CN113122619B - Method for tracing dominant strain source blocked by aquifer microorganisms in artificial recharge process - Google Patents

Abstract

The invention discloses a method for tracing the blockage of aquifer microorganisms in an artificial recharge process to dominant strain sources, which comprises the following steps: filling the sterilized quartz sand into an organic glass column; collecting water samples from a recharge well and river water respectively, and preparing a sterilization nutrient solution; simulating different recharging modes for recharging by using a manual recharging simulation device; recording the water outlet volume and the water heads of the pressure measuring pipes during recharging, further calculating the saturation permeability coefficient, and reflecting the blocking condition of the medium in the column body of the organic glass column through the change of the relative saturation permeability coefficient; performing microbiological analysis on the water sample I, the water sample III and the sand sample collected after the recharge test; by comparing the saturated permeability coefficient and microbial community characteristics of the aquifer medium in different recharging modes, the source tracing is carried out on the dominant bacteria in the blocking process. The invention can provide scientific reference for improving the efficiency of artificial recharge of the aquifer and preventing and treating the blockage of the aquifer.

Description

A method to trace the origin of the dominant strains for microbial clogging in aquifers during artificial recharge method

technical field

The invention belongs to the field of groundwater environmental protection, and in particular relates to a method for tracing the source of dominant bacteria species blocked by microorganisms in an aquifer during artificial recharge of groundwater.

Background technique

The artificial recharge of groundwater can not only effectively solve many environmental problems caused by over-exploitation of groundwater resources, but also is the only way to realize the joint scheduling of surface and underground water resources, optimize the allocation of water resources, and improve the comprehensive utilization rate of water resources. However, a large number of engineering practices have shown that due to the mismatch of the quality of the recharge water and the formation water, the recharge efficiency is reduced or even the recharge well is seriously blocked. According to the cause of clogging, aquifer clogging can be divided into physical clogging, chemical clogging and microbial clogging. Among them, once the microbial blockage is formed, the permeability of the aquifer is difficult to recover, and it is easy to form a compound effect with physical and chemical blockage, which accelerates the rate of aquifer blockage. Artificial recharge involves the joint scheduling of surface water and groundwater, and the characteristics of microbial communities in different water sources are quite different. At present, there is not enough research on the source of bacteria that cause aquifer clogging during artificial recharge.

SUMMARY OF THE INVENTION

Based on the above-mentioned technical problems, the present invention proposes a method for tracing the source of the dominant bacteria species blocked by microorganisms in the aquifer during the artificial recharge process.

The technical solution adopted by the present invention is:

A method for tracing the source of dominant bacterial species blocked by microorganisms in an aquifer during artificial recharge, which adopts an artificial recharge simulation device, the device comprises a plexiglass column, and the top inlet of the plexiglass column is connected with a water inlet tank through a water inlet pipe, and the device is connected to a water inlet tank through a water inlet pipe. The water pipe is provided with a water head device, the bottom outlet of the plexiglass column is connected to the water outlet through the water outlet pipe, an overflow port is arranged on the upper part of one side of the plexiglass column, and the other side of the plexiglass column is connected with a pressure measuring device at intervals up and down. A pressure measuring plate is provided at the end of the pressure measuring pipe;

The method includes the following steps:

(1) packing the sterilized quartz sand into the plexiglass column;

(2) Take two groundwater samples from the recharge well, one for groundwater microbial sequencing, denoted as water sample 1, and the other for return tank simulation, denoted as water sample 2; and from the river water Two river water samples were collected in 2019, one water sample was used for microbial sequencing of river water, recorded as water sample 3, and the other water sample was used for back-tank simulation, recorded as water sample 4;

(3) Prepare sterile nutrient solution in the laboratory;

(4) Using the water samples 2 and 4 in step (2) and the sterilized nutrient solution in step (3) as the refilling liquid, the artificial refilling simulation device is used to simulate different refilling methods for refilling ;

The refilling liquid in the water inlet tank enters from the top inlet of the plexiglass column through the fixed water head device, flows through the plexiglass column, and is discharged from the bottom outlet of the plexiglass column; every period of time, the effluent volume and The water head of each piezometer, and then the saturated permeability coefficient is calculated, and the blockage of the medium in the plexiglass column is reflected through the change of the relative saturated permeability coefficient;

(5) Carry out microbiological analysis on water sample 1, water sample 3 and sand samples collected after the recharge test;

(6) By comparing the saturated permeability coefficient and microbial community characteristics of the aquifer medium under different recharge methods, the dominant bacteria during the plugging process were traced.

Preferably, the plexiglass column is 20cm high and has an inner diameter of 5cm; the overflow port is set at a position 2cm from the left side of the plexiglass column from the top, and the right side of the plexiglass column is 4, 6, 8, 10 and 4 inches from the top. Ports communicated with the pressure measuring tube are respectively set at 20 cm; the effective filling height of the plexiglass column is 16 cm; the water head constant device is a peristaltic pump.

Preferably, in step (1): the particle size of the quartz sand is 0.5 mm, the sterilization process of the quartz sand is sterilization in an oven at 120-130 ° C for 6-8 hours, and the quartz sand is used as an aqueous medium for the simulation device, etc. Thickness wet packing into a plexiglass column.

Preferably, in step (1), the plexiglass column adopts the method of wet column protection in the process of filling quartz sand, and the specific steps are as follows: inject saturated column water into the plexiglass column with the water inlet and outlet valves closed, and weigh once A certain weight of sterilized quartz sand is filled with a plexiglass column, and the sand column is compacted with a constant pressure after two times, and the test is repeated.

Preferably, in step (3): the preparation of the nutrient solution adopts glucose, NaNO ₃ and K ₂ HPO ₄ as the only carbon, nitrogen and phosphorus sources for the growth of microorganisms; Sterilize for 10-15min.

Preferably, in step (4), there are four types of recharge methods: first, river water is used to recharge the groundwater-saturated aquifer, that is, a water sample bisaturated column is used, and four water samples are used to recharge; the second method is sterilization The nutrient solution recharges the groundwater-saturated aquifer, that is, the water sample bisaturated column is used, and the sterilized nutrient solution is used to recharge. For the saturated column, four water samples are used for refilling; the fourth type, the sterilized nutrient solution is used to refill the sterilized deionized water saturated aquifer, that is, the sterilized deionized water saturated column is used, and the sterilized nutrient solution is used for refilling.

Preferably, in step (4): every 4 hours, record the water outlet volume of the artificial recharge simulation device and the water head of each pressure measuring tube.

Preferably, in step (4): the formula for calculating the saturated permeability coefficient is

In the formula, the superscript i refers to a certain layer in the sand column; K _s ⁱ represents the K _s of the i-th layer, specifically K _s ¹ corresponds to the saturated permeability coefficient in the interval of 4 to 6 cm from the top of the sand column, K _s ² Corresponds to the saturated permeability coefficient from 6 to 8 cm from the top of the sand column, K _s ³ corresponds to the saturated permeability coefficient from 8 to 10 cm from the top of the sand column, K _s ⁴ corresponds to the saturated permeability coefficient from 10 to 20 cm from the top of the sand column, K _s ^1-4 corresponds to the saturated permeability coefficient from 4 to 20 cm from the top of the sand column; Q is the seepage flow, that is, the volume flow (mL/ ^s ) through each section of the sand column, and Li is the permeation path, that is, the water-passing section between the hydraulic heads distance (cm), A is the cross-sectional area (cm ² ) of the water-passing section, (ΔH) ⁱ is the head difference (cm) of the water-passing section.

Preferably, in step (4):

The relative saturation permeability coefficient (K _s ⁱ )' of the medium is used to characterize the recharge blockage, that is, the ratio of the K _s ⁱ value at t to its initial value, that is, the K _s ⁱ value at zero;

When the value of (K _s ⁱ )' dropped to 0.1, that is, when the saturated permeability coefficient of the sand column decreased to 10% of the initial value, the sand column was considered to be blocked; the sand column was removed and sampled for microbial community analysis.

Preferably, in step (5):

Filter water sample 1 and water sample 3 with a 0.45μm PES microporous membrane, transfer the microporous membrane to a sterile centrifuge tube immediately, and store at -80°C;

After the recharge test, use a sterilized spoon to collect the sand sample on the surface of the sand column, immediately transfer it to a sterile centrifuge tube, and store it at -80°C;

High-throughput sequencing was used, and the sequencing results were used to analyze and compare the diversity, richness and correlation of microbial communities, and screen out the sources of dominant strains.

The beneficial technical effects of the present invention are:

By comparing the saturated permeability coefficient and microbial community characteristics of the aquifer medium under different recharge methods, the invention traces the source of dominant bacteria in the clogging process, and can provide scientific reference for improving the efficiency of artificial recharging aquifers and preventing aquifer clogging.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments:

Fig. 1 is the schematic diagram of the structure of the artificial recharge simulation device involved in the present invention;

Fig. 2 is the use of hierarchical clustering heat map in the application example of the present invention;

Fig. 3 is the principal coordinate analysis diagram based on the weighted Unifrac distance in the application example of the present invention.

Detailed ways

In view of the lack of in-depth research on the source of the bacteria that cause the clogging of the aquifer during the current artificial recharge process, the present invention traces the cause of the clogging of the aquifer by simulating the artificial recharging process of the aquifer, through the calculation of the saturated permeability coefficient and the analysis of the characteristics of the microbial community. The source of microbial dominant strains.

The present invention will be described in detail below.

A method for tracing the source of dominant strains of aquifer blockage by microorganisms in the process of artificial recharge, using an artificial recharge simulation device, as shown in Figure 1, the device includes a plexiglass column 1, and the top inlet of the plexiglass column 1 passes through a water inlet pipe 2 is communicated with the water inlet tank 3, and a water head fixing device 4 is arranged on the water inlet pipe 2. The bottom outlet of the plexiglass column 1 is communicated with the water outlet tank 6 through the water outlet pipe 5 . An overflow port 7 is arranged on the upper part of one side of the plexiglass column 1 , a plurality of pressure measuring tubes 8 are connected up and down on the other side of the plexiglass column 1 , and a pressure measuring plate 9 is arranged at the end of the pressure measuring tube 8 .

The overflow port 7 communicates with the overflow groove 11 through the overflow pipe 10 .

Specifically, the height of the above-mentioned plexiglass column is 20cm, and the inner diameter is 5cm; the overflow port is arranged at the left side of the plexiglass column at a distance of 2cm from the top, and the right side of the plexiglass column is 4, 6, 8, 10, and 20cm from the top. Ports communicated with the pressure measuring tube are respectively arranged at each of the ports, and each port is communicated with a pressure measuring tube. The effective packing height of the plexiglass column is 16 cm.

The constant water head device is used to provide constant pressure, and specifically, a peristaltic pump can be selected.

The method includes the following steps:

(1) Fill the sterilized quartz sand into the plexiglass column.

Specifically, standard quartz sand with a particle size of 0.5 mm was sterilized in an oven at 121° C. for 6 h, and was used as an aqueous medium for the device to be packed into a plexiglass column with an effective filling height of 16 cm with a wet saturated column of equal thickness (about 2 cm). Inside.

Wet saturated column packing includes the following steps: inject saturated column water into the plexiglass column with the water inlet and outlet valves closed, weigh 92 g of sterilized quartz sand, fill the plexiglass column, and compact the sand column with constant pressure after twice, repeat In the test, each plexiglass column needs at least 920g of quartz sand to be saturated. The sand column is fully saturated for more than 12h.

(2) Take two groundwater samples from the recharge well, one for groundwater microbial sequencing, denoted as water sample 1, and the other for back-tank simulation, denoted as water sample 2. And two river water samples were collected from the river water, one water sample was used for river water microbial sequencing, which was recorded as water sample 3, and the other water sample was used for back-tank simulation, which was recorded as water sample 4.

(3) Prepare sterile nutrient solution in the laboratory.

When preparing the nutrient solution, glucose (2.20mg/L), NaNO ₃ (0.067mg/L) and K ₂ HPO ₄ (0.42mg/L) were used as the sole carbon, nitrogen and phosphorus sources for microbial growth, respectively. The nutrient solution was autoclaved (121°C at 0.987ATM) for 15min.

(4) Using the water samples 2 and 4 in step (2) and the sterilized nutrient solution in step (3) as the refilling liquid, the artificial refilling simulation device is used to simulate different refilling methods for refilling .

There are four types of recharge methods: first, river water recharges groundwater-saturated aquifers, that is, using water sample two-saturated column, using water sample four recharge methods, which is counted as GR method; second, sterilized nutrient solution recharge The groundwater-saturated aquifer, that is, the water sample bisaturated column is used, and the sterilized nutrient solution is recharged, which is regarded as the GN method; the third way, the river water is recharged to the sterilized deionized water-saturated aquifer, that is, the sterilized deionized water is used. For the water-saturated column, four refills of water samples are used, which is regarded as the DR method; the fourth method is to refill the sterilized nutrient solution into the sterilized deionized water-saturated aquifer, that is, the sterilized deionized water saturated column is used, and the sterilized nutrient solution is used. Liquid refilling is counted as Con mode.

Specifically, the test steps of each recharge method are as follows:

The refill liquid in the water inlet tank enters from the top inlet of the plexiglass column through the constant water head device (the constant water head difference is 15cm), flows through the plexiglass column, and is discharged from the bottom outlet of the plexiglass column. On the basis of Darcy's law and hydraulic changes, every 4h, that is, from the 0th, 4th, 8th, 12th, 16th... The saturated permeability coefficient in the layer reflects the blockage of the medium in the plexiglass column through the change of the relative saturated permeability coefficient.

The formula for calculating the saturated permeability coefficient is:

In the formula, the superscript i refers to a certain layer in the sand column; K _s ⁱ represents the K _s of the i-th layer, specifically K _s ¹ corresponds to the saturated permeability coefficient in the interval of 4 to 6 cm from the top of the sand column, K _s ² Corresponds to the saturated permeability coefficient from 6 to 8 cm from the top of the sand column, K _s ³ corresponds to the saturated permeability coefficient from 8 to 10 cm from the top of the sand column, K _s ⁴ corresponds to the saturated permeability coefficient from 10 to 20 cm from the top of the sand column, K _s ^1-4 correspond to the saturated permeability coefficient from 4 to 20 cm from the top of the sand column; Q is the seepage flow, that is, the volume flow (mL/ ^s ) through each section of the sand column, and Li is the permeation path, that is, the water-passing section between the hydraulic heads distance (cm), A is the cross-sectional area (cm ² ) of the water-passing section, (ΔH) ⁱ is the head difference (cm) of the water-passing section.

The relative saturation permeability coefficient (K _s ⁱ )' of the medium is used to characterize the recharge blockage, that is, the ratio of the K _s ⁱ value at t to its initial value, that is, the K _s ⁱ value at zero. When the value of (K _s ⁱ )' dropped to 0.1, that is, when the saturated permeability coefficient of the sand column decreased to 10% of the initial value, the sand column was considered to be blocked; the sand column was removed and sampled for microbial community analysis.

(5) Microbiological analysis of water sample 1, water sample 3 and sand samples collected after the recharge test.

The sampling steps for water sample 1 and water sample 3 are as follows: filter water sample 1 and water sample 3 with a 0.45μm PES microporous filter membrane, transfer the microporous filter membrane to a sterile centrifuge tube immediately, and store them in - 80°C.

The sampling steps for sand samples are as follows: after the percolation experiment, use a sterilized spoon to sample about 0.5g of sand in the first layer of each sand column (the sand layer 2 to 4 cm from the top of the sand column), and then immediately transfer it to a non-toxic area. bacteria in centrifuge tubes and store at -80°C.

High-throughput sequencing of sand samples and filtered river and groundwater to compare microbial community diversity, richness, and correlation.

A brief introduction to high-throughput sequencing is as follows:

Final DNA concentration and purity were determined by UV-Vis spectrophotometer and DNA quality was checked on 1% agarose gel electrophoresis by thermocycler PCR system with universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5' '-GGACTACHVGGGTWTCTAAT-3') amplifies the V3-V4 hypervariable region of the bacterial 16S rRNA gene. PCR reaction conditions: initial denaturation at 95 °C for 3 min, followed by 27 cycles of reaction (30 s at 95 °C, 30 s at 55 °C, 45 s at 72 °C), and finally extension at 72 °C for 10 min. The PCR reaction system was: 20 μL mixture containing 5×FastPfu buffer (4 μL), 2.5 mM dNTP (2 μL), primers (5 μM, 0.8 μL), FastPfu polymerase (0.4 μL) and 10 ng template DNA. The product of the PCR reaction was extracted from a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit. A heatmap with Bray-Curtis distance differences was generated based on the most abundant OTUs of bacterial communities using the R package, as shown in Figure 2. Based on OTU data, alpha diversity indices including richness (Chao), bacterial community diversity (Shannon index), evenness and coverage were obtained using Mothur. Based on OTU data, co-ordinate analysis (PCoA), analysis of similarity (ANOSIM) using weighted Unifrac distances were used to test for population differences between bacterial populations.

(6) By comparing the saturated permeability coefficient and microbial community characteristics of aquifer media under different recharge methods, including microbial community diversity (Shannon index), richness (Chao) and correlation analysis (cluster heat map), etc. The similarity between the microbial community and the original groundwater and river water under different recharge methods can trace the source of the dominant bacteria during the plugging process, that is, screen out the source of the dominant bacteria.

The present invention is further described below by specific application example:

Experimental samples and experimental site: In situ groundwater was collected from a recharge well in the lower reaches of the Dagu River (36.370318°N, 120.159088°E), and river water was collected from the nearby Xiaoqing River (36.370203°N, 120.159526°E). The Dagu River is 179.9km long, with a drainage area of 4161.9km ² . The average gradient of the river is 1.2/1000, the total drop is about 200m, the average width of the river is 460m, and the river network density is 0.34km/km ² . This experiment was carried out in Shandong University of Science and Technology Geology and Groundwater Physics Simulation Laboratory and Water Chemistry Laboratory.

Recharge device setup: use standard quartz sand (d ₆₀ = 0.5 mm; Guangzhou Vigorous Medical Equipment Co., Ltd., Guangzhou, China), sterilize in an oven at 121 °C for 6 h, and use as the device porous medium at equal increments (approx. 2 cm) into a plexiglass column with an effective height of 16 cm and an inner diameter of 5 cm.

Test treatment: The present invention simulates 4 different recharge modes: (1) The river water recharges the groundwater-saturated aquifer, which is referred to as the GR mode; (2) The sterilized nutrient solution recharges the groundwater-saturated aquifer, which is referred to as the GN mode (3) River water refills the sterilized deionized water-saturated aquifer, which is regarded as the DR mode; (4) The sterilized nutrient solution refills the sterilized deionized water-saturated aquifer, which is regarded as the Con mode. Two groups of parallel experiments were set up for the four recharge methods.

Blockage monitoring: During the recharge process, the head pressure is monitored every 4 hours, and the relative saturated permeability coefficient is calculated.

Microbial sequencing analysis: when the relative saturated permeability coefficient reaches 0.1, remove the column, use a sterilizing spoon to sample about 0.5g of sand in the first layer of each sand column (the sand layer 2-4cm from the top of the sand column), and transfer it immediately into sterile centrifuge tubes and store at -80°C. High-throughput sequencing was performed on sand samples in four sand columns and filtered river water and groundwater to compare the diversity, richness and correlation of microbial communities in four recharge methods and in situ groundwater and river water.

results and analysis:

1. The permeability coefficient of the test group in each time period

The relative saturated permeability coefficient can characterize the plugging degree of artificial recharge, as shown in Table 1, the reduced (K _s ¹ )' value, indicating that the sand column is obviously plugged under different recharge methods. Taking the GR column as an example, when the recharge test was carried out for 16 hours, the relative saturated permeability coefficient (K _s ¹ )' value of the surface layer of the sand column gradually decreased from 1.0 to 0.736, with a decrease rate of 26.4%, and then decreased from 0.736 to 0.103. The permeability test Finish. In the GR', GN, GN', DR and DR' columns, the relative saturated permeability coefficients were 0.117, 0.110, 0.041, 0.083 and 0.002, respectively. On the contrary, the (K _s ¹ )' values in the Con and Con' sand columns fluctuated around 1.0, indicating that there was no plugging in the control group; in different sand layers, the relative saturated permeability coefficient decreased, which further proved that in the combined layer of the sand column Blockage also occurs. At the end of the infiltration experiment, the (K _s ^1-4 )' values of the GR, GR', GN, GN', DR and DR sand columns were 0.355, 0.301, 0.419, 0.237, 0.371 and 0.017, respectively, less than the first in the corresponding sand columns. layer value. This result shows that the degree of clogging gradually decreases as the sand column goes from top to bottom.

2. Diversity, richness and correlation of microbial communities in each experimental group

Table 2 shows the alpha diversity indices (ie, samG and samR) of bacterial communities in in situ groundwater and river water. For samG and samR, the mean values of the observed OTU numbers were 1158 and 219, the OUT number index mean values were 1410 and 240, the diversity index mean values were 4.22 and 1.73, and the homogeneity index mean values were 0.60 and 0.32, respectively, It shows that the diversity and uniformity of bacterial community in groundwater is much higher than that in river water. Table 2 also shows the α-diversity index of bacterial communities in biofilms under different feeding regimes. For samGR, samGN and samDR, the mean values of the observed OTUs were 219, 178, and 130, the OUT number index mean values were 312, 250, and 150, and the diversity index mean and homogeneity index mean were 3.05, 2.48, respectively. , 0.31 and 0.57, 0.48, and 0.48, indicating that the diversity and uniformity of bacterial communities in the method of river water recharging groundwater-saturated aquifers are much higher than those of sterilized nutrient solution recharging groundwater-saturated aquifers and river water recharging sterilization and deionization. Water-saturated aquifer approach.

Table 3 shows the bacterial community composition in in situ groundwater and river water. Among the phyla, Proteobacteria is the most dominant phylum, followed by Bacteroidetes, Actinobacteria, Durabacteria, Rodobacteria, and Campylobacter aeruginosa. In samG, the main phyla are Proteobacteria, Bacteroidetes, Actinobacteria, and subterranean environments, and in samR, the first four main phyla are Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, These bacteria are broadly similar to other communities in freshwater aquatic life. At the class level, samG is dominated by species C bacteria, Bacteroidetes and Actinomycetes, but only gamma Proteobacteria is the dominant class in samR, indicating that the diversity and homogeneity of bacterial communities in groundwater is much higher in river water. At the genus level, the four genera dominated by samG were Hydrogenobacter, Acetobacter, Pseudomonas, and Rhodococcus, and in samR, Acinetobacter was absolutely dominant, followed by Exiguobacterium, indicating that the genus in groundwater More genera than in river water. Table 3 also shows the bacterial community composition of biofilms with different recharge methods. At the phylum level, Proteobacteria is the most important phylum, followed by Bacteroidetes, Actinobacteria and Bacteroides. In samGR and samGN, Proteobacteria were absolutely dominant, followed by Bacteroidetes and Actinobacteria. Compared with samGR and samGN, the average relative abundance of bacteria in samDR was lower, while the average relative abundance of Bacteroides was higher. At the class level, the dominant Proteobacteria are subdivided into alpha-proteobacteria and gamma-proteobacteria. Among the nonproteobacterial species, Bacillus predominates in samGR and samGN. At the genus level, the dominant genera in samGR and samGN were Acinetobacter, Exiguobacterium and Porphyromonas. This result shows that the bacterial community structures in samGR and samGN are similar. Compared with these samples, there were differences in bacterial community composition in samDR, with the top three being Clostridium, Sphingobacter, and Acinetobacter, respectively.

Figure 2 uses a hierarchical clustering heatmap to clearly show bacterial community structure. Genus-level-based heatmap showing the distribution of fifty bacteria-rich genera found in groundwater, river water, and biofilms. As can be seen, samGR and samGN aggregate preferentially, indicating that the bacterial community structure in these samples is extremely similar. For samDR, the greatest caloric value occurred in Clostridium, followed by Sphingosine, Acinetobacter, Bacillus and Brevibacterium, which were also in the region of samGR and samGN, while the caloric value was relatively low.

Figure 3 is a principal coordinate analysis (PCoA) based on weighted Unifrac distance, revealing differences in bacterial community composition between different samples. The results showed that PC1 accounted for 48.27% of the variance, and PC2 accounted for 37.88% of the variance. The results showed that the samples were divided into different groups due to different sources of samples, and the difference between groups was significantly higher than the difference within groups (ANOSIM, R=0.9837, P=0.001). Compared with the bacterial community structure in samDR, The bacterial community structures in samGR and samGN were more similar. Figure 3 also shows that the bacterial community structures of in situ groundwater and river water, namely samG and samR, are far away from those of biofilms, namely samGR, samGN and samDR. This result shows that the bacterial community structure in natural systems is completely different from that in artificial conditions, and river water is the most important factor affecting bacterial community composition.

According to the above results, it is concluded that there is blockage in each layer of the sand column, and the degree of blockage gradually decreases with the sand column from top to bottom. By comparing the bacterial community characteristics of in situ groundwater and river water under different recharge methods, the diversity and uniformity of bacterial community in groundwater are much higher than those in river water, and the bacterial community composition after simulated recharge is similar to that in in situ river water. , it was traced that the in situ river water was the source of the dominant strains for microbial blockage in the aquifer during the artificial recharge process.

Table 1

Table 2

table 3

Claims (6)

1. A method for tracing the source of a dominant strain blocked by aquifer microorganisms in the process of artificial recharge is characterized in that an artificial recharge simulation device is adopted, the device comprises an organic glass column, the top inlet of the organic glass column is communicated with a water inlet tank through a water inlet pipe, a constant water head device is arranged on the water inlet pipe, the bottom outlet of the organic glass column is communicated with a water outlet tank through a water outlet pipe, an overflow port is arranged at the upper part of one side of the organic glass column, pressure measuring pipes are connected to the other side of the organic glass column at intervals from top to bottom, and a pressure measuring plate is arranged at the tail end of each pressure measuring pipe;

the method comprises the following steps:

(1) filling the sterilized quartz sand into an organic glass column;

(2) two groundwater water samples are extracted from the recharge well, one water sample is used for groundwater microbial sequencing and is marked as a water sample I, and the other water sample is used for recharge simulation and is marked as a water sample II; collecting two river water samples from river water, wherein one water sample is used for river microorganism sequencing and is marked as a water sample three, and the other water sample is used for recharge simulation and is marked as a water sample four;

(3) preparing a sterilization nutrient solution in a laboratory;

(4) taking the water sample II and the water sample IV in the step (2) and the sterilized nutrient solution in the step (3) as recharging solutions, and simulating different recharging modes for recharging by using a manual recharging simulation device;

the recharging liquid in the water inlet tank enters from the top inlet of the organic glass column through the constant water head device, flows through the organic glass column and is discharged from the bottom outlet of the organic glass column; recording the water outlet volume of the artificial recharge simulating device and the water heads of the pressure measuring tubes at intervals, further calculating the saturation permeability coefficient, and reflecting the blocking condition of the medium in the column body of the organic glass column through the change of the relative saturation permeability coefficient;

(5) performing microbiological analysis on the water sample I, the water sample III and the sand sample collected after the recharge test;

(6) tracing the dominant bacteria in the plugging process by comparing the saturated permeability coefficient and microbial community characteristics of the aquifer medium in different recharging modes;

in the step (4), the recharging method comprises four steps: firstly, river water is recharged to the groundwater saturated aquifer, namely a water sample secondary saturation column is adopted, and a water sample is recharged for four times; secondly, recharging the underground water saturated aquifer by using the sterilizing nutrient solution, namely adopting a water sample secondary saturated column and recharging by using the sterilizing nutrient solution; thirdly, the river water is refilled with the sterilized deionized water saturated aquifer, namely, a sterilized deionized water saturated column is adopted, and a water sample is refilled for four times; fourthly, recharging the sterilized nutrient solution into the sterilized deionized water saturated water aquifer, namely adopting a sterilized deionized water saturated column and recharging the sterilized nutrient solution;

in the step (4), the relative saturation permeability coefficient (K) of the medium is adopted _s ⁱ ) ' characterise recharge blocks, i.e. K at t _st ⁱ The value and its initial value, K at zero _s ⁱ The ratio of the values;

when (K) _s ⁱ ) When the value is reduced to 0.1, namely the saturation permeability coefficient of the sand column is reduced to 10 percent of the initial value, the sand column is considered to be blocked; the sand column is disassembled and sampled for microbial community analysis;

in the step (5), filtering the water sample I and the water sample III by using a 0.45 mu m PES microfiltration membrane, immediately transferring the microfiltration membrane into a sterile centrifuge tube, and storing at-80 ℃;

after the recharging test is finished, collecting sand samples on the surface layers of the sand columns by using a disinfection spoon, immediately transferring the sand samples into a sterile centrifuge tube, and storing the sand samples at-80 ℃;

adopting high-throughput sequencing, and utilizing sequencing results to analyze and compare the diversity, the abundance and the correlation of microbial communities so as to screen out dominant strain sources;

in the step (4): the saturated permeability coefficient is calculated by the following formula

In the formula, the superscript i refers to a certain layer in the sand column; k _s ⁱ K representing the i-th layer _s In particular K _s ¹ Corresponding to a saturation permeability coefficient, K, in the interval of 4 to 6cm from the top of the sand column _s ² Corresponding to a saturation permeability coefficient, K, of 6 to 8cm from the top of the sand column _s ³ Corresponding to a saturation permeability coefficient, K, of 8 to 10cm from the top of the sand column _s ⁴ Corresponding to a saturation permeability coefficient, K, of 10 to 20cm from the top of the sand column _s ^1-4 Corresponding to a saturation permeability coefficient of 4 to 20cm from the top of the sand column; q is the osmotic flow, i.e., the volume flow through each section of the sand column, mL/s, L ⁱ Is a penetration path, i.e. the distance of the cross-section between the hydraulic heads is cm, and A is the cross-sectional area of the cross-section ² ，(ΔH) ⁱ Is the water head difference c of the water passing sectionm。

2. The method for tracing the blockage of the aquifer microorganism on the source of the dominant species in the artificial recharge process as claimed in claim 1, wherein: the height of the organic glass column is 20cm, and the inner diameter of the organic glass column is 5 cm; the overflow port is arranged at the position 2cm away from the top of the left side of the column body of the organic glass column, and ports communicated with the pressure measuring pipe are respectively arranged at the positions 4, 6, 8, 10 and 20cm away from the top of the right side of the column body of the organic glass column; the effective filling height of the organic glass column is 16 cm; the constant head device is a peristaltic pump.

3. The method for tracing the blockage of the aquifer microorganism with the dominant species source in the artificial recharge process as claimed in claim 1, wherein in the step (1): the particle size of the quartz sand is 0.5mm, the sterilization process of the quartz sand is to sterilize in an oven at the temperature of 120-130 ℃ for 6-8h, and the quartz sand is used as an aqueous medium of a simulation device and is filled into an organic glass column in a wet mode with equal thickness.

4. The method for tracing the blockage of the aquifer microorganisms with the dominant bacterial source in the artificial recharge process according to claim 1, wherein in the step (1), the organic glass column is filled with quartz sand in a wet column saturation manner, and the specific steps are as follows: and (3) injecting the saturated column water into an organic glass column with the water inlet and outlet valves closed, weighing a certain weight of sterilized quartz sand once, filling the organic glass column, compacting the sand column with constant pressure after twice, and repeating the test.

5. The method for tracing the blockage of the aquifer microorganism with the dominant species source in the artificial recharge process as claimed in claim 1, wherein in the step (3): the nutrient solution is prepared from glucose and NaNO ₃ And K ₂ HPO ₄ As the only carbon, nitrogen and phosphorus sources for microbial growth; sterilizing the nutrient solution at the temperature of 120-.

6. The method for tracing blockage of the source of dominant species by aquifer microorganisms in an artificial recharge process according to claim 1, wherein in step (4): and recording the water outlet volume of the manual recharge simulating device and the water heads of the pressure measuring pipes every 4 hours.

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