CN102507885B - Method for forecasting comprehensive quality of complex water environment by applying number of microorganisms in bottom sediment - Google Patents
Method for forecasting comprehensive quality of complex water environment by applying number of microorganisms in bottom sediment Download PDFInfo
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Abstract
A method for forecasting the comprehensive quality of a complex water environment by applying the number of microorganisms in bottom sediment belongs to the field of water treatment. Types of advantageous bacteria in the bottom sediment are identified by means of PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis), the quantitative response relation between the number of the types of the advantageous bacteria in the bottom sediment of a surface layer and a subsurface layer and the comprehensive quality of the water environment is finally found, a traditional wide gap between microscopic molecular biology research and macroscopic water environment quality evaluation is bridged, and the method is applicable to forecasting and evaluating the comprehensive quality of the complex water environment with various water bodies and various contamination degrees. Besides, according to related national standards of Chinese water environment quality, a quantitative/qualitative evaluation mode for the comprehensive quality of the complex water environment is built for reference, and the reference can also be independently used for forecasting the comprehensive quality of the water environment.
Description
Technical field
The present invention relates to water treatment field, specifically apply the method for bottom mud microbe quantity forecast Complex Water Environment overall quality.
Background technology
Physico-chemical method is the main method of monitoring water environment and evaluation, the content that traditional chemistry or physical index can quantitative test principal ingredients, but be difficult to reflect directly, all sidedly the combined influence of these compositions.From the eighties in 20th century so far, biological indicator is applied increasingly extensive in water quality evaluation, and the feature long with life cycle, sensitivity is high, combination property is strong and become indispensable, even standardized water quality assessment and monitoring index.So far, fish, periphyton and the macroinvertebrate being most widely used have formed main water quality assessment and monitoring bio monoid, although these biologies are monitored to the variation that can reflect more truly, all sidedly water environment, exist time lag, be difficult to quantize, disturb the defects such as biological factor is many.Thereby widely distributed, more responsive water environment microorganism monitors and passes judgment on the overall quality of Complex Water Environment in necessary employing.
In aquatic ecosystem, microorganism is analyst and the conversion person of nutrition/polluter, is again the storage person of material/energy, while or the important producer of food chain.Water environment microorganism is the most responsive to quality of water environment, affected by it, the kind of water environment microorganism and composition can change along with the pollution level of water environment: general quality of water environment is degenerated or impaired the kind to microorganism, quantity had a negative impact, but some patience microbe species quantity after contaminated environment may increase on the contrary.In general, the microorganism that is suspended in water body can only characterize the instantaneous mass of water environment; And stable bed mud is the terminal storage storehouse of various polluters, can provide long-term, a diverse ecological niche for microorganism, therefore bottom mud microbe can react the quality of water environment of long term, and application bottom mud microbe can play better indicative function to the overall quality/pollution situation of water environment.
Traditional microorganism (form) taxonomy need to separately carry out laboratory pure culture by different types of microorganism, the possibility of result only has the aerobic type kind of < 1% to survive, and the death because not obtaining strict anaerobism habitat of most anaerobism kind, the conclusion that therefore only relies on traditional morphological classification sometimes may lead to errors.Since the eighties in 20th century, utilize molecular biology method the details of genotype/Phenotypic Diversity in complicated microbe groups can be detected unprecedentedly, but the microorganism that a lot, even great majority are identified out can not carry out laboratory pure culture, thereby cannot study its ecological functions or metabolic mechanism, sometimes between microbe species, the exchange of edge gene also may confuse evaluation, the classification results of molecular biology method.Therefore,, although molecular biology method can identify the microbe species of various aerobic or anaerobism, due to the ABC lacking about its physiological ecological aspect, be also difficult to for the quality of water environment in habitat, place is passed judgment on.
Summary of the invention
For the defect in above-mentioned field, the invention provides a kind of method of applying bottom mud microbe quantity forecast Complex Water Environment overall quality, by index and microbiological data, predict the overall quality of water environment, more comprehensively, and more there is realistic meaning.
The method of application bottom mud microbe quantity forecast Complex Water Environment overall quality, comprises the steps:
(1) measure the top layer of waters bed mud and the superior microorganism quantity of subsurface stratum, (2) calculate the probability of occurrence of passing judgment on water environment overall quality by following formula,
P(bad)/P(medium)＝Exp[-2.987+8.049×ln(surf/sub)]
P(good)/P(medium)＝Exp[-0.944-0.269×ln(surf/sub)]
P(medium)＝e
0＝1
Wherein, the probability of occurrence of P (bad)=" water environment overall quality is for poor " event, the probability of occurrence of P (good)=" water environment overall quality is good " event, the probability of occurrence of P (medium)=" water environment overall quality be in " event, predominant bacteria kind number in Surf=sediments, predominant bacteria kind number in Sub=subsurface stratum bed mud.
Described top layer is the mud layer of dark 5cm, the mud layer that described subsurface stratum is dark 20cm.
Described step (1) identifies that by PCR-DGGE the kind of the superior microorganism in bed mud top layer and subsurface stratum realizes.
The method step of described PCR-DGGE is as follows:
Microbial DNA in A, extraction bed mud,
B, use adopt universal primer to GC-F338 and R518, pcr amplification object fragment,
Described primer GC-F338:5 '-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GTAC GGG AGG CAG CAG-3 ',
R518：5’-ATT ACC GCG GCT GCT GG-3’；
C, DGGE electrophoretic separation PCR product,
Again PCR after the cutting glue and reclaim of D, DGGE band, described primer is F338 and R518,
Described primers F 338:5 '-TAC GGGAGG CAG CAG-3 ',
R518：5’-ATT ACC GCG GCT GCT GG-3’；
E, recovery secondary PCR product, be connected with expression vector, transforms, and cultivates, and extracts plasmid, and PCT detects, and described in described PCR detection, primer is M13-47 and RV-M,
Described primer M13-47:5 '-CGC CAG GGT TTT CCC AGT CAC GAC-3 ',
RV-M：5′-GAG CGG ATA ACA ATT TCA CAC ACA AGG-3′。
F, positive plasmid check order, and determine the kind of contained microorganism.
Described expression vector is Pmd18-T carrier.
Described conversion used organism is DH5 α competent cell.
Described step also comprises that the water environment overall quality of evaluating take chemical index in water is as reference.
The method of the water environment overall quality of chemical index evaluation in described water: measure chemical index in water, with take < < GB3838-2002 > > V class as benchmark, calculate every water-quality guideline that each sampling point is corresponding and the business of V class standard value, then each business's weighting is averaged
Make GX=X/VX
Wherein, GX is the business of each water-quality guideline and < < GB3838-2002 > > V class standard, i.e. TP/V
tP, TN/V
tN, NH
4-N/V
nH4-N, Cr/V
cr..., Pb/V
pb, Cd/V
cd; VX is < < GB3838-2002 > > V class standard corresponding to each water-quality guideline;
Make again Gaverage=(GTP+GTN × 0.5+GNH
4-N × 0.5+GCr+...+GZn+GCu+GPb+GCd)/i
Wherein, i is water-quality guideline quantity;
And then pass judgment on classification, specific as follows:
GIII/V＝(III
TP/V
TP+III
TN/V
TN×0.5+III
NH4-N/V
NH4-N×0.5+III
Cr/V
Cr+...+III
Zn/V
Zn+III
Cu/V
Cu+III
Pb/V
Pb+III
Cd/V
Cd)/i；
GV/V＝(V
TP/V
TP+V
TN/V
TN×0.5+V
NH4-N/V
NH4-N×0.5+V
Cr/V
Cr+...+V
Zn/V
Zn+V
Cu/V
Cu+V
Pb/V
Pb+V
Cd/V
Cd)/i＝1；
GB/V＝(B
TP/V
TP+B
TN/V
TN×0.5+B
NH4-N/V
NH4-N×0.5+B
Cr/V
Cr+...+B
Zn/V
Zn+B
Cu/V
Cu+B
Pb/V
Pb+B
Cd/V
Cd)/i。
Wherein, B is < < GB18918-2002 > > one-level (B) standard, if Gaverage < is GIII/V, water environment overall quality is judged to excellent; If GIII/V < Gaverage < is GV/V, be judged to good; If GV/V < Gaverage < is GB/V, in being judged to; If Gaverage > is GB/V, it is poor to be judged to.
Method of the present invention is specific as follows:
One, applied chemistry index is passed judgment on water environment overall quality, in contrast.
With reference to grade scale and the corresponding function of " water environment quality standard (GB3838-2002) " and " urban wastewater treatment firm pollutant emission standard (GB18918-2002) ", set up first the Complex Water Environment quality overall evaluation pattern of suitable various water quality, Different Waters, specific as follows.
Take < < GB3838-2002 > > V class as benchmark, calculate every water-quality guideline that each sampling point is corresponding and the business of V class standard value, then each business's weighting is averaged.
Make GX=X/VX
Wherein, GX is the business of each water-quality guideline and < < GB3838-2002 > > V class standard, i.e. TP/V
tP, TN/V
tN, NH
4-N/V
nH4-N, Cr/V
cr..., Pb/V
pb, Cd/V
cd; VX is < < GB3838-2002 > > V class standard corresponding to each water-quality guideline.
Make again Gaverage=(GTP+GTN × 0.5+GNH
4-N × 0.5+GCr+...+GZn+GCu+GPb+GCd)/i
Wherein, i is water-quality guideline quantity.
And then pass judgment on classification, specific as follows.
GIII/V＝(III
TP/V
TP+III
TN/V
TN×0.5+III
NH4-N/V
NH4-N×0.5+III
Cr/V
Cr+...+III
Zn/V
Zn+III
Cu/V
Cu+III
Pb/V
Pb+III
Cd/V
Cd)/i；
GV/V＝(V
TP/V
TP+V
TN/V
TN×0.5+V
NH4-N/V
NH4-N×0.5+V
Cr/V
Cr+...+V
Zn/V
Zn+V
Cu/V
Cu+V
Pb/V
Pb+V
Cd/V
Cd)/i＝1；
GB/V＝(B
TP/V
TP+B
TN/V
TN×0.5+B
NH4-N/V
NH4-N×0.5+B
Cr/V
Cr+...+B
Zn/V
Zn+B
Cu/V
Cu+B
Pb/V
Pb+B
Cd/V
Cd)/i
Wherein, B is < < GB18918-2002 > > one-level (B) standard.
Therefore,, if Gaverage < is GIII/V, water environment overall quality is judged to excellent; If GIII/V < Gaverage < is GV/V, be judged to good; If GV/V < Gaverage < is GB/V, in being judged to; If Gaverage > is GB/V, be judged to poor (being inferior to urban wastewater treatment firm water outlet).
Two, application PCR-DGGE means are identified bottom mud microbe
It is stand-by that collection bed mud sample is put into-20 ℃ of Refrigerator stores.
1. extract the microbe genome DNA in bed mud
Concrete operations are with reference to Fast DNA SPIN Kit for Soil kit step (www.mpbio.com).
2.PCR amplification object fragment
(wherein universal primer F338R518 is synthetic by the design of Bo Mai get biotech firm to adopt GC-F338 primer, and at F338 end, add one section of GC clip according to the separation characteristic of DGGE electrophoresis), by PCR by required 16SRNA fragment amplification out, gained PCR product is the fragment of length in 180bp left and right.
Primer GC-F338:5 '-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GTAC GGG AGG CAG CAG-3 ',
R518：5’-ATT ACC GCG GCT GCT GG-3’。
The purifying of 3.PCR product
Concrete operation step is with reference to PCR product purification kit instructions (Beijing Bo Mai get company product).
4.DGGE electrophoretic separation PCR product
The principle of DGGE current separation is, according to the difference of gene basic sequence, DNA sequence dna is separated.In double-stranded DNA molecular, between A-T base, there are 2 hydrogen bonds, and between G-C base, have 3 hydrogen bonds to connect, therefore A-T base-pair will be lower than G-C base-pair to the tolerance of denaturant.Due to composition and the arrangement difference of these 4 kinds of bases, make different sequence double chain DNA molecules there is different melting temperatures.When in the polyacrylamide gel that double chain DNA fragment is containing gradient denaturant (urea, first phthalein amine) during electrophoresis, speed, degree and its sequence of unwinding are closely related, when a certain double chain DNA sequence moves to the certain position of denaturant gel and reaches melting temperature, be to unwind in beginning, the migration velocity of the DNA fragmentation that part is unwind reduces with the increase of the degree of unwinding, thereby make not homotactic DNA fragmentation be stranded in the diverse location of gel, after dyeing, gel-forming is fingerprint band spectrum.In theory, as long as the deposition condition (denaturant gradient, electrophoresis time, voltage etc.) of selecting is enough meticulous, only there is the DNA fragmentation of single base difference also can be separated.The glue of cutting of 5.DGGE band reclaims
By the clear band of DGGE gel, cut with disinfecting forceps, band is cut to such an extent that fine and smoothly evenly by cutting adhesive tape band, be placed in a clean EP pipe, and add 40 μ L sterilized waters as far as possible, 4 ℃, place 12～24h.
6.DGGE band cut glue reclaim after PCR again
Take the sterilized water of the 5th step as template, amplimer is used F338 and R518.
Primers F 338:5 '-TAC GGG AGG CAG CAG-3 ',
R518：5’-ATT ACC GCG GCT GCT GG-3’。
7. the recovery of PCR product again (using Bo Mai get kit purifying)
Concrete operation step is with reference to Beijing Bo Mai get biotech firm gel purification kit instructions.
8. the 7th step reclaims being connected of product and Pmd18-T carrier (Takara).
9. transform DH5 α competent cell.
10. single bacterium colony of picking white, shakes bacterium, extracts plasmid, and PCR detects.
11. positive are sent company's order-checking (examining order is completed by Beijing Bo Mai get biotech firm).
Because the PCR-DGGE conventional method to conventional is optimized, because the predominant bacteria kind number of its detection and in esse more approaching.
Three, application bottom mud microbe quantity is forecast Complex Water Environment overall quality
In water environment bed mud top layer (5cm) and subsurface stratum (20cm) bed mud, predominant bacteria kind number is than the key factor that is sign quality of water environment, and has following quantitative relationship.
P(bad)/P(medium)＝Exp[-2.987+8.049×ln(surf/sub)]
P(good)/P(medium)＝Exp[-0.944-0.269×ln(surf/sub)]
P(medium)＝e
0＝1
Wherein, the probability of occurrence of P (bad)=" water environment overall quality is for poor " event, the probability of occurrence of P (good)=" water environment overall quality is good " event, the probability of occurrence of P (medium)=" water environment overall quality be in " event.Predominant bacteria kind number in Surf=sediments (dark 5cm), predominant bacteria kind number in Sub=subsurface stratum bed mud (dark 20cm).
Tool of the present invention has the following advantages:
1. the application is take latest national standards one < < water environment quality standard (GB3838-2002) > > as basis, both with Chinese characteristics, also tally with the national condition; And introduced < < urban wastewater treatment firm pollutant emission standard (GB18918-2002) > > relevant criterion, the scope of application is wide, from clean water body to the water environment of serious pollution, all can be used as with reference to being suitable for.
2. the water quality physical and chemical index quantity that the application selects and option are unfixing, can be according to passing judgment on object, choosing from relevant national standard selectively; The water environment composite quality index of the existing quantification of result of calculation, also can carry out qualitative evaluation according to national standard result of calculation, and this judgment criteria can also upgrade along with the renewal of national standard, has very strong adaptability and vitality.
3., although in water environment, microbe species is various, accounting for the most anaerobism kind of total monoid cannot carry out pure culture according to traditional mode under normal experiment condition; Although molecular biosciences means can identify microbe species, often can only be to superior microorganism Qualitative Identification, be difficult to know its ecological functions; Even bacterial classification that can pure culture, often also can only find this bacterial classification place genus, the physiological characteristic of section, ecological functions, due to the greatest differences that may exist between equal/genus bacterial classification, also be just difficult to pass judgment on according to physiological characteristic or ecological functions the quality of water environment in its habitat, place.The application has got around bacterial classification pure culture, bacterial classification physiological characteristic or ecological functions these data gully or technology barriers, directly adopt predominant bacteria kind number as examination index, successfully find first the quantification response relation between bed mud in river predominant bacteria kind and quality of water environment, broken through the traditional wide gap between microcosmic molecular biology research and macroscopical water quality evaluation.
Accompanying drawing explanation (is filled in figure title below, and detailed information is placed with below the map title.
Fig. 1 genome extracts: 94# 97# 20# 76# 83# 86# 78# 62 # 175a 64a 79a 67# 77#|# represents that sampling point comprises top layer and subsurface stratum from left to right, and a represents that top layer b represents subsurface stratum
Fig. 2 Genomic PCR: 20# 62# 76# from left to right
Fig. 3 liquid purifying: 39# 123# 20# 35# 62# 64# 76# 79# 83# 86# 94# 97# 175a from left to right
Fig. 4 DGGE electrophoresis pattern
PCR after Fig. 5 DGGE: 62# (b1 b2 b3 b4 ab1 ab2 ab3 ab4 ab5 ab6 ab7 ab8 ab9 ab10) 64# (1a-8a) from left to right
To cut adhesive tape band corresponding with DGGE in bracket, and a represents that top layer b represents that subsurface stratum ab represents that top layer and subsurface stratum have.
Fig. 6 solid purifying: to cut adhesive tape band corresponding with DGGE in 35# (ab1 ab2 b1 b2 ab3 b3 ab4) 62# (b1 b2 b3 b4 ab1 ab2 ab3 ab4 ab5ab6 ab7 ab8 ab9 ab10) bracket from left to right, and a represents that top layer b represents that subsurface stratum ab represents that top layer and subsurface stratum have.
Fig. 7 clones rear bacterium colony
Fig. 8 bacterium colony PCR: to cut adhesive tape band corresponding with DGGE in 175# (1a-6a) 62# (b1 b2 b3 b4 ab1 ab2 ab3 ab4 ab5 ab6 ab7 ab8 ab9ab10) bracket from left to right, and a represents that top layer b represents that subsurface stratum ab represents that top layer and subsurface stratum have
Fig. 9 collection of illustrative plates (Beijing Bo Mai get biotech firm) that checks order
Embodiment
Below by embodiment, the present invention is described in further detail.
One, according to hydrochemistry index, certain sampling point (Caobai River 62#) water environment overall quality is passed judgment on
At Caobai River Beijing Section, gather water sample and analyze TP, TN, NH
3-N, Cr, Zn, Cu, Pb, Cd index are as shown in table 1.
The Baihe, table 1 lake Beijing Section sampling point water quality
| No. | Longitude | Latitude | TP | TN | NH 4-N | Cr | Zn | | Pb | Cd | |
| 62# | 114.28 | 35.51 | 1.629 | 7.889 | 6.339 | 0.051 | 0.047 | 0.072 | 0.058 | 0.031 |
The classification of concerned countries water quality standard and standard are as shown in table 2.
The national water quality standard classification of table 2 and relevant criterion
| National standard | TP | TN | NH 4 +-N | Cr | Zn | Cu | Pb | Cd |
| GB3838-2002III level | 0.2 | 1.0 | 1.0 | 0.05 | 1.0 | 1.0 | 0.05 | 0.005 |
| GB3838-2002V level | 0.4 | 2.0 | 2.0 | 0.1 | 2.0 | 1.0 | 0.1 | 0.01 |
| GB18918-2002- |
1 | 20 | 15 * | 0.1 | 1.0 | 0.5 | 0.1 | 0.01 |
According to above-mentioned formula,
GIII/V＝(III
TP/V
TP+III
TN/V
TN×0.5+III
NH4-N/V
NH4-N×0.5+III
Cr/V
Cr+III
Zn/V
Zn+III
Cu/V
Cu+III
Pb/V
Pb+III
Cd/V
Cd)/7＝0.5714；
GV/V＝(V
TP/V
TP+V
TN/V
TN×0.5+V
NH4-N/V
NH4-N×0.5+V
Cr/V
Cr+V
Zn/V
Zn+V
Cu/V
Cu+V
Pb/V
Pb+V
Cd/V
Cd)/7＝1；
GB/V＝(B
TP/V
TP+B
TN/V
TN×0.5+B
NH4-N/V
NH4-N×0.5+B
Cr/V
Cr+B
Zn/V
Zn+B
Cu/V
Cu+B
Pb/V
Pb+B
Cd/V
Cd)/7＝2.1786。
The water environment composite quality index Gaverage=1.7023 of this sampling point, is less than GB/V but is greater than G (V/V), thereby during water environment overall quality is judged as.
Two, application PCR-DGGE means are identified this sampling point (Caobai River 62#) bottom mud microbe
1. get these sampling point-20 ℃ freezing mud sample, according to the Fast DNA SPIN Kit for Soil listed operation steps of kit instructions (www.mpbio.com), extract the microbe genome DNA (Fig. 1) in bed mud.
2. by PCR by required 16SRNA fragment amplification out, gained PCR product is length 180bp left and right fragment (figure below), the primer is that (wherein universal primer F338 R518 is synthetic by the design of Bo Mai get biotech firm, because the separation characteristic of DGGE electrophoresis adds one section of GC clip at F338 end for GC-F338.Fig. 2
The purifying of 3.PCR product, operation steps, with reference to PCR product purification kit instructions (Bo Mai get biotech firm), the results are shown in Figure 3.
4. according to the difference of the upper base-pair sequence of PCR product D NA, by DGGE, DNA sequence dna is separated to (seeing Fig. 4).
5. choose in DGGE gel band clearly, cut (band will cut as far as possible fine and smoothly evenly) with clean tweezers, then by cutting adhesive tape band, be placed in clean EP pipe, and add the sterilized water of 40 μ L, place 12～24h for 4 ℃.
6. take the sterilized water of the 5th step as template, select F338 and R518 as primer, the glue of cutting of DGGE band is reclaimed, then re-start pcr amplification.See Fig. 5
7. use gel purification kit (Bo Mai get biotech firm, operation steps is referring to this product description), PCR product is again carried out to purifying, recovery.See Fig. 6
8. the 7th step is reclaimed to (the connection of (buy from Takara company, there is preservation in the applicant's laboratory in colleges and universities, can provide the public) of product and Pmd18-T carrier.
9. transform DH5 α competent cell.See Fig. 7
10. single bacterium colony of picking white, shakes bacterium, extracts plasmid, and the performing PCR of going forward side by side detects.See Fig. 8
11. select positive send Beijing Bo Mai get biotech firm to check order, and the collection of illustrative plates that partly checks order is as follows.See Fig. 9.
Predominant bacteria kind inquiry in 12. this sampling point (Caobai River 63#) bed muds (showing that through genebank comparison bacterial classification inquiry sequence number and part are through pure culture bacterial classification) result is as shown in the table.
Three, application bottom mud microbe quantity is forecast Complex Water Environment overall quality
At this sampling point, in water environment bed mud top layer (5cm) and subsurface stratum (20cm) bed mud, predominant bacteria kind number is than being 10/16=0.625, and substitution quantitative model has:
P(bad)/P(medium)＝Exp[-2.987+8.049×ln(0.625)]＝0.001148
P(good)/P(medium)＝Exp[-0.944-0.269×ln(0.625)]＝0.041504
P(medium)＝e
0＝1
Wherein, the probability of occurrence of P (bad)=" water environment overall quality is for poor " event, the probability of occurrence of P (good)=" water environment overall quality is good " event, the probability of occurrence of P (medium)=" water environment overall quality be in " event.Know thus, this sampling point water quality be good, in, the probability that occurs of assignment part is respectively 30.6%, 69.3%, 0.08% (quality of water environment be in probability approximately 70%), quite well the result of determination of making according to chemical index (" water environment overall quality be in ").
The prediction of an above-mentioned just sampling point, if relate to this waters, measures multiple sampling points, on average plant number and calculate, and sampling point obtains manyly with predominant bacteria, and result of determination is more accurate.
Claims (8)
1. the method for application bottom mud microbe quantity forecast Complex Water Environment overall quality, comprises the steps:
(1) measure the top layer of waters bed mud and the superior microorganism kind number of subsurface stratum, (2) calculate the probability of occurrence of passing judgment on water environment overall quality by following formula,
P(bad)/P(medium)=Exp[-2.987+8.049×ln(surf/sub)]，
P(good)/P(medium)=Exp[-0.944-0.269×ln(surf/sub)]，
P(medium)=e
0=1，
Wherein, the probability of occurrence of P (bad)=" water environment overall quality is for poor " event, the probability of occurrence of P (good)=" water environment overall quality is good " event, the probability of occurrence of P (medium)=" water environment overall quality be in " event, predominant bacteria kind number in Surf=sediments, predominant bacteria kind number in Sub=subsurface stratum bed mud.
2. method according to claim 1, the mud layer that described top layer is dark 5cm, the mud layer that described subsurface stratum is dark 20cm.
3. method according to claim 1, described step (1) identifies that by PCR-DGGE the kind of the superior microorganism in bed mud top layer and subsurface stratum realizes.
4. method according to claim 3, the method step of described PCR-DGGE is as follows:
Microbial DNA in A, extraction bed mud,
B, use adopt universal primer to GC-F338 and R518, pcr amplification object fragment,
Described primer GC-F338:5'-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GTAC GGG AGG CAG CAG-3',
R518：5’-ATT ACC GCG GCT GCT GG-3’；
C, DGGE electrophoretic separation PCR product,
Again PCR after the cutting glue and reclaim of D, DGGE band, described primer is F338 and R518,
Described primers F 338:5 '-TAC GGG AGG CAG CAG-3',
R518：5’-ATT ACC GCG GCT GCT GG-3’；
E, recovery secondary PCR product, be connected with expression vector, transforms, and cultivates, and extracts plasmid, and PCT detects, and described primer is M13-47 and RV-M,
Described primer M13-47:5'-CGC CAG GGT TTT CCC AGT CAC GAC-3',
RV-M:5'-GAG CGG ATA ACA ATT TCA CAC ACA AGG-3'；
F, positive plasmid check order, and determine the kind of contained microorganism.
5. method according to claim 4, described expression vector is Pmd18-T carrier.
6. method according to claim 4, described conversion used organism is DH5 α competent cell.
7. method according to claim 1, described step also comprises that the water environment overall quality of evaluating take chemical index in water is as reference.
8. method according to claim 7, chemical index in the method for the water environment overall quality of chemical index evaluation: a, mensuration water in described water, b, with take < < GB3838-2002 > > V class as benchmark, calculate every water-quality guideline that each sampling point is corresponding and the business of V class standard value, then each business's weighting is averaged
Make GX=X/ V X
Wherein, GX is the business of each water-quality guideline and < < GB3838-2002 > > V class standard, i.e. TP/ V
tP, TN/ V
tN, NH
4-N/V
nH4-N, Cr/V
cr..., Pb/ V
pb, Cd/ V
cd; V X is < < GB3838-2002 > > V class standard corresponding to each water-quality guideline;
Make again Gaverage=(GTP+GTN × 0.5+GNH
4-N × 0.5+GCr+ ... + GZn+GCu+GPb+GCd)/i
Wherein, i is water-quality guideline quantity;
C and then pass judgment on classification, specific as follows:
GⅢ/Ⅴ=(Ⅲ
TP/Ⅴ
TP+Ⅲ
TN/Ⅴ
TN×0.5+Ⅲ
NH4-N/Ⅴ
NH4-N×0.5+Ⅲ
Cr/Ⅴ
Cr+…+Ⅲ
Zn/Ⅴ
Zn+Ⅲ
Cu/Ⅴ
Cu+Ⅲ
Pb/Ⅴ
Pb+Ⅲ
Cd/Ⅴ
Cd)/i；
GⅤ/Ⅴ=(Ⅴ
TP/Ⅴ
TP+Ⅴ
TN/Ⅴ
TN×0.5+Ⅴ
NH4-N/Ⅴ
NH4-N×0.5+Ⅴ
Cr/Ⅴ
Cr+…+Ⅴ
Zn/Ⅴ
Zn+Ⅴ
Cu/Ⅴ
Cu+Ⅴ
Pb/Ⅴ
Pb+Ⅴ
Cd/Ⅴ
Cd)/i=1；
GB/Ⅴ=(B
TP/Ⅴ
TP+B
TN/Ⅴ
TN×0.5+B
NH4-N/Ⅴ
NH4-N×0.5+B
Cr/Ⅴ
Cr+…+B
Zn/Ⅴ
Zn+B
Cu/Ⅴ
Cu+B
Pb/Ⅴ
Pb+B
Cd/Ⅴ
Cd)/i；
Wherein, B is < < GB18918-2002 > > one-level (B) standard, if Gaverage<G III/V, water environment overall quality is judged to excellent; If G III/V <Gaverage<G V/V, is judged to good; If G V/V <Gaverage<GB/ V, in being judged to; If Gaverage>GB/ V, it is poor to be judged to.
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