natural Attenuation: 1) having the characterization nature to decline
Subtract the environmental geochemistry index of process；2) Historical Monitoring is statistics indicate that pollutant load has decreasing trend, and underground water pollution
Caused by the reduction of plumage concentration is not only Pollution Plume migration；3) microcosm experiment directly proves the generation of Natural Attenuation process.
Currently, can be degraded by anaerobe using reductive dehalogenation bacterium assessment chlorobenzene class organic contamination place with generation Natural Attenuation
There is not been reported for appraisal procedure.
Summary of the invention
In view of the above-mentioned problems, the present invention provides a kind of occurring by anaerobe degradation for chloro- benzenes contamination place
The technology evaluation method of Natural Attenuation.
To test above-mentioned goal of the invention, the technical solution adopted by the present invention are as follows:
The appraisal procedure of Natural Attenuation can occur by anaerobe degradation for a kind of chlorobenzene class organic contamination place, including
Following steps:
(1) place is investigated first, specifies pollutant range, then carry out ground to Polluted areas different in contaminated site
The acquisition of lower water sample, pickup area include pollution in a steady stream with Pollution Plume region (i.e. pollutant with Groundwater movement area
Domain) and the pollution-free region of background, it is as shown in Figure 1 to sample range；Wherein, there may be high concentrations for pollution source area and Pollution Plume region
Contaminant degradation bacterium, the sample of each Polluted area has been both needed to 3 Duplicate Samples, and every part of groundwater sample at least contains the underground 1L
Water.
(2) groundwater sample is centrifuged 15 minutes with the speed of 10000g, abandons supernatant, collect centrifuge tube bottom deposit
Object carries out DNA extraction；Or aseptic filter membrane filtering underground sample is used directly in place, abandon filtered underground
Water collects filter membrane and carries out DNA extraction.
Above-mentioned DNA extraction step is this field routine techniques, and MO BIO Laboratories company can be usedDNA Isolation Kit kit or other DNA extraction methods generally in the art.
(3) it is ready to containing Dehalobacter, Dehalogenimonas and Dehalococcoides 16S rRNA base
The plasmid of cause carries out qPCR experiment as table sample,
(4) prepare primer as shown in table 1, bacterial strain Dehalococcoides upstream primer 1f (its nucleotide sequence such as SEQ
Shown in ID NO.1)；Downstream primer 264r (its nucleotide sequence is as shown in SEQ ID NO.2)；The upstream bacterial strain Dehalobacter
Primer 477f (its nucleotide sequence is as shown in SEQ ID NO.3), downstream primer 647r (its nucleotide sequence such as SEQ ID
Shown in NO.4)；Bacterial strain Dehalogenimonas upstream primer 273f (its nucleotide sequence is as shown in SEQ ID NO.5), downstream
Primer 537r (its nucleotide sequence is as shown in SEQ ID NO.6).
1 Chlorobenzens microorganism remediation of table tests qPCR and tests primer
Dehalobacter bacterial strain, Dehalogenimonas bacterial strain and Dehalococcoides bacterial strain be this field
Open bacterial strain, wherein primer involved in table 1 can be found in following documents disclosure: 1) Dehalococcoides primer is joined
Examining document is Hendrickson, E.R.；Payne,J.A.；Young,R.M.；Starr,M.G.；Perry,M.P.；
Fahnestock,S.；Ellis,D.E.；Ebersole,R.C.,Molecular analysis of Dehalococcoides
16S ribosomal DNA from chloroethene-contaminated sites throughout North
America and Europe.Appl.Environ.Microbiol.2002,68, (2), 485-495 and Grostern, A.；
Edwards,E.A.,Characterization of a Dehalobacter coculture that dechlorinates
1,2-dichloroethane to ethene and identification of the putative reductive
dehalogenase gene.Appl.Environ.Microbiol.2009,75,(9),2684-93.12。
2) bibliography of Dehalobacter primer is Grostern, A.；Edwards,E.A.,Growth of
Dehalobacter and Dehalococcoides spp.during degradation of chlorinated ethan
es.Appl.Environ.Microbiol.2006,72,(1),428-436.13.3) reference of Dehalogenimonas primer
Document is Manchester, M.J.；Hug,L.A.；Zarek,M.；Zila,A.；Edwards,E.A.,Discovery of a
trans-dichloroethene-respiring Dehalogenimonas species in the 1,1,2,2-
tetrachloroethane-dechlorinating WBC-2consortium.Appl.Environ.Microbiol.2012,
78,(15),5280-7.
Above-mentioned nucleotide sequence primer as shown in SEQ ID NO.1-SEQ ID NO.6 can also be bought by commercial sources
(such as sigma company).
(5) qPCR reaction is carried out, each reaction must be provided with 2-3 Duplicate Samples, and all operations are all in aseptic operating platform
It is carried out on (bibliography: ESCO Technologies, Hatboro, PA), the substance such as the following table 2 for being included in each reactant
It is shown；
2 Chlorobenzens microorganism remediation of table tests qPCR reaction system
Involved water is the ultrapure water without nucleic acid in the application.
(6) it is as shown in table 3 to react temperature program setting by qPCR.QPCR tests instrument reference: PTC-200Peltier
Thermal Cycler(MJ Research Inc.,Waltham,MA).Wherein, Tm reference table 1.
3 Chlorobenzens microorganism remediation of table tests qPCR response procedures
(7) pollution source area, Pollution Plume region and background area can get by the above experimental procedure
The quantity of Dehalobacter, Dehalogenimonas and Dehalococcoides, it is possible thereby to obtain different Polluted areas
Chlorobenzens anaerobic reduction dehalogenation bacterium number amount and its variation tendency: if the dehalogenation bacterium of pollution source area and Pollution Plume region
The quantity of (Dehalobacter, Dehalogenimonas and Dehalococcoides) an order of magnitude higher than background area,
Or the total quantity of dehalogenation bacterium is higher than 104A/L underground water then illustrates there are the potentiality for carrying out anaerobe reparation in place
(USA Department of defense,Application of Nucleic Acid-Based Tools for
Monitoring Monitored Natural Attenuation(MNA),Biostimulation,and
Bioaugmentation at Chlorinated Solvent Sites,January 2011)；If the quantity of dehalogenation bacterium is higher than
106A/L underground water illustrates reduction dechlorination reaction (the USA Department of that Chlorobenzens are certainly existed in place
defense,Application of Nucleic Acid-Based Tools for Monitoring Monitored
Natural Attenuation(MNA),Biostimulation,and Bioaugmentation at Chlorinated
Solvent Sites, January 2011), as shown in table 4；Otherwise, then it is assumed that the contaminated site, which does not have, utilizes indigenous anaerobism
The feasibility of microbiological deterioration generation Natural Attenuation.
Relationship in 4 underground water of table between dehalogenation bacterium number amount and anaerobe reparation
The present invention is based on Protocols in Molecular Biology means, the chloro- benzenes contamination object in determining place is provided to fast, economical
Whether the appraisal procedure of Natural Attenuation can be occurred by anaerobe degradation.Under anaerobic condition, dehalogenation bacterium is with organochlorine
Substance is that final electron acceptor carries out dehalogenation respiration acquisition energy, to maintain cell to grow or movement.By the end of currently,
Known Chlorobenzens reductive dehalogenation bacterium only have Dehalobacter, Dehalogenimonas and Dehalococcoides this
Three Pseudomonas.Present invention firstly provides using in Protocols in Molecular Biology quantitative PCR (qPCR) detection contaminated site underground water
The quantity of Dehalobacter, Dehalogenimonas and Dehalococcoides, whether to assess chloro- benzenes contamination place
It can occur Natural Attenuation by anaerobe degradation, it is visual result, accurate, it is suitable for the use of Contamination Assessment place.
Detailed description of the invention
Fig. 1 is that polluted underground water samples schematic diagram.
Fig. 2 is Nanjing six directions contaminated site plane figure；
In Fig. 2, with representing slag, BR represents boiler room, the production of F1 generation table 2-nitro-paracresol and adjacent amino paracresol to SP
Workshop, F2 represent the workshop of fluorescent whitening agent PF, and WWB represents wastewater disposal basin, and CWB represents circulating water pool, and LAT represents liquid alkaline
Tank, SH represent warehouse.
Fig. 3 is each pollutant concentration 2012-2016 concentration at peephole S1；
In Fig. 3, TCB, DCB, MCB and Benzene are trichloro-benzenes, dichloro-benzenes, chlorobenzene and benzene respectively.
Wherein, 1, pollution sources；2, sample point；3, Pollution Plume, 4, ground, 5, groundwater level/phreatic surface；6, phreatic water
Flow direction；7, deeper subsurface water flow direction.
Specific embodiment
Below by way of specific embodiment, technical scheme is further illustrated.
Embodiment 1
Whether it is Nanjing Liuhe District waste pollution place that the present embodiment, which assesses target, assess the place and can be implemented and detest
Oxygen animalcule restorative procedure.
For the place plane figure as shown in Fig. 2, the plant area north is primary producing region, the south is Office Area.Long-term place tune
Look into the results show that Polluted area is concentrated mainly on raw wastewater pool area, pollutant 1,2,4- trichloro-benzenes, dichloro-benzenes, chlorobenzene and
Benzene.The contaminated site anaerobic condition is the silty clay medium of infiltration coefficient very small (0.002-0.007 meters/day), pollution
Object is concentrated mainly on phreatic aquifer, covers one layer of continuous clay on phreatic surface.It follows that can ignore due to underground water
Pollutant concentration caused by the diluting effects such as flowing, rainfall is lower.The contaminated site has been fallen into disuse 3 years before the first sub-sampling,
Therefore ignore anaerobic condition to organic matter absorption and caused by material concentration reduce.Therefore effective restorative procedure is not carried out in judgement
Before, the reduction of the regional pollution object concentration is mainly caused by microbial degradation obtains.
For the contaminated site, it is as follows to assess the specific implementation step that the whether implementable anaerobe in place is repaired:
In June, 1.2012, pollution-free region W4 point takes three parts of undergrounds 1L respectively outside contaminated area S1 point and place
Water removes supernatant after offline 10000 × g in laboratory, uses MO BIO Laboratories company
DNA Isolation Kit kit carries out DNA extraction.
2. respectively for three kinds of Chlorobenzens dehalogenation bacterium Dehalococcoides (positive strand primer and negative strand primer difference
As shown in SEQ ID NO.1 and SEQ ID NO.2), (positive strand primer and negative strand primer are respectively such as SEQ ID by Dehalobacter
Shown in NO.3 and SEQ ID NO.4) and Dehalogenimonas (positive strand primer and negative strand primer respectively such as SEQ ID NO.5 and
Shown in SEQ ID NO.6) carry out qPCR experiment.
QPCR reaction system (20 μ L): 10 μ L of Eva Green, positive strand primer (10 μM) 1 μ L, negative strand primer (10 μM) 1 μ
L, 2 μ L of DNA profiling, Yi Shui (ultrapure water without nucleic acid) supply surplus；
QPCR response procedures are as shown in table 3:
3 Chlorobenzens microorganism remediation of table tests qPCR response procedures
The quantity of three kinds of dehalogenation bacterium is below detection limit 10 at W4 point as the result is shown3A/(L underground water), and contaminated area
The dehalogenation bacterium number amount of S1 point is 104-105Between a/(L underground water), as shown in table 5.
5 contaminated area S1 point Chlorobenzens dehalogenation bacterium number amount of table
3. by qPCR result it is found that Polluted area (S1 point) Chlorobenzens dehalogenation bacterium number amount is higher than 104A/(underground L
Water), determine there are the potentiality for carrying out anaerobe reparation in the place in conjunction with table 4.
4. after in June, 2012 is using Chlorobenzens dehalogenation bacterium in qPCR detection underground water, in Septembers, 2014 and 2016
October in year extracts underground water at S1 again, and using headspace gas chromatography, (Zhou Ni, Qiao Wenjing, Ye Shujun .1,2,4- trichloro-benzenes are detested
Hydrogen reduction dechlorination process and fluorescent whitening agent PF influence research [J] 2018.38 (10): 3954-3963 to dechlorination process), detection
Pollutant concentration at hole, as a result as shown in Figure 3.The contaminated site is from after 2012 and any underground Organic substance in water is not carried out
Recovery technique, and pollutant concentration highest at 2012 in underground water at peephole S1 in Fig. 3, are being not carried out any reparation skill
In the case of art, each pollutant concentration reduces at any time, is finally eventually declined to defined level of security at 2016.Consider
Microbial degradation activity to the hydrogeologic condition of the contaminated site, only anaerobism can explain the reduction of this concentration.
5. therefore, for Nanjing six directions contaminated site, by being carried out to the soil and groundwater sample in contaminated site
Real-time quantitative PCR (qPCR) in fact detect, obtain be currently known all Chlorobenzens reductive dehalogenation bacterium (including
Dehalobacter, Dehalogenimonas and Dehalococcoides) quantity, fast, economical determine place in chlorine
Whether benzene pollutant can carry out anaerobe reparation.
Sequence table
<110>Nanjing University
<120>appraisal procedure of Natural Attenuation can occur by anaerobe degradation for chlorobenzene class organic contamination place
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gatgaacgct agcggcg 17
<210> 2
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cctctcagac cagctaccga tcgaa 25
<210> 3
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gattgacggt acctaacgag g 21
<210> 4
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tacagtttcc aatgctttac g 21
<210> 5
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tagctcccgg tcgcccg 17
<210> 6
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
cctcaccagg gtttgacatg ttagaag 27
Claims (3)
1. the appraisal procedure of Natural Attenuation can occur by anaerobe degradation for a kind of chlorobenzene class organic contamination place,
It is characterized in that, the specific steps are as follows:
(1) it acquires Polluted area groundwater sample in contaminated site and extracts sample DNA；
The Polluted area includes pollution sources region, background is pollution-free region and Pollution Plume region；
(2) qPCR reaction is carried out to the sample DNA that step (1) is extracted, expanded respectivelyDehalobacterBacterial strain,DehalogenimonasBacterial strain andDehalococcoidesBacterial strain；Simultaneously with known concentrationDehalobacterBacterial strain,DehalogenimonasBacterial strain andDehalococcoidesThe standard specimen that the 16S rRNA gene plasmid of bacterial strain is tested as qPCR；
Obtain total bacterium,DehalobacterBacterial strain、DehalogenimonasBacterial strain andDehalococcoidesThe quantity of bacterial strain；
Wherein,DehalobacterThe positive strand primer and negative strand primer of bacterial strain are respectively such as SEQ ID NO.1 and SEQ ID NO.2 institute
Show；DehalogenimonasThe positive strand primer and negative strand primer of bacterial strain are respectively as shown in SEQ ID NO.3 and SEQ ID NO.4；DehalococcoidesThe positive strand primer and negative strand primer of bacterial strain are respectively as shown in SEQ ID NO.5 and SEQ ID NO.6；
(3) to total bacterium,DehalobacterBacterial strain、DehalogenimonasBacterial strain andDehalococcoidesBacterial strain quantity
Assessment, if in pollution source area and Pollution Plume regionehalobacterBacterial strain、DehalogenimonasBacterial strain andDehalococcoidesThe total quantity of bacterial strain is higher than 104A/L underground water then illustrates there is progress anaerobe reparation in place
Potentiality；If in pollution source area and Pollution Plume regionehalobacterBacterial strain、DehalogenimonasBacterial strain andDehalococcoidesThe total quantity of bacterial strain is higher than 106A/L underground water, then illustrate the reduction in place there are Chlorobenzens
Dechlorination reaction.
2. by anaerobe degradation nature can occur for a kind of chlorobenzene class organic contamination place according to claim 1
The appraisal procedure of decaying, which is characterized in that in step (1), in the contaminated area, under each region at least acquires 3 in parallel
Water sample, every part of groundwater sample at least contain 1L underground water.
3. by anaerobe degradation nature can occur for a kind of chlorobenzene class organic contamination place according to claim 1
The appraisal procedure of decaying, which is characterized in that step (2) the qPCR reaction system includes: 10 μ L of Eva Green, 10 μM
1 μ L of positive strand primer, 10 μM of 1 μ L of negative strand primer, 2 μ L of DNA profiling, Yi Shui complement to 20 μ L；
QPCR response procedures are as follows: 98 DEG C of preheating 2min；98 DEG C of heating 5s, Tm value temperature heat 10s, 65 DEG C of extension 10s, repeat 39
It is secondary；4 DEG C of preservations.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113772804A (en) * | 2021-09-15 | 2021-12-10 | 宝航环境修复有限公司 | Groundwater pollution monitoring natural attenuation restoration prediction method, system and device |
| CN114354786A (en) * | 2021-12-25 | 2022-04-15 | 生态环境部南京环境科学研究所 | Method for analyzing benzene series spatial distribution of underground water in polluted site based on pollution plume |
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2019
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Non-Patent Citations (6)
| Title |
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| ARIEL GROSTERN,ET AL: "Characterization of a Dehalobacter Coculture That Dechlorinates 1,2-Dichloroethane to Ethene and Identification of the Putative Reductive Dehalogenase Gene", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| CARMEN LEBRON: "Application of Nucleic Acid-Based Tools for Monitoring Monitored Natural Attenuation（MNA），Biostimulation，and Bioaugmentation at Chlorinated Solvent Sites", 《ENGINEERING SERVICE》 * |
| JENNIFER L. NELSON，ET AL: "Dehalogenation of Chlorobenzenes, Dichlorotoluenes, and Tetrachloroethene by Three Dehalobacter spp.", 《ENVIRONMENTAL SCIENCE&TECHNOLOGY》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113772804A (en) * | 2021-09-15 | 2021-12-10 | 宝航环境修复有限公司 | Groundwater pollution monitoring natural attenuation restoration prediction method, system and device |
| CN114354786A (en) * | 2021-12-25 | 2022-04-15 | 生态环境部南京环境科学研究所 | Method for analyzing benzene series spatial distribution of underground water in polluted site based on pollution plume |
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