CN109913563B - Evaluation method for natural attenuation of chlorobenzene organic pollution site through anaerobic microorganism degradation - Google Patents
Evaluation method for natural attenuation of chlorobenzene organic pollution site through anaerobic microorganism degradation Download PDFInfo
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Abstract
The invention discloses an evaluation method for natural attenuation of chlorobenzene organic polluted sites through anaerobic microbial degradation, which is characterized in that real-time quantitative PCR (qPCR) detection is carried out on underground water samples in the polluted sites to obtain the absolute quantity of all currently known chlorobenzene substance reduction dehalogenation bacteria (including dehalogacter, Dehalogenimonas and dehaococcoides), and whether chlorobenzene pollutants in the sites can be naturally attenuated through anaerobic microbial degradation is rapidly and economically determined.
Description
Technical Field
The invention relates to the field of groundwater pollution remediation, in particular to an assessment method for natural attenuation of chlorobenzene organic pollution sites through anaerobic microorganism degradation.
Background
Underground water is an important strategic resource in China and plays an irreplaceable role in maintaining the healthy development of urban economy and society and the like. 65 percent of domestic water, 50 percent of industrial water and 33 percent of agricultural irrigation water in northern areas of China come from underground water, and in 655 cities in the country, about 400 cities use the underground water as drinking water sources (Ministry of environmental protection, national underground water pollution prevention and control program, 2011). In recent years, with the rapid development of economic society, the environmental pressure of underground water is gradually increased, the underground water of main cities in China, especially the underground water of shallow layers, is polluted to different degrees (gorgeous, handsome 2011. 69 studies on the characteristics of organic pollution of the urban underground water in China. the globe bulletin [ J ],32: 581-containing 591), and the pollution is developed from local expansion to regional, recessive to dominant, inorganic pollution to organic pollution. Groundwater pollution has the characteristics of durability, concealment, complexity, difficulty in treatment and the like, poses great threats to ecology and human health (QIU J2010, China faces up to group water crisis. Nature [ J ],466:308), and is highly concerned by researchers and government agencies at home and abroad. However, groundwater pollution remediation in China is still in the initial stage, and various economic, effective, green and environment-friendly remediation technologies are urgently needed to be developed.
Chlorobenzene is a chemical substance formed by replacing hydrogen atoms on a benzene ring with chlorine atoms, and 12 chlorobenzene substances are available in total according to the quantity of substituted chlorine and the position on the benzene ring. The organic matters are used as chemical raw materials and intermediates, and are widely applied to the industries of dye, medicine, pesticide, rubber and electrical products (Wang Fang, DRFLER U, SCHMID M, et al.2007.1,2, 4-trichlorobenzene mineralized bacteria identification and functional analysis. environmental science [ J ],28: 1082-. Due to the large amount of use and unreasonable treatment, chlorobenzene pollutants are detected in soil, underground water, sediments, vegetables and other environmental media (Song, Wang Fang, Jianxin 2011. research progress on microbial degradation of 1,2, 4-trichlorobenzene. soil [ J ],43: 343-. In addition, hexachlorobenzene (DOESBURG W, EEKERT M H, MIDDELDORP P J, et al 2005. reduced dechlorination of beta-hexachlorocyclohexane (beta-HCH) by a dehalogenator species in culture with a section reactor 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 of gamma-hexachlorocyclohexane Sci Technol [ J ],37: 1822-. The pollutants are also common pollutants in soil and underground water of industrial sites in China (natural, congratulatory, forest jade lock and the like 2015. monitoring natural attenuation restoration initial exploration of chlorine organic polluted sites and chemical engineering report [ J ],66: 2361-. The chlorobenzene substances have stable chemical properties, can be retained in the environment for a long time, are easy to be biologically enriched, can cause the damage of the liver and the kidney of a human, have potential carcinogenicity, and pose a threat to the ecological environment and the health of a human body.
Therefore, the restoration of the chlorobenzene polluted site has important practical significance. In the existing repair technology, the natural attenuation method has the advantages of low operation cost, high benefit, small site disturbance, capability of being used as a long-term repair technology for low-concentration polluted sites and the like, and is increasingly paid more attention. Microbial degradation is the most important, destructive, natural attenuation. Microbial degradation is generally considered to be critical to the success of natural decay methods. Aerobic microbial remediation is generally limited by several factors: 1) the oxygen content of underground water is low; 2) a small amount of oxygen dissolved in underground water is easily degraded by aerobic degradation bacteria and molecules with simple structures are quickly consumed; 3) sufficient oxygen is pumped into underground water, so that the engineering is complex and the cost is high; 4) chlorobenzene substances belong to heavy non-aqueous phase organic matters and are easy to migrate downwards to a deep anaerobic confined aquifer under the action of gravity. Therefore, anaerobic microbial degradation is critical in the natural attenuation of chlorobenzene-contaminated sites. Then, when the natural attenuation method by the degradation by anaerobic microorganisms is carried out, the contaminated site is first evaluated for the feasibility of the degradation by anaerobic microorganisms. Before in-situ anaerobic microbial remediation, microbial remediation feasibility assessment needs to be carried out on a polluted site.
Currently, the method published in 1998 (Technical Protocol for Evaluating Natural Attenuation of Chlorinated solutions in group Water) is being evaluated for Natural Attenuation of a field. The method suggests confirming the occurrence of natural decay of the field from evidence in three areas: 1) the environment geochemical index representing the natural attenuation process; 2) historical monitoring data shows that the content of pollutants tends to decrease, and the decrease of the concentration of the pollution plume of the underground water is not only caused by the migration of the pollution plume; 3) the occurrence of the natural decay process is directly proved by a microcosm experiment. At present, an evaluation method for evaluating natural attenuation of chlorobenzene organic polluted sites through anaerobic microorganism degradation by using reductive dehalogenation bacteria is not reported.
Disclosure of Invention
In order to solve the problems, the invention provides a technical evaluation method for natural attenuation of chlorobenzene polluted sites through anaerobic microbial degradation.
For the purpose of experiment, the invention adopts the technical scheme that:
a method for evaluating natural attenuation of chlorobenzene organic polluted sites through anaerobic microbial degradation comprises the following steps:
(1) firstly, investigating a field, defining the range of pollutants, and then collecting underground water samples in different polluted areas in the polluted field, wherein the collection areas comprise a pollution source, a pollution plume area (namely an area where the pollutants move along with underground water) and a background pollution-free area, and the sampling range is shown in figure 1; wherein, pollutant degrading bacteria with high concentration may exist in the pollution source area and the pollution plume area, each sample in the pollution area needs 3 parallel samples, and each groundwater sample at least contains 1L of groundwater.
(2) Centrifuging the underground water sample at 10000g for 15 minutes, discarding supernatant, and collecting sediment at the bottom of a centrifuge tube for DNA extraction; or directly filtering the underground water sample in the field by using a sterile filtering membrane, discarding the filtered underground water, and collecting the filtering membrane for DNA extraction.
The above DNA extraction step is a conventional technique in the art, and may be carried out by MO BIO Laboratories
DNA Isolation Kit, or a Kit thereofThe DNA extraction methods commonly used in the art.
(3) Plasmids containing Dehalobacter, Dehalogenimonas and Dehalococcoides 16S rRNA genes were prepared, and qPCR experiments were performed as surface samples,
(4) preparing primers shown in Table 1, strain Dehalococcoides upstream primer 1f (the nucleotide sequence is shown in SEQ ID NO. 1); a downstream primer 264r (the nucleotide sequence of which is shown as SEQ ID NO. 2); the strain Dehalobacter upstream primer 477f (the nucleotide sequence is shown in SEQ ID NO. 3) and the strain Dehalobacter downstream primer 647r (the nucleotide sequence is shown in SEQ ID NO. 4); the strain Dehalogenimonas upstream primer 273f (the nucleotide sequence is shown in SEQ ID NO. 5) and the strain Dehalogenimonas downstream primer 537r (the nucleotide sequence is shown in SEQ ID NO. 6).
TABLE 1 Chlorobenzene substance microbial remediation experiment qPCR experiment primer
The strains Dehalobacter, Dehalogenimonas and Dehalococcoides are all strains disclosed in the art, wherein the primers referred to in Table 1 can be found in the following publications: 1) the dehalocccoides primer reference 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 dehalocccoides 16S ribosomal DNA from chloroethylene-conjugated sites through northern America and europe.app. environ. microbiol.2002,68, (2), 485-; edwards, E.A., Characterisation of a Dehalobacterium focus at dechlorinated 1, 2-dichloethane to ethene and identification of the reactive reductive gene, application, environ, Microbiol.2009,75, (9), 2684-93.12.
2) References to dehaobacter primers are Grostern, a.; edwards, E.A., Growth of dehalogacter and dehaloglycoides sp.during degradation of chlorinated ethanes.appl.environ.Microbiol.2006,72, (1), 428-436.13. 3) References to Dehalogenimonas primers are Manchester, m.j.; hug, l.a.; zarek, M.; zila, a.; edwards, E.A., Discovery of a trans-dichloro-lubricating WBC-2consortium, appl.environ.Microbiol.2012,78, (15),5280-7.
Primers having the nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.6 can also be purchased commercially (e.g., sigma).
(5) The qPCR reactions were performed, each reaction had to be set up in 2-3 replicates, all performed on a sterile operating table (ref: ESCO Technologies, Hatboro, Pa.), and the substances contained in each reaction are shown in Table 2 below;
TABLE 2 Chlorobenzene substance microbiological repair experiment qPCR reaction System
The water referred to in this application is ultrapure water containing no nucleic acid.
(6) The qPCR reaction temperature control program settings are shown in table 3. Instrument reference for qPCR experiments: PTC-200Peltier Thermal Cycler (MJ Research Inc., Waltham, Mass.). Wherein, Tm is shown in Table 1.
TABLE 3 Chlorobenzene-based microbial remediation experiment qPCR reaction procedure
(7) The numbers of Dehalobacter, Dehalogenimonas and Dehalococcoides in a pollution source region, a pollution plume region and a background region can be obtained through the experimental steps, so that the number of chlorobenzene substances in different pollution regions for anaerobic reduction dehalogenation and the change trend thereof can be obtained: if the number of dehalogenation bacteria (Dehalobacter, Dehalogenimonas and Dehalococcoides) in the contaminated source region and the contaminated feather region is one order of magnitude higher than that in the background region, or the total number of dehalogenation bacteria is higher than 104The individual/L groundwater indicates the potential for anaerobic microbial remediation in the field (USA Department of defense, Application of Nucleic acid)Acid-Based Tools for Monitoring Monitored Natural Attention (MNA), biostimation, and bioauthentication at Chlorinated Solvent Sites, January 2011); if the number of dehalogenation bacteria is more than 106groundwater/L, indicating that reductive dechlorination of chlorobenzene-Based substances must be present in the field (USA Department of damage, Application of Nucleic Acid-Based Tools for Monitoring national institutes (MNA), Biostimulation, and bioauthentication at Chlorinated solvents, January 2011), as shown in Table 4; otherwise, the contaminated site is considered to have no feasibility of natural attenuation by indigenous anaerobic microbial degradation.
TABLE 4 relationship between number of dehalogenating bacteria in groundwater and anaerobic microbial remediation
The invention provides an evaluation method for determining whether chlorobenzene pollutants in a field can be naturally attenuated through anaerobic microorganism degradation based on a molecular biology technical means. Under the anaerobic condition, the dehalogenation bacteria take an organic chlorine substance as a final electron acceptor to perform dehalogenation respiration to obtain energy so as to maintain the growth or movement of cells. Up to now, only three genera, Dehalobacter, Dehalogenimonas and Dehalococcoides, have been known as chlorobenzene-reducing dehalogenation bacteria. The invention firstly proposes that the quantity of Dehalobacter, Dehalogenimonas and Dehalococcoides in underground water of a polluted site is detected by utilizing molecular biology technology quantitative PCR (qPCR) to evaluate whether the chlorobenzene polluted site can be naturally attenuated through anaerobic microorganism degradation, and the result is visual and accurate and is suitable for the pollution evaluation site.
Drawings
FIG. 1 is a schematic view of a sample of contaminated groundwater.
FIG. 2 is a layout view of a ground plane of a pollution field of Nanjing Liuhe;
in FIG. 2, SP represents slag, BR represents boiler room, F1 represents a production plant for o-nitro-p-cresol and o-amino-p-cresol, F2 represents a production plant for fluorescent whitening agent PF, WWB represents a wastewater tank, CWB represents a circulating water tank, LAT represents a caustic soda tank, and SH represents a warehouse.
FIG. 3 shows the concentration of each contaminant at well S1 in year 2012-2016;
in FIG. 3, TCB, DCB, MCB and Benzene are trichlorobenzene, dichlorobenzene, chlorobenzene and Benzene, respectively.
Wherein, 1, a pollution source; 2. sampling points; 3. pollution feather, 4, ground, 5, underground water surface/diving surface; 6. shallow groundwater flow direction; 7. deep underground water flow direction.
Detailed Description
The technical solution of the present application is further described below by specific examples.
Example 1
In this example, the evaluation target was a waste contaminated site in the Liuhe district of Nanjing City, and whether the site can be subjected to the anaerobic microorganism remediation method was evaluated.
The floor plan layout is shown in fig. 2, the north side of the factory area is the original production area, and the south side is the office area. Long-term site survey results show that the polluted area is mainly concentrated in the original wastewater pool area, and the pollutants are 1,2, 4-trichlorobenzene, dichlorobenzene, chlorobenzene and benzene. The aquifer medium of the polluted site is a silty clay medium with very small permeability coefficient (0.002-0.007 m/day), pollutants are mainly concentrated in the diving aquifer, and a layer of continuous clay is covered on the diving surface. It can be seen that the low concentration of contaminants due to dilution effects such as groundwater flow and rainfall can be ignored. The contaminated site was wasted for 3 years before the first sampling, so the decrease in concentration of the material due to the adsorption of the organic material by the aquifer medium was ignored. It was therefore judged that the reduction in the concentration of contaminants in this area was mainly due to the acquisition of microbial degradation before effective remediation methods were carried out.
Specific implementation steps for evaluating whether the site can be subjected to anaerobic microorganism remediation or not are as follows:
in 6 months in 1.2012, taking three 1L underground water from the S1 point of the polluted area and the W4 point of the pollution-free area outside the site, taking off the water in a laboratory at 10000 Xg, removing the supernatant, and using MOBIO Laboratories Inc
DNA Isolation Kit for DNA extraction.
2. qPCR experiments were performed on three kinds of chlorobenzene dehalogenation bacteria Dehalococcides (the forward and reverse primers are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively), Dehalobacter (the forward and reverse primers are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively), and Dehalogenimonas (the forward and reverse primers are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively).
qPCR reaction (20 μ L): eva Green 10. mu.L, plus strand primer (10. mu.M) 1. mu.L, minus strand primer (10. mu.M) 1. mu. L, DNA template 2. mu.L, the balance being water (ultrapure water containing no nucleic acid);
the qPCR reaction program is shown in table 3:
TABLE 3 Chlorobenzene-based microbial remediation experiment qPCR reaction procedure
The results show that the number of three dehalogenation bacteria at the W4 point is less than the detection limit of 103The number of the dehalogenation bacteria at the S1 point of the pollution area is 10/(L of underground water)4-105counts/(L groundwater) as shown in Table 5.
TABLE 5 contaminated site S1 number of dehalogenating bacteria of chlorobenzene substances
3. From the results of qPCR, it was found that the number of the dehalogenating bacteria of chlorobenzene substances in the contaminated area (S1 point) was more than 104and/(L groundwater), and the potential for anaerobic microbial remediation in the field is judged in combination with table 4.
4. After the chlorobenzene dehalogenation bacteria in the groundwater were detected by qPCR in 6 months of 2012, the groundwater was extracted at S1 in 9 months of 2014 and 10 months of 2016, and the concentrations of the pollutants at the pores were detected by headspace gas chromatography (Zhou Ni, Joweng Jing, leaf gentlemen.1, 2, 4-trichlorobenzene anaerobic reduction dechlorination process and fluorescent brightener PF influence study on dechlorination process [ J ]2018.38(10): 3954-. The contaminated site has not been subjected to any organic remediation technology for groundwater since 2012, whereas the concentration of contaminants in groundwater at observation hole S1 in fig. 3 was highest at 2012, and without any remediation technology, the concentration of each contaminant decreased with time and finally decreased to a prescribed safe level at 2016. Given the hydrogeological conditions of the contaminated site, only anaerobic microbial degradation activity could account for this reduction in concentration.
5. Therefore, aiming at a certain polluted site of Nanjing Liuhe, real-time quantitative PCR (qPCR) real detection is carried out on soil and underground water samples in the polluted site, the quantity of all currently known chlorobenzene substance reduction dehalogenation bacteria (including Dehalobacter, Dehalogenimonas and Dehalococcides) is obtained, and whether the chlorobenzene pollutants in the site can be subjected to anaerobic microbial remediation or not is rapidly and economically determined.
Sequence listing
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Claims (3)
1. An assessment method for natural attenuation of chlorobenzene organic polluted sites through anaerobic microorganism degradation is characterized by comprising the following specific steps:
(1) collecting underground water samples of a pollution area in a pollution site and extracting DNA of the samples;
the pollution area comprises a pollution source area, a background pollution-free area and a pollution plume area;
(2) carrying out qPCR reaction on the sample DNA extracted in the step (1), and respectively amplifyingDehalobacterBacterial strain,DehalogenimonasStrains andDehalococcoidesa strain;while at a known concentrationDehalobacterBacterial strain,DehalogenimonasStrains andDehalococcoidesthe 16S rRNA gene plasmid of the strain is used as a standard sample of a qPCR experiment; obtaining the total bacteria,DehalobacterBacterial strains、DehalogenimonasStrains andDehalococcoidesthe number of strains;
wherein the content of the first and second substances,Dehalobacterthe positive strand primer and the reverse strand primer of the strain are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;Dehalogenimonasthe positive strand primer and the reverse strand primer of the strain are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4;Dehalococcoidesthe positive strand primer and the reverse strand primer of the strain are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6;
(3) for the total bacteria,DehalobacterBacterial strains、DehalogenimonasStrains andDehalococcoidesevaluating the number of strains, if the strains pollute a source region and a pollution plume regionehalobacterBacterial strains、DehalogenimonasStrains andDehalococcoidesthe total number of strains is more than 104The groundwater/L indicates that the field has the potential for anaerobic microorganism remediation; if the pollution source area and the pollution plume area are pollutedehalobacterBacterial strains、DehalogenimonasStrains andDehalococcoidesthe total number of strains is more than 106And (4) indicating that the chlorobenzene substances exist in the site for reductive dechlorination by using one/L of underground water.
2. The method for evaluating the natural attenuation of chlorobenzene organic polluted site through anaerobic microbial degradation according to claim 1, wherein in the polluted area in the step (1), at least 3 parallel groundwater samples are collected in each area, and each groundwater sample contains at least 1L of groundwater.
3. The method for evaluating the natural attenuation of chlorobenzene organic polluted sites through anaerobic microbial degradation according to claim 1, wherein the qPCR reaction system in the step (2) comprises: eva Green 10. mu.L, 10. mu.M plus strand primer 1. mu.L, 10. mu.M minus strand primer 1. mu. L, DNA template 2. mu.L, made up to 20. mu.L with water;
the qPCR reaction procedure was: preheating at 98 deg.C for 2 min; heating at 98 deg.C for 5s, heating at Tm value for 10s, extending at 65 deg.C for 10s, and repeating 39 times; preserving at 4 ℃.
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