WO2023098239A1 - River ecosystem health assessment method based on microbial community specific response - Google Patents

Abstract

A river ecosystem health evaluation method based on microbial community specific response. The method comprises: a: respectively setting sampling points of rural areas, urban suburban areas and urban city centers along a river; b: measuring the water body to determine 12 indicators of pH, water temperature (WT), dissolved oxygen (DO), COD, TN, total organic carbon, nitrate nitrogen, ammonium nitrogen, nitrite nitrogen, total phosphorus, sulfide and fluoride, and calculating a water quality index (IWQ); c: extracting microbial DNA from the water body and carrying out bacterial 16s rRNA amplicon sequencing; d: obtaining a bacterial genus level information table, and screening for aeffective bacterial genus information; e: performing microbial sensitivity division on the basis of an ecological level model; and f: performing evaluation result reliability analysis. By means of the present method, the health condition of an urban river ecosystem can be quickly, accurately and objectively reflected.

Description

A Health Assessment Method for River Ecosystem Based on Specific Response of Microbial Community Technical field:

The invention belongs to the technical field of water body environment monitoring and protection, and in particular relates to a river ecosystem health evaluation method based on the specific response of microbial communities.

Background technique:

River ecosystems provide important service functions such as water resources, food, and shipping for human and urban development. With the intensification of human disturbance, water pollution and deterioration occur frequently, seriously threatening the health of river ecosystems. Researching and establishing health assessment methods for river ecosystems is an important basic support for long-term monitoring, development and utilization of river ecosystems, as well as pollution control and restoration.

Microorganisms are the decomposers and important producers of the ecosystem. They play an important role in the control and restoration of environmental pollution. They are very sensitive to changes in the surrounding environmental conditions. Their diversity and community structure are closely related to the health of the ecosystem. The use of microbial communities as an indicator of ecosystem health has the following advantages: First, different types of microorganisms have different tolerances and resistances to different pollutants and environmental changes, and changes in the abundance and functional activities of specific microorganisms can be used for health evaluation. Provide important information; secondly, because microorganisms in the ecosystem function in the form of communities, changes in the structure and function of microbial communities can more accurately reflect changes in the health of the ecosystem. However, due to the limited understanding of the specific response characteristics and related mechanisms of microbial communities in river ecosystems, the division standards of microbial indicators and the weighting method of the integrity index in water body health assessment mainly depend on decision makers and experts. knowledge and experience, which introduces more preference and randomness to the evaluation results.

With the rapid development of microbial analysis and detection technology and high-throughput sequencing technology, the specific response mechanism of microbial communities to ecosystem changes has been further elucidated, providing important theoretical and technical support for evaluating the health of river ecosystems. The present invention utilizes the specific response characteristics of sensitive microorganisms to environmental changes and combines the uniqueness of microbial ecological niches to establish a scientific and reasonable index system to ensure the objectivity and practicability of index weights.

Invention content:

The purpose of the present invention is to provide a method for evaluating the health of river ecosystems based on the specific response of microbial communities. The method of the present invention is highly directional, indicating characteristic pollutants by utilizing changes in the structure and function of specific microbial groups.

The river ecosystem health evaluation method based on microbial community specific response of the present invention, comprises the following steps:

a: Set up sampling points in rural areas, urban suburbs and urban centers along the river;

b: Determination of water quality indicators such as pH, water temperature (WT), dissolved oxygen (DO), COD, TN, total organic carbon, nitrate nitrogen, ammonium nitrogen, nitrite nitrogen, total phosphorus, sulfide and fluoride , to calculate the water quality index IWQ;

c: Extract water microbial DNA and perform bacterial 16S rRNA gene amplicon sequencing;

d: Obtain the bacterial genus level information table, and screen the effective bacterial genus information;

e: Classification of microbial sensitivity based on the niche model;

f: Reliability analysis of evaluation results.

Preferably, described step b is:

Using the water quality calculation formula, 5 parameters were selected to evaluate the water quality, including pH, dissolved oxygen (DO), chemical oxygen demand (COD), phosphate, and nitrate nitrogen; the factor scores are as follows:

pH:

If 4.5≤pH<7.0 SI _pH ＝1.9EXP((pH-1)*0.66)

If 7.0≤pH≤7.6 SI _pH = 100

If 7.6<pH≤10.5 SI _pH ＝100exp((pH-7.65)*-0.528)

If 10.5<pH<4.5 SI _pH =10

DO:

If DO<3.3 SI _DO =10

If DO>12.5 SI _DO = 100

If 3.3≤DO≤12.5 SI _DO ＝-59.6+24.9*DO-0.98*DO^2

COD:

SI _COD ＝100*0.86^COD

TP:

If TP<0.03 SI _TP = 100

If TP>1.2 SI _TP = 10

If 0.03<TP<1.2 SI _TP ^- ＝99.5*0.17^(PO ₄ ^3- )

NO _3- ^:

NO ₃ ^- ≤1 SI _NO3- ＝100

NO ₃ ^- >1 SI _NO3- ＝102*0.8887^(NO ₃ ^- )

WQI calculation formula: n is the number of selected factors, SIi is the factor score

Preferably, microbial susceptibility is classified as follows:

A. Statistics on the relative abundance of microorganisms at the genus level, and those whose relative abundance is higher than 1% in at least one sample point are effective species, which are used for screening of sensitive microorganisms;

B. Fit the response curves of various species of microorganisms to environmental factors based on the generalized additive model, and select the ones with a significance lower than 0.01 as the effective fitting curves;

C. Extension of generalized additive model and generalized linear model;

Taking two environmental factors as an example, the calculation method is as follows:

log(E(yi)=a+s1(x1i)+s2(x2i)

Where yi is the relative abundance of microorganisms, xi is an environmental parameter, s1(x1), s2(x2) are non-parametric smooth functions; Log() is an example of a correlation function;

D. According to the fitting curve type of the generalized additive model, respectively estimate the ecological niche of each genus: for the single-peak fitting curve, the value of the environmental parameter corresponding to the peak value is the most suitable ecological niche for the microorganism;

E. For the fitting curve of monotonous increase or monotonous decrease, select the microorganism with the largest correlation coefficient as the sensitive microorganism, and the microorganism with sudden increase or decrease in the fitting curve as the sensitive microorganism of the environmental factor;

F. Utilize the effective bacterial genera information table for screening sensitive microorganisms; fit the response curves of various species of microorganisms to environmental factors based on the generalized additive model, and select the effective fitting curve with a significance lower than 0.01; according to the generalized additive model The fitted curve type of the model estimates the niche sensitivity of various microorganisms (genus level) separately.

A second object of the present invention is to provide the use of an increase in g__Jeotgalicoccus as an indicator of water quality deterioration.

The third object of the present invention is to provide the reduction of g__Sphingobium in indicating the deterioration of water body and the application of the reduction of dissolved oxygen.

The fourth object of the present invention is to provide the reduction of g__Treponma to reflect the application of COD reduction in water bodies.

The fifth object of the present invention is to provide an increase in g_Yersinia to reflect the application of an increase in total phosphorus in water bodies.

A sixth object of the present invention is to provide the use of an increase in g__Pleomorphomomas as an indicator of water quality deterioration.

Compared with the prior art, the present invention has the following technical effects:

1. This method is highly directional, indicating characteristic pollutants by using changes in the structure and function of specific microbial groups;

2. Using microbial niche screening, scientific and reasonable sensitive microbial groups can be obtained;

3. This method uses microbial indicators to reflect changes in water quality, and at the same time has an indicative effect on characteristic pollutants;

4. This method is not limited to special environmental indicators, and has good applicability to urban river ecosystems;

5. The method can quickly, accurately and objectively reflect the health status of urban river ecosystems.

Description of drawings

Figure 1 is a sampling map of river ecosystems in Guangzhou;

Fig. 2 is sensitive microorganism type 1-environmental factor water temperature (WT);

Fig. 3 is sensitive microorganism type 2-environmental factor nitrate nitrogen (NO ₃ ^-N );

Fig. 4 is sensitive microorganism type 3-environmental factor pH;

Figure 5 shows the relative abundance of sensitive microorganisms in different water quality grades.

Detailed ways:

The following examples are to further illustrate the present invention, but not to limit the present invention.

Example 1

Sampling point selection: This sampling point is located in Guangzhou and surrounding rivers, mainly concentrated in the Pearl River Delta, which is a typical river city.

1. The sampling site is shown in Figure 1.

According to the flow direction of the river, it is mainly divided into urban areas, suburban areas, and rural areas far away from the city. Sampling points 8, 8, and 8 were selected respectively, and 3 repetitions were taken each time. A total of 10 samples were taken, with a total of 240 samples. The scale of repeated sampling was Differences, data based on science. The distance between the urban area, the suburban area and the rural area is about 100km, and the total span is 200km, which is used to cover the urban area of Guangzhou and the surrounding river plots.

2. Water sample collection and water quality analysis:

The collection time of water samples is 1 year, with a total of 10 collections in monthly units, and the time span is April, May, June, July, September, October, November, December and January 2019 in 2018 ,February. Determination of 12 indicators of water quality environment indicators pH, water temperature (WT), dissolved oxygen (DO), COD, TN, total organic carbon, nitrate nitrogen, ammonium nitrogen, nitrite nitrogen, total phosphorus, sulfide and fluoride .

3. Main types of water pollution and WQI classification:

Using the classic water quality calculation formula, 5 parameters were selected to evaluate the water quality, including pH, dissolved oxygen (DO), chemical oxygen demand (COD), phosphate, and nitrate nitrogen; the factor scores are as follows:

pH:

If 4.5≤pH<7.0 SI _pH ＝1.9EXP((pH-1)*0.66)

If 7.0≤pH≤7.6 SI _pH = 100

If 7.6<pH≤10.5 SI _pH ＝100exp((pH-7.65)*-0.528)

If 10.5<pH<4.5 SI _pH =10

DO:

If DO<3.3 SI _DO =10

If DO>12.5 SI _DO = 100

If 3.3≤DO≤12.5 SI _DO ＝-59.6+24.9*DO-0.98*DO^2

COD:

SI _COD ＝100*0.86^COD

TP:

If TP<0.03 SI _TP = 100

If TP>1.2 SI _TP = 10

If 0.03<TP<1.2 SI _TP ^- ＝99.5*0.17^(PO ₄ ^3- )

NO _3- ^:

NO ₃ ^- ≤1SINO ₃ ^- ＝100

NO ₃ ^- >1SI _NO3- ＝102*0.8887^(NO ₃ ^- )

WQI calculation formula: n is the number of selected factors, SIi is the factor score

Through calculation and statistics, V and VI were 55 and 76 times in a total of 240 tests (Table 1), and the characteristic pollutants were the main contribution frequencies: 0 times for pH, 42 times for DO, 45 times for COD, TP A total of 38 times, NO ₃ ^- a total of 39 times (Table 2).

Table 1 Statistical table of water quality division of rivers in Guangzhou

WQI range	classification	Water Quality Statistics	water quality
<45	VI	60	very bad
45-60	V	19	Difference
61-69	IV	32	generally
70-79	III	twenty three	General/Excellent
80-90	II	106	excellent
91-100	I	0	excellent

Table 2 Statistical Table of Water Quality Characteristics Pollutants of Rivers in Guangzhou

Types of water pollution	WQI division	SI interval	Statistics
pH	VI and V	SI pH =10	0
do	VI and V	SI DO =10	42
COD	VI and V	SI COD = 10	45
TP	VI and V	SITP = 10	38
NO3-	VI and V	SI NO3- = 10	39

4. Water sample microbial DNA extraction and high-throughput sequencing:

The 2L water sample was filtered through a 0.22 μm filter membrane, and the PowerWater DNA extraction kit (power

Isolation Kit) to extract the microbial DNA of the filter membrane, and carry out DNA extraction according to the operation steps of the kit, and the extracted DNA sample adopts an ultramicro spectrophotometer to measure the concentration and purity of the DNA. Qualified DNA samples were sent to Biomark Biotechnology Co., Ltd. for sequencing. The pCR amplification test of the microbial 16S rRNA fragment was performed by using the bacterial universal primer 338F/806R to amplify the bacterial V3-V4 hypervariable region. The primer sequence used is: pre- Primer 338F (5'-ACTCCTACGGGAGGCAGCA-3') and back primer 806R (5'-GGACTACHVGGGATCTWTCTAAT-3') were sequenced by paired-end sequencing using the Illumina HiSeq 2500 system (Illumina, Unite States) desktop sequencer.

5. Microbial community information analysis:

The high-quality 16S rRNA gene amplicon sequence was analyzed using the open-source quantitative analysis software for microbial ecology (QIIME2), and the sequence reads of DATA2 in the QIIME2 system were used for denoising, and the denoised sequences were normalized with 100% similarity. It is a type of OTU, and finally use SILVA 132 to perform taxonomic classification analysis on the OTU sequence (http://www.mothur.org/wiki/Taxonnomy_outline). When performing bacterial diversity analysis and statistics, the number of amplicon sequences obtained after the determination is equalized, that is, the same number of sequences is randomly selected for all samples based on the lowest sequence number in the sample (flattened) After obtaining 12365 sequences), and obtain the genus level information table.

6. Information table of effective bacterial genera:

In the genus level information table, statistics are made according to the relative abundance of the genus level higher than 1%, 0.5% and 0.01%. After the total relative abundance is higher than 90%, it is divided into an independent table, and then the unnamed OTU of the genus hit is removed , or change the nomenclature to the species level (both classes and orders are acceptable), so as to obtain the effective bacterial genera information table.

7. Screening of sensitive microorganisms:

Microbial susceptibility is classified as follows:

A. Statistics on the relative abundance of microorganisms at the genus level, and those whose relative abundance is higher than 1% in at least one sample point are effective species, which are used for screening of sensitive microorganisms;

B. Fit the response curves of various species of microorganisms to environmental factors based on the generalized additive model, and select the ones with a significance lower than 0.01 as the effective fitting curves;

C. The extension of the generalized additive model and the generalized linear model. Taking two environmental factors as an example, the calculation method is as follows:

log(E(yi)=a+s1(x1i)+s2(x2i)

Where yi is the relative abundance of microorganisms, xi is an environmental parameter, s1(x1), s2(x2) are non-parametric smooth functions, and Log() is an example of a related function;

D. According to the fitting curve type of the generalized additive model, respectively estimate the ecological niche of each genus: for the single-peak fitting curve, the value of the environmental parameter corresponding to the peak value is the most suitable ecological niche for the microorganism;

E. For the fitting curve of monotonous increase or monotonous decrease, select the microorganism with the largest correlation coefficient as the sensitive microorganism, and the microorganism with sudden increase or decrease in the fitting curve as the sensitive microorganism of the environmental factor;

F. Use the effective bacterial genera information table for screening sensitive microorganisms; fit the response curves of various genera microorganisms to environmental factors based on the generalized additive model, and select the effective fitting curves with a significance lower than 0.01. According to the fitting curve type of the generalized additive model, the niche sensitivities of various microorganisms (genus level) were estimated separately:

For the unimodal fitting curve, the value of the environmental parameter corresponding to the peak value is the most suitable ecological niche for the microorganism. After data fitting, the environmental factor water temperature (TW) screening results are g__Altererythrobacter and g__Facklamia, which are fitted to a unimodal model (as shown in Figure 2a ,b); if there are multiple species in a similar niche, the species with the narrowest niche will be used as the sensitive microorganism. Taking TW results as an example, the peak value of g__Altererythrobacter is 8.85, while the peak value of g__Facklamia is 7.98. Altererythrobacter has a stronger response, and the niche of Altererythrobacter is narrower, and Altererythrobacter is more suitable as a sensitive microorganism for TW than Facklamia.

For the fitting curve of monotonically increasing or monotonically decreasing, the microorganism with the largest correlation coefficient was selected as the sensitive microorganism. Take the environmental factor nitrate nitrogen screening results as an example: g__Nitrospira and g__Trichococcus obtained in the results are one-way linear fitting (as shown in Figure 3a, b); The fitted r value was 0.173, the significance level was <0.05, and Trichococcu had a stronger response than Nitrospira.

For the fitting curve with a gentle trend but a sudden increase or drop in a certain interval, the microorganism is taken as the sensitive microorganism in the mutation interval of the environmental factor. Take the environmental factor pH screening results as an example: g__Jeotgalicoccus suddenly increases when pH>8.2 is obtained in the results (Figure 4), and g__Jeotgalicoccus can be regarded as a pH-sensitive microorganism as a factor.

8. Sensitive microorganism statistics:

Use the effective bacterial genus information table for the screening of sensitive microorganisms to determine the sensitive microorganisms of 12 environmental factors (including the characteristic pollutants in the water quality calculation), and obtain the sensitive microbial species of each factor through the sensitive microorganism screening, and obtain them by comparison The most suitable sensitive microorganisms, the results are shown in Table 3: a total of 11 kinds of sensitive microorganisms of environmental factors were obtained, of which 11 were used for total nitrogen, only 1 was used for dissolved oxygen and COD, and fluoride was not effective for sensitive microorganisms, because 10 Fluoride levels in the monthly samples were low and the microbes did not respond to it.

Table 3 Statistics table of sensitive microorganisms of various environmental factors

9. Reliability analysis of evaluation results

In order to verify that this method has an evaluation effect on water quality, the relative abundance of sensitive microorganisms obtained in this study and the water quality IWQ were divided and counted. The results are shown in Figure 5. The representative sensitive microorganisms (g__Jeotgalicoccus) of pH increased sharply in VI water body , relative to other water quality increased by 1190%, the sharp increase of g__Jeotgalicoccus indicated water quality deterioration (Fig. 5a); the relative abundance of dissolved oxygen representative sensitive microorganisms (g__Sphingobium) in V and VI water bodies was low, and the average decrease was 73% compared to other water quality % and 77%, the drastic decrease of g__Sphingobium not only represents the deterioration of the water body, but also reflects the decrease of dissolved oxygen (Fig. 5b); It reflects the reduction of COD (Figure 5c); the detection value of the representative sensitive microorganism (g__Yersinia) of total phosphorus (TP) in IV, V and VI water bodies is extremely low, indicating that it has a good indicator effect on the increase of TP (Figure 5d); The relative abundance of nitrate-nitrogen-sensitive microorganisms (g__Pleomorphomomas) increased sharply in VI water body, indicating that the increase of nitrate-nitrogen can promote its growth, and its abundance change can represent the change of water quality (Fig. 5e).

The health level of urban water ecosystems is affected by many factors, such as: urban development progress, domestic sewage discharge, land use and management, etc., which cause complex pollutants in urban water bodies, which leads to the deterioration of the overall water environment. Therefore, rapid, An accurate and objectively feasible evaluation system requires the identification of water quality deterioration and characteristic pollutants. Therefore, the present invention can quickly judge the water quality and specify the pollution type by screening the sensitive microbial groups of environmental factors (including water body characteristic pollutants) through the change of their relative abundance.

Claims (8)

1. A method for assessing the health of river ecosystems based on the specific response of microbial communities, characterized in that it comprises the following steps:

   a: Set up sampling points in rural areas, urban suburbs and urban centers along the river;

   b: Determination of water quality indicators such as pH, water temperature (WT), dissolved oxygen (DO), COD, TN, total organic carbon, nitrate nitrogen, ammonium nitrogen, nitrite nitrogen, total phosphorus, sulfide and fluoride, Calculate the water quality index IWQ;

   c: Extract water microbial DNA and perform bacterial 16S rRNA gene amplicon sequencing;

   d: Obtain the bacterial genus level information table, and screen the effective bacterial genus information;

   e: Classification of microbial sensitivity based on the niche model;

   f: Reliability analysis of evaluation results.
2. The river ecosystem health assessment method based on microbial community specific response according to claim 1, characterized in that, described step b is:

   Using the water quality calculation formula, 5 parameters were selected to evaluate the water quality, including pH, dissolved oxygen (DO), chemical oxygen demand (COD), phosphate, and nitrate nitrogen; the factor scores are as follows:

   pH:

   If4.5≤pH<7.0 SI _pH ＝1.9EXP((pH-1)*0.66)

   If7.0≤pH≤7.6 SI _pH ＝100

   If7.6<pH≤10.5 SI _pH ＝100exp((pH-7.65)*-0.528)

   If10.5<pH<4.5 SI _pH ＝10

   DO:

   If DO < 3.3 SI _DO = 10

   If DO>12.5 SI _DO = 100

   If3.3≤DO≤12.5 SI _DO ＝-59.6+24.9*DO-0.98*DO^2

   COD:

   SI _COD ＝100*0.86^COD

   TP:

   IfTP< _0.03SITP ＝100

   IfTP> _1.2SITP ＝10

   If0.03<TP<1.2 SI _TP ^- ＝99.5*0.17^(PO ₄ ^3- )

   NO _3- ^:

   NO ₃ ^- ≤1 SI _NO3 -＝100

   NO ₃ ^- >1 SI _NO3 -＝102*0.8887^(NO ₃ ^- )

   WQI calculation formula: n is the number of selected factors, SIi is the factor score

   
3. The river ecosystem health evaluation method based on microbial community specific response according to claim 1, is characterized in that, microbial sensitivity is divided as follows:

   A. Statistics on the relative abundance of microorganisms at the genus level, and those whose relative abundance is higher than 1% in at least one sample point are effective species, which are used for screening of sensitive microorganisms;

   B. Fit the response curves of various species of microorganisms to environmental factors based on the generalized additive model, and select the ones with a significance lower than 0.01 as the effective fitting curves;

   C. Extension of generalized additive model and generalized linear model;

   Taking two environmental factors as an example, the calculation method is as follows:

   log(E(yi)=a+s1(x1i)+s2(x2i)

   Where yi is the relative abundance of microorganisms, xi is an environmental parameter, s1(x1), s2(x2) are non-parametric smooth functions; Log() is an example of a related function;

   D. According to the fitting curve type of the generalized additive model, respectively estimate the ecological niche of each genus: for the single-peak fitting curve, the value of the environmental parameter corresponding to the peak value is the most suitable ecological niche for the microorganism;

   E. For the fitting curve of monotonous increase or monotonous decrease, select the microorganism with the largest correlation coefficient as the sensitive microorganism, and the microorganism with sudden increase or decrease in the fitting curve as the sensitive microorganism of the environmental factor;

   F. Utilize the effective bacterial genera information table for screening sensitive microorganisms; fit the response curves of various species of microorganisms to environmental factors based on the generalized additive model, and select the effective fitting curve with a significance lower than 0.01; according to the generalized additive model The fitted curve type of the model estimates the niche sensitivity of various microorganisms (genus level) separately.
4. Use of an increase in g__Jeotgalicoccus as an indicator of water quality deterioration.
5. Application of the reduction of g__Sphingobium in indicating the deterioration of water bodies and the reduction of dissolved oxygen.
6. The reduction of g__Treponma is used to reflect the reduction of COD in water bodies.
7. The increase of g__Yersinia reflects the application of the indicator of the increase of total phosphorus in the water body.
8. Use of an increase in g__Pleomorphomomas as an indicator of water quality deterioration.

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