CN104962620A - Microflora-based ecology health evaluation method - Google Patents

Microflora-based ecology health evaluation method Download PDF

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CN104962620A
CN104962620A CN201510317051.1A CN201510317051A CN104962620A CN 104962620 A CN104962620 A CN 104962620A CN 201510317051 A CN201510317051 A CN 201510317051A CN 104962620 A CN104962620 A CN 104962620A
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microorganism
quality guideline
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CN104962620B (en
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张德民
朱建林
王凯
李建峰
陈和平
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Ningbo University
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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract

The invention discloses a microflora-based ecology health evaluation method. The method comprises the following steps: 1, sampling water and microbes, and analyzing the water quality; 2, calculating the correlation coefficient between the water quality indexes and the relative abundance of the microbes; 3, screening a microbial flora correlated with a specific water quality index and executing an opposite function; 5, establishing a relationship between the total relative abundance of the correlated microbial flora and the specific water quality index; and 5, mapping with one water quality index of all samples an x axis and Sija-Sijb as a y axis, allowing an S-shaped curve to have two corresponding inflection points when the value of Sija-Sijb is 0 and does not increase, and respectively evaluating three sections divided by the two inflection points to be ecologically healthy, sub-healthy and unhealthy. The method has the advantages of simplicity, fastness, accuracy, impersonality, and clear environment ecology significance, and provides an objective criterion for calculation of the marine site environment capacity.

Description

A kind of environmental health evaluation method based on microflora
Technical field
The invention belongs to marine eco-environment protection field, especially relate to a kind of marine ecology health assessment method based on microflora.
Background technology
Environmental health refers to that the ecosystem keeps its natural character, maintenance species diversity and crucial ecological process to stablize and continues to play the ability of its service function.Offshore Marine ecosystems are divided into the type of ecosystem such as karang, sea grass bed, mangrove forest, river mouth and bay by " coastal ocean environmental health evaluation guide " that National Bureau of Oceanography issues.Its basic skills is: (1), to various type of ecosystem, investigates its all kinds of water surrounding index, habitat index, Biological indicators, bio-toxicity index; (2) respectively assignment is carried out to these indexs artificially by certain rule; (3) the assignment phase adduction of all indexs is averaged, using this average as environmental health index; (4) in advance environmental health rank is divided into 3 grades, namely healthy, subhealth state and unhealthy, and specify environmental health index range (i.e. judgement criteria) at different levels; (5) when the environmental health index value of evaluation object falls into the standard range value of certain rank, namely this evaluation object belongs to this health level.
The advantage of this evaluation method is: (1) distinguishes the dissimilar ecosystem, specifies different indicators and standards to different system, is conducive to the basic feature of outstanding different system; (2) investigation index is comprehensive, can reflect system state comparatively all sidedly.But its defect is: the index related to needed for (1) evaluation is too many, and in fact a lot of index is difficult to obtain, or cost is too large; (2) its judgement criteria is formulated in all obtainable situation of all indexs of acquiescence, and therefore when lacking some index, then it is obviously inappropriate for applying mechanically original judgement criteria; (3) assignment of each index and judgement criteria value are all artificially given, and whether its value is suitable does not have objective basis.The existence of these problems, make Marine ecosystems health assessment business carry out difficulty, its reliability is also extensively queried.
We think: (1) microorganism is the decomposer in the ecosystem, is again producers and consumers, is in the least significant end of food chain, occupies very important status in the ecosystem.The change of microorganism structure and function can reflect the most basic feature of the ecosystem, namely can carry out the change of the ecological state of evaluation system with the change of biological community structure and function; (2) the healthy essence of water ecology is the balance of all kinds of microbial function, in water ecosystem, the result of the removal of pollutent mainly microbial process, the balance between the microorganism performing types of functionality is that the ecosystem maintains balance and healthy basic assurance; (3) for certain quasi-microorganism, the fabulous or extreme difference of water quality correspond respectively to two extreme, two extreme between should there is a condition of water quality, make such bacterial abundance maximum herein and progressively decline to the two poles of the earth or become flat; (4) microorganism relative abundance and environmental factor closely related, by the indicator microoraganism of the stronger microbe groups of screening dependency as the specific environment factor or specific pollution load; (5), under different waters, Different ecosystems type and different time conditions, the microorganism of state particularly in order to characterize ecosystem health state of the ecosystem also has larger difference.
In addition our research finds, to a certain environmental factor or total pollution load, all exists and associates close microbe groups with this factor or pollution load.These monoids can be roughly divided into two types: one is the monoid be proportionate with it, and two is the monoids in negative correlation; The microbe groups performing reverse functions can be interpreted as respectively; Positively related microorganism, its abundance increases with Pollutant levels and increases, but no longer increases when being increased to a certain threshold value, or declines on the contrary; The microorganism of negative correlation, its abundance increases with Pollutant levels and reduces, but no longer declines when reducing to a certain threshold value, or increases on the contrary, meets the middle potential theory in ecology and Law of Tolerance.Therefore, the state of health of water ecosystem can be evaluated by the change of monitoring microflora.The develop rapidly of Protocols in Molecular Biology, makes to become possibility by adopting the technique means such as high-flux sequence or gene chip to realize the fast monitored of microorganism in water body, thus can realize water ecosystem health assessment by the change of monitoring microorganism.
Summary of the invention
Technical problem to be solved by this invention is to provide and a kind ofly realizes the method for marine ecology health assessment according to the change of microflora in ocean water body or settling.
The present invention solves the problems of the technologies described above adopted technical scheme:
Based on an environmental health evaluation method for microflora, comprise the following steps:
(1) sampling analysis water quality and microorganism
In certain evaluation region, base area domain space or Pollutant levels gradient are arranged nthe sample of individual sampling point i, i=1,2 ..., n, gather water sample and/or sediment sample at each sampling point; To all sample analyses gindividual water-quality guideline j, j=1,2 ..., g, obtain the water-quality guideline of each sample jvalue x ij ; Each sample is drawn by the order-checking of high-flux sequence, gene chip or high-throughput quantification PCR method mplant microorganism k, k=1,2 ..., m, calculate the relative abundance of each microorganism in each sample y ik ; Wherein the relative abundance of a certain microorganism is the per-cent that the number of a certain microorganism in certain sample accounts for all microorganism numbers in this sample;
(2) relation conefficient of water-quality guideline and microorganism relative abundance is calculated
By the water-quality guideline of all samples jvalue x ij with respective sample mplant the relative abundance of microorganism y ik carry out correlation analysis, obtain the water-quality guideline of all samples jwith respective sample mplant the relation conefficient of microorganism r jk and significance index pvalue p jk ; ;
(3) microbe groups that screening is relevant to specific water-quality guideline
Set a certain significance index pvalue standard p s , from p jk in find out and be less than and equal p s microbe species, be and corresponding water-quality guideline jrelevant microbe groups; If filtered out aplant microorganism and this water-quality guideline jbe proportionate, have bplant microorganism and this water-quality guideline jin negative correlation; Definition akind of positive correlation microorganism and bplanting negative correlation microorganism is the microbe groups performing reverse functions;
(4) total relative abundance of related microorganisms monoid and contacting of specific water-quality guideline is set up
To each sample i, calculate all and a certain water-quality guideline jbe proportionate aplant the relative abundance sum of microorganism s ija , and in negative correlation bplant the relative abundance sum of microorganism s ijb ; With this water-quality guideline of all samples jvalue x ij for x-axis, with s ija with s ijb for y-axis mapping, total the relative abundance setting up positive and negative two class functional microorganism monoids is with water-quality guideline jvariation diagram;
(5) to divide between environmental health state area and to determine judgement criteria
With a certain water-quality guideline of all samples jvalue x ij for x-axis, with s ija - s ijb for y-axis mapping, x ij with s ija - s ijb logic appears being similar in figure, and this forms the sigmoid curve of (Logistic) growth curve; Work as difference s ija - s ijb when=0 and s ija - s ijb when no longer increasing, there are corresponding two flex points in this sigmoid curve, and namely the value of the water-quality guideline that two flex points are corresponding respectively can be used as judgement criteria; Namely with difference s ija - s ijb the value of water-quality guideline when=0 is as function balance point e 1 , with difference s ija - s ijb the value of water-quality guideline when no longer increasing is as environmental capacity point e 2 ; When the water-quality guideline of a certain sampling point jvalue x ij for x ij ≤ e 1 , then this sampling point region is judged as environmental health; When the water-quality guideline of a certain sampling point jvalue x ij for e 1 ﹤ x ij ≤ e 2 , then this sampling point region is judged as subhealth state; When the water-quality guideline of a certain sampling point jvalue x ij for e 2 ﹤ x ij , then this sampling point region is judged as unhealthy.
Compared with prior art, the invention has the advantages that: evaluate marine ecology health according to microflora's change in ocean water body or settling, from microbial ecological angle sea area evaluation environmental health state objectively, and can be marine environment calculation of capacity objective quantitative standard is provided; Method simple and fast, objective and accurate, Environmental ecology meaning is clear and definite.
Accompanying drawing explanation
Fig. 1 is the variation diagram of total abundance with water-quality guideline of the positive and negative two class functional microorganism monoids of the present invention;
Fig. 2 is the variation diagram of difference with water-quality guideline of total abundance of the positive and negative two class functional microorganism monoids of the present invention.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Specific embodiment
In August, 2013,98 sampling points are set respectively in marine site, Ningbo, gather water sample, settling sample and carry out chemical analysis, and to sample after filtration, carry out high-flux sequence after the pre-treatment such as DNA extraction, pcr amplification, obtain the relative abundance of each microorganism OTU of each sample.During with the data evaluation of different categorization levels (doors, classes, orders, families, genera and species), except data difference, its flow process and technical solution of the present invention method completely the same.With the data instance that following microorganism belongs to, water-quality guideline PO 4 -for example, idiographic flow and method are described:
(1) sample ihave 98 i=1,2 ..., 98; To all samples analyze 10 water-quality guideline ( j=1,2 ..., 10), wherein PO 4 -be the 6th water-quality guideline, namely its value is x i6 , and calculate the equivalent pollution loads value of each sample z i ; Each sample is drawn by the order-checking of the method such as high-flux sequence or quantitative PCR mindividual microorganism belonging to genus k, k=1,2 ..., m, calculate the relative abundance of each microorganism in each sample y ik ; Wherein the relative abundance of a certain microorganism is the per-cent that the number of a certain microorganism in certain sample accounts for all microorganism numbers in this sample;
(2) to water-quality guideline PO 4 -( j=6), by the water-quality guideline PO of all samples 4 -value x ij with respective sample mthe relative abundance of individual microorganism belonging to genus y ik carry out correlation analysis, must this index PO 4 -with all mplant the relation conefficient of microorganism r jk and significance index pvalue p jk ; ;
(3) given a certain pvalue standard p s =0.01, from p jk in find out and be less than and equal p s microorganism belong to, be and water-quality guideline PO 4 -relevant microbe groups; If filtered out aplant microorganism and water-quality guideline PO 4 -be proportionate, have bplant microorganism and water-quality guideline PO 4 -in negative correlation; Definition akind of positive correlation microorganism and bplanting negative correlation microorganism is the microbe groups performing reverse functions;
(4) to each sample i, calculate all and PO 4 -be proportionate aplant the relative abundance sum of microorganism s ija and all and PO 4 -in negative correlation bplant the relative abundance sum of microorganism s ijb ; With all sample PO 4 -value x ij for x-axis, with s ija with s ijb for y-axis mapping, total the relative abundance that can obtain positive and negative two class functional microorganism monoids is with PO 4 -variation diagram (see figure 1);
(5) with this water-quality guideline PO of all samples 4 -value x ij for x-axis, with s ija - s ijb for y-axis mapping, x ij with s ija - s ijb there is the sigmoid curve being similar to Logistic growth curve in figure; Work as difference s ija - s ijb when=0 and s ija - s ijb when no longer increasing, the corresponding appearance of this sigmoid curve two flex points, two flex points the value of corresponding respectively water-quality guideline namely can be used as judgement criteria; Namely with difference s ija - s ijb the value of water-quality guideline when=0 is as function balance point e 1 , this example e 1 =0.03 mg/L; With difference s ija - s ijb the value of water-quality guideline when no longer increasing is as environmental capacity point e 2 , this example e 2 =0.05 mg/L; By two flex points e 1 with e 2 three intervals distinguishing are judged as environmental health, subhealth state and unhealthy respectively; This figure is environmental health judgement criteria figure (see figure 2).If the PO of the new sample of certain needs assessment 4 -value be 0.04mg/L, can find from Fig. 2, belonging to this sample, the environmental health grade in marine site is " subhealth state ".
Each sea area evaluation, type of ecosystem and evaluation time should have different standards.Sea area evaluation can be larger range scale, as the East Sea, the South Sea, Bohai and Yellow Seas etc., also can be less marine site scope, as Xiangshan Bay, nutrients etc.; Type of ecosystem by " coastal ocean environmental health evaluation guide ", or can be determined by special requirement; Evaluation time can be divided into season and month;
Above-mentioned sample can be settling, and corresponding Environmental indicators is settling respective environment level of factor.
Mentioned microorganism kind can be the categorization levels such as doors, classes, orders, families, genera and species or OTU in significance for taxonomy respectively; The result being calculated gained by different categorization levels can mutually compare and confirm.
Above-mentioned water-quality guideline can also be the free state ammonia content of water body, total phosphorous, pH value and suspended solids content.
Can by equivalent pollution loads z i as an overall target, do identical evaluation by above method.
Certainly, above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited to above-mentioned citing.The change that those skilled in the art make in essential scope of the present invention, remodeling, interpolation or replacement, also should belong to scope.

Claims (1)

1., based on an environmental health evaluation method for microflora, it is characterized in that comprising the following steps:
(1) sampling analysis water quality and microorganism
In certain evaluation region, base area domain space or Pollutant levels gradient are arranged nthe sample of individual sampling point i, i=1,2 ..., n, gather water sample and/or sediment sample at each sampling point; To all sample analyses gindividual water-quality guideline j, j=1,2 ..., g, obtain the water-quality guideline of each sample jvalue x ij ; Each sample is drawn by the order-checking of high-flux sequence, gene chip or high-throughput quantification PCR method mplant microorganism k, k=1,2 ..., m, calculate the relative abundance of each microorganism in each sample y ik ; Wherein the relative abundance of a certain microorganism is the per-cent that the number of a certain microorganism in certain sample accounts for all microorganism numbers in this sample;
(2) relation conefficient of water-quality guideline and microorganism relative abundance is calculated
By the water-quality guideline of all samples jvalue x ij with respective sample mplant the relative abundance of microorganism y ik carry out correlation analysis, obtain the water-quality guideline of all samples jwith respective sample mplant the relation conefficient of microorganism r jk and significance index pvalue p jk ; ;
(3) microbe groups that screening is relevant to specific water-quality guideline
Set a certain significance index pvalue standard p s , from p jk in find out and be less than and equal p s microbe species, be and corresponding water-quality guideline jrelevant microbe groups; If filtered out aplant microorganism and this water-quality guideline jbe proportionate, have bplant microorganism and this water-quality guideline jin negative correlation; Definition akind of positive correlation microorganism and bplanting negative correlation microorganism is the microbe groups performing reverse functions;
(4) total relative abundance of related microorganisms monoid and contacting of specific water-quality guideline is set up
To each sample i, calculate all and a certain water-quality guideline jbe proportionate aplant the relative abundance sum of microorganism s ija , and in negative correlation bplant the relative abundance sum of microorganism s ijb ; With this water-quality guideline of all samples jvalue x ij for x-axis, with s ija with s ijb for y-axis mapping, total the relative abundance setting up positive and negative two class functional microorganism monoids is with water-quality guideline jvariation diagram;
(5) to divide between environmental health state area and to determine judgement criteria
With a certain water-quality guideline of all samples jvalue x ij for x-axis, with s ija - s ijb for y-axis mapping, x ij with s ija - s ijb logic appears being similar in figure, and this forms the sigmoid curve of growth curve; Work as difference s ija - s ijb when=0 and s ija - s ijb when no longer increasing, there are corresponding two flex points in this sigmoid curve, and namely the value of the water-quality guideline that two flex points are corresponding respectively can be used as judgement criteria; Namely with difference s ija - s ijb the value of water-quality guideline when=0 is as function balance point e 1 , with difference s ija - s ijb the value of water-quality guideline when no longer increasing is as environmental capacity point e 2 ; When the water-quality guideline of a certain sampling point jvalue x ij for x ij ≤ e 1 , then this sampling point region is judged as environmental health; When the water-quality guideline of a certain sampling point jvalue x ij for e 1 ﹤ x ij ≤ e 2 , then this sampling point region is judged as subhealth state; When the water-quality guideline of a certain sampling point jvalue x ij for e 2 ﹤ x ij2 , then this sampling point region is judged as unhealthy.
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