CN113122619B - Method for tracing dominant strain source blocked by aquifer microorganisms in artificial recharge process - Google Patents
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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
Technical Field
The invention belongs to the field of underground water environment protection, and particularly relates to a method for tracing dominant strain sources blocked by aquifer microorganisms in an underground water artificial recharge process.
Background
The artificial recharge of the underground water can effectively solve a plurality of environmental problems caused by excessive exploitation of the underground water resources, and is a necessary way to realize the combined dispatching of the surface and the underground of the water resources, optimize the water resource allocation and improve the comprehensive utilization rate of the water resources. However, a great deal of engineering practice shows that the quality of the recharge water and the formation water is not matched, so that the recharge efficiency is reduced and even the recharge well is seriously blocked. Aquifer plugging can be classified into physical plugging, chemical plugging, and microbial plugging, depending on the cause of the plugging. Wherein, once the microorganism blockage is formed, the permeability of the aquifer is difficult to recover, and the composite effect is easily formed with physical and chemical blockage, thereby accelerating the blockage rate of the aquifer. Manual recharge involves the joint scheduling of surface and ground water, with widely varying microbial community characteristics in different water sources. At present, the research aiming at the problem of strain source which causes water-bearing layer blockage in the process of artificial recharge is not deep enough.
Disclosure of Invention
Based on the technical problems, the invention provides a method for tracing the blockage of aquifer microorganisms in the artificial recharge process to the dominant strain source.
The technical solution adopted by the invention is as follows:
a method for tracing the source of a dominant strain blocked by aquifer microorganisms in the process of artificial recharge adopts an artificial recharge simulation device, 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 upper part and the lower part of the other side of the organic glass column at intervals, 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 samples are extracted from the recharge well, one groundwater sample is used for groundwater microbial sequencing and is marked as a water sample I, and the other groundwater sample is used for tank returning 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 water microorganism sequencing and is marked as a water sample three, and the other water sample is used for tank returning 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) 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.
Preferably, the height of the organic glass column is 20cm, and the inner diameter 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.
Preferably, in 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.
Preferably, in the step (1), the organic glass column is protected in a wet manner in the process of filling the quartz sand, and the method comprises the following specific steps: 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.
Preferably, in 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 ℃ and 130 ℃ and under the pressure of 0.987ATM for 10-15 min.
Preferably, in the step (4), the recharging method includes four methods: 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, the sterilizing nutrient solution is recharged to the sterilizing deionized water saturated aquifer, namely a sterilizing deionized water saturated column is adopted, and the sterilizing nutrient solution is recharged.
Preferably, 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.
Preferably, in 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 permeate flow, i.e., the volume flow (mL/s) through each section of the sand column, L i Is a permeation path, i.e. the distance (cm) of the water passing section between the hydraulic heads, A is the cross-sectional area (cm) of the water passing section 2 )，(ΔH) i Is the water head difference (cm) of the water cross section.
Preferably, in step (4):
relative saturation permeability coefficient (K) of the medium 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 decreases to 0.1, i.e. the saturation permeability coefficient of the sand column decreases to 10% of the initial value, the sand column is considered to be clogged; the sand column was removed and sampled for microbial community analysis.
Preferably, in step (5):
respectively filtering the water sample I and the water sample III by using a 0.45 mu mPES 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 ℃;
and (3) adopting high-throughput sequencing, and utilizing sequencing results to analyze and compare the diversity, the abundance and the correlation of the microbial communities so as to screen out dominant bacterial sources.
The beneficial technical effects of the invention are as follows:
according to the invention, by comparing the saturated permeability coefficient and microbial community characteristics of the aquifer medium in different recharging modes, the dominant bacteria in the plugging process are traced, and scientific reference can be provided for improving the efficiency of the aquifer in artificial recharging and preventing and treating the aquifer plugging.
Drawings
The invention will be further described with reference to the following detailed description and drawings:
FIG. 1 is a schematic diagram of the structure of a manual recharge simulation device according to the present invention;
FIG. 2 is a diagram illustrating the use of hierarchical clustering heatmaps in an exemplary embodiment of the present invention;
FIG. 3 is a graph of principal coordinate analysis based on weighted Unifrac distance in an example application of the present invention.
Detailed Description
Aiming at the problem that the source of the strains causing the water-bearing layer blockage in the current artificial recharge process is not deeply researched, the method traces the dominant strain source of the microorganisms causing the water-bearing layer blockage through simulating the process of the artificial recharge water-bearing layer and through means of saturated permeability coefficient calculation, microbial community characteristic analysis and the like.
The present invention will be described in detail below.
A method for tracing the source of dominant bacteria blocked by aquifer microorganisms in the process of artificial recharge adopts an artificial recharge simulation device, as shown in figure 1, the device comprises an organic glass column 1, the top inlet of the organic glass column 1 is communicated with a water inlet tank 3 through a water inlet pipe 2, and a constant water head device 4 is arranged on the water inlet pipe 2. The bottom outlet of the organic glass column 1 is communicated with a water outlet groove 6 through a water outlet pipe 5. An overflow port 7 is arranged at the upper part of one side of the organic glass column 1, a plurality of pressure measuring pipes 8 are connected to the other side of the organic glass column 1 at intervals up and down, and a pressure measuring plate 9 is arranged at the tail end of each pressure measuring pipe 8.
The overflow port 7 communicates with the overflow tank 11 via an overflow pipe 10.
Specifically, the height of the organic glass column is 20cm, and the inner diameter is 5 cm; the overflow mouth sets up in the cylinder left side of organic glass post apart from top 2cm department, and the cylinder right side of organic glass post is apart from top 4, 6, 8, 10 and 20cm punishment and is set up the port that is linked together with the pressure-measuring pipe respectively, and each port all is linked together with a pressure-measuring pipe. The effective packing height of the plexiglas column is 16 cm.
The constant water head device is used for providing constant pressure, and a peristaltic pump and the like can be selected.
The method comprises the following steps:
(1) and filling the sterilized quartz sand into the organic glass column.
Specifically, standard quartz sand with a particle size of 0.5mm was sterilized in an oven at 121 ℃ for 6h, and filled as an apparatus aqueous medium into an organic glass column with an effective packing height of 16cm in a wet saturated column of equal thickness (about 2 cm).
The wet type column filling method comprises the following steps: and (3) injecting column saturation water into the organic glass columns with the water inlet and outlet valves closed, weighing 92g of sterilized quartz sand, filling the organic glass columns, compacting the sand columns with constant pressure after twice, and repeating the test, wherein each organic glass column at least needs 920g of quartz sand column saturation. The complete saturation time of the sand column is more than 12 h.
(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 tank returning simulation and is marked as a water sample II. And collecting two river water samples from the river water, wherein one water sample is used for river water microorganism sequencing and is marked as a water sample three, and the other water sample is used for tank returning simulation and is marked as a water sample four.
(3) And (4) preparing the sterilization nutrient solution in a laboratory.
When preparing the nutrient solution, glucose (2.20mg/L) and NaNO are respectively adopted 3 (0.067mg/L) and K 2 HPO 4 (0.42mg/L) as the sole source of carbon, nitrogen and phosphorus for microbial growth. Pressurizing the nutrient solutionSterilizing (121 deg.C, at 0.987ATM) for 15 min.
(4) And (3) 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 method comprises four steps: firstly, river water is reinjected into an underground water saturated aquifer, namely a water sample two-saturated column is adopted, a water sample four-reinjection is adopted, and the GR mode is adopted; secondly, recharging the saturated aquifer of the underground water by using the sterilizing nutrient solution, namely adopting a water sample secondary saturated column, recharging by using the sterilizing nutrient solution and counting as GN mode; thirdly, river water is refilled to sterilize the deionized water saturated aquifer, namely a sterilized deionized water saturated column is adopted, a water sample is refilled for four times, and the DR mode is calculated; fourthly, the sterilizing nutrient solution is recharged to the sterilizing deionized water saturated aquifer, namely a sterilizing deionized water saturated column is adopted, and the sterilizing nutrient solution is recharged, and the Con mode is adopted.
Specifically, the test steps for each recharging method are as follows:
the recharging liquid in the water inlet tank enters from the top inlet of the organic glass column through a constant water head device (the constant water head difference is 15cm), flows through the organic glass column and is discharged from the bottom outlet of the organic glass column. On the basis of Darcy's law and hydraulic change, every 4h, namely 0, 4, 8, 12, 16 … from the beginning of the experiment, record the effluent volume and each piezometer tube head of the artificial recharge analog device, and then calculate the saturated permeability coefficient in different layers, reflect the blocking condition of the medium in the column body of the organic glass column through the change of the relative saturated permeability coefficient.
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 8 to 10cm from the top of the sand columnSaturated permeability coefficient, K 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 permeate flow, i.e., the volume flow (mL/s) through each section of the sand column, L i Is a permeation path, i.e. the distance (cm) of the water passing section between the hydraulic heads, A is the cross-sectional area (cm) of the water passing section 2 )，(ΔH) i Is the water head difference (cm) of the water cross section.
Relative saturation permeability coefficient (K) of the medium 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 was removed and sampled for microbial community analysis.
(5) And (4) performing microbiological analysis on the water sample I, the water sample III and the sand sample collected after the recharge test.
Wherein the sampling steps of the water sample I and the water sample III are as follows: respectively filtering the water sample I and the water sample III by using a 0.45 mu mPES microfiltration membrane, immediately transferring the microfiltration membrane into a sterile centrifuge tube, and storing at-80 ℃.
The sand sample sampling steps are as follows: after the percolation experiment, approximately 0.5g of sand in the first layer of each column (2-4 cm sand from the top of the column) was sampled with a sterile spoon and immediately transferred to a sterile centrifuge tube and stored at-80 ℃.
And (3) carrying out high-throughput sequencing on the sand sample and the filtered river water and underground water, and comparing the diversity, the abundance and the relevance of the microbial communities.
High throughput sequencing is briefly introduced as follows:
the final DNA concentration and purity were determined by UV-visible spectrophotometer and DNA quality was checked on 1% agarose gel electrophoresis, and the V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified by a thermal cycler PCR system with universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5 '-GGACTACHVGGGTWTCTAAT-3'). And (3) PCR reaction conditions: initial denaturation at 95 ℃ for 3min, followed by 27 cycles of reaction (30 s at 95 ℃, 30s at 55 ℃, 45s at 72 ℃) and final extension at 72 ℃ for 10 min. The PCR reaction system is as follows: 20 μ L of a mixture containing 5 XFastPfu buffer (4 μ L), 2.5mM dNTPs (2 μ L), primers (5 μ M, 0.8 μ L), FastPfu polymerase (0.4 μ L) and 10ng template DNA. The products of the PCR reaction were extracted from a 2% agarose gel and further purified using the AxyPrep DNA gel extraction kit. The use of package R generates a heat map with Bray-Curtis distance differences based on the most abundant OTUs of the bacterial community, as shown in figure 2. From the OTU data, an alpha diversity index including abundance (Chao), bacterial community diversity (Shannon index), uniformity and coverage was obtained using Mothur. Population differences between bacterial populations were tested based on OTU data using coordinate analysis (PCoA) with weighted Unifrac distance, similarity Analysis (ANOSIM).
(6) Through saturated osmotic coefficient and the microbial community characteristic of the aquifer medium under the different recharge modes of contrast, including microbial community diversity (Shannon index), richness (Chao) and correlation analysis (cluster chart) etc. compare the microbial community under the different recharge modes and the similarity between native groundwater and river to trace to the source, select the dominant bacteria source promptly to the dominant bacteria of jam in-process.
The invention is further illustrated by the following specific application examples:
experimental samples and experimental sites: in situ groundwater was collected from 1 recharge well downstream of the stilling river (36.370318 ° N, 120.159088 ° E) and river water was collected from a nearby small clear river (36.370203 ° N, 120.159526 ° E). The length of the river is 179.9km, the area of the watershed is 4161.9km 2 The average river ratio is reduced by 1.2/1000, the total fall is about 200m, the average river width is 460m, and the river network density is 0.34km/km 2 . The experiment is carried out in geological and underground water physical simulation laboratory and hydrochemical laboratory of Shandong science and technology university.
The recharging device is provided with: standard quartz sand (d) was used 60 0.5 mm; guangzhou city active medical equipment Co., Ltd, Guangzhou, China), sterilized in an oven at 121 ℃ for 6 hours, and filled in equal increments (about 2cm) as a device porous medium to an effective height of 16cm,In a plexiglas column with an internal diameter of 5 cm.
And (3) test treatment: the invention simulates 4 different recharging modes: (1) river water is recharged into the groundwater saturated aquifer, and the GR mode is adopted; (2) recharging the underground water saturated aquifer with the sterilizing nutrient solution, and counting as GN mode; (3) the river water recharging sterilization deionized water saturated water-containing layer is counted as a DR mode; (4) the sterilized nutrient solution is filled back into the sterilized deionized water saturated water-containing layer, and is designed in a Con mode. And 2 groups of parallel tests are respectively set in 4 recharging modes.
And (3) blockage monitoring: in the recharging process, the water head pressure is monitored every 4h, and the relative saturation permeability coefficient is calculated.
Sequencing analysis of microorganisms: when the relative saturation permeability coefficient reaches 0.1, disassembling the columns, sampling about 0.5g of sand in the first layer (a sand layer 2-4 cm away from the top of the sand column) of each sand column by using a disinfection spoon, immediately transferring the sand into a sterile centrifuge tube, and storing at-80 ℃. And (3) carrying out high-throughput sequencing on the sand samples in the four sand columns and the filtered river water and underground water, and comparing the diversity, the abundance and the correlation of the microbial communities in the 4 recharging modes and the in-situ underground water and river water.
Results and analysis:
1. permeability coefficient of test group at each time period
The relative saturation permeability coefficient may be indicative of the degree of clogging of the artificial recharge, as shown in Table 1 as decreasing (K) s 1 ) ' values indicate significant plugging of the sand column under different recharge modes. Taking GR column as an example, when the recharge test is carried out for 16h, the relative saturation permeability coefficient (K) of the surface layer of the sand column s 1 ) ' the value was gradually decreased from 1.0 to 0.736 by 26.4%, followed by a decrease from 0.736 to 0.103, and the permeation experiment was completed. In the GR ', GN ', DR and DR ' columns, the relative saturation permeability coefficients are 0.117, 0.110, 0.041, 0.083 and 0.002, respectively. In contrast, (K) in Con and Con' sand columns s 1 ) ' values fluctuated around 1.0, indicating no clogging in the control group; the relative saturation permeability coefficient decreased in the different sand layers, further demonstrating that plugging also occurred in the combined layers of sand columns. At the end of the permeation experiment, of GR, GR ', GN, GN', DR and DR sand columns (K) s 1-4 ) ' value0.355, 0.301, 0.419, 0.237, 0.371 and 0.017, respectively, are less than the values of the first layer in the corresponding sand column. The results show that the degree of clogging gradually decreased from top to bottom as the sand column was moved.
2. Diversity, abundance and relatedness of microbial communities in each experimental group
Table 2 shows the alpha diversity index (i.e., samG and samR) of the bacterial communities in the in situ groundwater and river water. The observed mean values of OTU numbers for samG and samR were 1158 and 219, respectively, for OUT number indices 1410 and 240, for diversity indices 4.22 and 1.73, and for homogeneity indices 0.60 and 0.32, respectively, indicating that the diversity and homogeneity of bacterial communities in groundwater were much higher than in river water. Table 2 also shows the alpha diversity index of the bacterial community in the biofilm under different feeding regimes. For samGR, samGN and samDR, the observed average values of OTU are 219, 178 and 130 respectively, the average value of OUT number indexes is 312, 250 and 150, and the average values of diversity index and uniformity index are 3.05, 2.48, 0.31 and 0.57, 0.48 and 0.48 respectively, which indicates that the diversity and uniformity of bacterial communities in the mode of the river water recharging groundwater saturated aquifer are far higher than those in the mode of the sterilized nutrient solution recharging groundwater saturated aquifer and the river water recharging sterilized deionized water saturated aquifer.
Table 3 shows the bacterial community composition in situ in groundwater and river water. Among phyla, proteus is the most predominant phylum, followed by bacteroides, actinobacillus, dura mater, baculobacter and campylobacter viridis. In samG, the main phyla are Proteobacteria, Bacteroides, Actinomycetes, and in the underground environment, in samR, the first four main phyla are Proteobacteria, Chaetobacter, Bacteroides, and Actinomycetes, which are roughly similar to other communities in freshwater aquatic organisms. On the class level, samG predominates with C.sp., Bacteroides and Actinomycetes, in contrast to only gamma Proteus, which is the dominant species in samR, indicating that the diversity and uniformity of bacterial communities in groundwater is much higher than in river water. On the genus level, the four genera that predominate with samG are hydrogenophiles, Acetobacter, Pseudomonas and Rhodococcus, with Acinetobacter predominating absolutely in samR, followed by Microbacterium, indicating that there are more genera in groundwater than in river water. Table 3 shows the bacterial community composition of biofilms with different recharging modes. At the phylogenetic level, Proteobacteria are the most predominant phylogenetic, and secondarily Bacteroides, Actinomycetes and Bacteroides. In samGR and samGN, Proteobacteria are absolutely predominant, followed by Bacteroides and Actinomycetes. The average relative abundance of bacteria is lower in samDR and higher in Bacteroides compared to those in samGR and samGN. On the class level, the predominant proteobacteria are subdivided into α -proteobacteria and γ -proteobacteria. In the non-proteus class, bacillus predominates in samGR and samGN. On the genus level, of samGR and samGN, the predominant genera are Acinetobacter, Microbacterium and Porphyromobacter. The results show that the bacterial community structures in samGR and samGN have similarities. The composition of the bacterial community in samDR was different compared to these samples, the first three being Clostridium, Sphingobacterium and Acinetobacter, respectively.
FIG. 2 uses a hierarchical clustering heatmap to clearly show bacterial community structure. Based on the heat map at the genus level, a distribution of fifty bacteria-rich genera found in groundwater, river water and biofilms is shown. As can be seen in the figure, samGR and samGN preferentially aggregate, indicating that the bacterial community structures in these samples are very similar. For samDR, the maximum calorific value occurs in Clostridia, followed by sphingosine, Acinetobacter, Bacillus and Brevibacterium, which are also in the region of samGR and samGN, while the calories are relatively low.
FIG. 3 is a principal coordinate analysis (PCoA) based on weighted Unifrac distance, revealing differences in bacterial community composition between different samples. The results show that: PC1 accounted for 48.27% of the variation, and PC2 accounted for 37.88% of the variation. The results show that: due to different sources of samples, the samples are divided into different groups, and the difference between each group is significantly higher than the intra-group difference (ANOSIM, R is 0.9837, P is 0.001), and the bacterial community structures in samGR and samGN are more similar than those in samDR. FIG. 3 also shows that the bacterial community structures of the in situ groundwater and river water, samG and samR, are far from the bacterial community structures of the biofilms, samGR, samGN and samDR. The results show that the structure of the bacterial community in the natural system is completely different from that under artificial conditions, and river water is the most important factor influencing the composition of the bacterial community.
According to the results, the blockage of each layer of the sand column is concluded and the blockage degree is gradually reduced along with the sand column from top to bottom. By comparing the characteristics of the bacterial communities with the characteristics of the in-situ underground water and the characteristics of the in-situ river water under different recharging modes, the diversity and the uniformity of the bacterial communities in the underground water are far higher than those of the river water, wherein the composition of the bacterial communities after simulated recharging is similar to that of the in-situ river water bacterial communities, and the in-situ river water is traced as a dominant strain source for the blockage of aquifer microorganisms in the artificial recharging 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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