Microbial Response of Fe and Mn Biogeochemical Processes in Hyporheic Zone Affected by Groundwater Exploitation Along Riverbank

Abstract

In order to explore the co-evolutionary relationship between the functions of microbial communities and the chemical composition of groundwater in a hyporheic zone affected by groundwater exploitation along riverbank, we have taken the Huangjia water source area on the Liao River main stream in Shenyang as an example. DNA was extracted from microorganisms in the hyporheic zone affected by groundwater exploitation along the riverbank, and we conducted high-throughput sequencing to select the dominant bacterial strains from the indigenous bacteria. They are classified as the Proteobacteria phylum, the Actinobacteria phylum, the Firmicutes phylum, the Bacteroidetes phylum, the Chloroflexi phylum, and the Acidobacteria phylum. The dominant bacteria have a good correlation with Fe, Mn, and environmental factors (such as DO—dissolved oxygen, Eh—oxidation-reduction potential, etc.) in the hyporheic zone. The functions and activities of the superior bacterial strains exhibit a feature of co-evolution with the water’s chemical environment, which has certain response characteristics to redox zoning. Studying the co-evolution relationship between the microbial community structure and function in the hyporheic zone and the chemical composition of the groundwater can provide a microbiological theoretical basis for the redox zonation. It also offers reference for understanding the process of Fe and Mn migration and transformation in the hyporheic zone under the hydrodynamic conditions of groundwater exploitation along the riverbank.

1. Introduction

The hyporheic zone is a critical transition area for the interaction between surface water and groundwater [1], serving as the core hub for material exchange and energy transfer between the two. The key issue in the interaction between surface water and groundwater lies in the hydraulic relationship between rivers and aquifers. As the central region for their interaction, the delineation of the hyporheic zone’s scope and its flow characteristics hold significant research value. Harvey, Boano, and others have proposed that the surface water–groundwater mixing zone includes “groundwater not reaching the riverbed,” while hyporheic zone flow, characterized by bidirectionality, differs from groundwater discharging into the river—after infiltrating the riverbed, surface water mixes with groundwater and recharges the river. This area exhibits active solute transport and transformation, as well as intense biogeochemical processes. By regulating microbial nutrient supply and energy acquisition, it shapes a unique transitional biological community [2,3]. The flow and mixing conditions in the surface water–groundwater mixing zone are complex. Kotowski pointed out that gaseous tracers (CFCs, SF6, and noble gases) can better help understand the inflow and mixing characteristics of surface water and groundwater in the hyporheic zone, assess dynamic changes under natural and exploitation disturbances, and determine the mixing ratio of the two and the scope of the hyporheic exchange zone. Additionally, the abiotic/biotic component contents of the mixed water in this area may differ from those of river water and groundwater [4]. Its core importance is reflected in maintaining hydrological connectivity, purifying water pollutants, and regulating regional water quality. It acts not only as a “buffer zone” for mutual recharge between surface water and groundwater, but also as an “active engine” driving element cycling and supporting ecosystem stability, playing an irreplaceable role in maintaining watershed hydrological and ecological balance.

Changes in the biological community structure directly or indirectly cause changes in the hydrochemical composition of groundwater, and affect the migration and transformation pathways of substances such as iron and manganese [5,6]. Research by Park et al. has shown that, when river water infiltrates to recharge groundwater, the microbial community structure is an important influencing factor in biogeochemical processes, and differences in the activity of dominant and functional bacteria affect the migration behavior of pollutants in the groundwater [7]. So Jeong et al., focusing on the riparian zone of the Geum River in South Korea, found that IRB (iron-reducing bacteria) exhibit spatial heterogeneity. Redox zonation and acid–base conditions influence their species distribution—strong iron reduction zones (near the river) are dominated by the genus Geobacter, while weak iron reduction zones (far from the river) are dominated by the genus Rhodoferax ferrireducens [8]. Research by Medihala et al. on the infiltration scenario of the North Saskatchewan River in Canada showed that the structure and function of groundwater microbial communities have significant spatial heterogeneity, with environmental changes such as groundwater exploitation being the main influencing factors. The area near the river is dominated by iron- and manganese-reducing bacteria, while the area 1–2 m around pumping wells is dominated by iron- and manganese-oxidizing bacteria [9].

At the present stage, research on the structure and function of microbial communities in hyporheic zones is mostly based on areas with stable groundwater environments. There is relatively little research on the structure and function of microbial communities in areas where hydrodynamic conditions, acid–base, and unstable redox conditions are caused by groundwater exploitation along riverbanks [10,11]. Exploring the co-evolutionary relationship between the microbial community structure and function in hyporheic zones affected by groundwater exploitation along riverbanks, as well as the groundwater chemical composition, makes it possible to deeply explore the biochemical processes and migration transformation rules of Fe-Mn transformation in hyporheic zone, and reveal the biogeochemical processes and microbial ecological processes of Fe-Mn during the infiltration of river water. It provides certain references and bases for the water supply safety of drinking water sources.

In many areas of northern China, there is a widespread distribution of groundwater with high Fe and Mn content. Fe and Mn are typical elements in the underground environment that exhibit multiple forms, multiple valences, and high abundance. They share similar geochemical behaviors. However, when Fe and Mn occur in a combined form, the biogeochemical processes become particularly complex [12,13]. In this study, by extracting DNA from the microorganisms in the hyporheic zone affected by groundwater exploitation along riverbanks of the Liaohe River, and with detection using high-throughput sequencing technology, we identify superior indigenous bacterial strains, and the relationship between strains and the redox zone; moreover, we discuss the co-evolution relationship between the microbial community structure and function in the hyporheic zone affected by groundwater exploitation along riverbanks, as well as the chemical composition of the groundwater. It can be seen that the superior strain has a good correlation with Fe and Mn in the groundwater, as well as with environmental factors (DO—dissolved oxygen, Eh—oxidation reduction potential, and others). The feature of co-evolution is significant for the functions and activities of dominant bacterial species in water-containing media and the water chemical environment, as it has certain response characteristics to redox zoning. Herein, we study the collaborative evolutionary relationship between the structure and function of microbial communities and the chemical composition of groundwater in the hyporheic zone.

2. Materials and Methods

2.1. Study Area and Sample Collection

2.1.1. Overview of the Study Area

This study area selected includes groundwater source areas along riverbank of the Liao River in Shenyang, as shown in Figure 1 [14]. The designed water supply capacity of this water source is 50,000 m³/d, consisting of 6 groups with 12 wells (PW1–PW12), where each group comprises a combination of shallow and deep wells for phased water extraction—shallow wells at 35 m depth and deep wells at 55 m depth. The water source is located in the high floodplain area of the Liaohe River, with the well cluster 350–500 m away from the left bank of the river. Moreover, the groundwater in the study area has high iron and manganese contents: the total iron concentration ranges from 1.0 to 29.9 mg/L, total manganese concentration from 0.38 to 11.61 mg/L, NH₄-N from 0.001 to 2.58 mg/L, NO₃-N from 0.001 to 1.615 mg/L, and DOC concentration from 1.01 to 14.85 mg/L. The water source is mainly recharged by lateral river water recharge and lateral groundwater runoff, while the upper part of the aquifer in the study area is dominated by grayish-yellow, brownish-yellow, and brown medium-fine sand, medium-coarse sand, and gravel-bearing coarse sand, intercalated with thin fine sand layers with a thickness of 8.6–16.0 m. The lower part is mainly composed of gravel-bearing coarse sand and gravel, locally interbedded with thin discontinuous mucky clay layers with a thickness of 17.0–30.0 m, and the aquifer has close vertical hydraulic connectivity and a unified groundwater level.

Based on seismic geophysical exploration, hydrogen and oxygen stable isotope tracing, and the analysis of the response characteristics of the groundwater level and temperature dynamics to river water infiltration, the horizontal width of the hyporheic zone on the right bank of the Liaohe River in this section is determined to be approximately 17 m, with a vertical burial depth of about 20 m, as shown in Figure 2.

2.1.2. Sampling of Riverbed Sediments and Hyporheic Zones of Aquatic Media

In the study area, 11 sampling points were arranged along the vertical Liaohe River direction to collect media samples, including the south bank of the lower reaches of the river (PW12, PW13), the south bank of the middle reaches of the river (PW3, PW4), the south bank of the lower reaches of the river upstream river (PW1, PW2), the bed sediments on the north bank of the Xixiaohe River (GS12), the bed sediments on the south bank of the seepage point (RS3), the aquifer media on the seepage zone on the bank of the river (RD—series of monitoring points), and the aquifer media on the profile (GS1, GS2, GS3, PW5, PW6), respectively. The depth of each layered monitoring well is 10.0 m, 20.0 m, 35.0 m, and 45.0 m below the ground surface. The layout of the monitoring holes in the hyporheic zone of the profile is shown in Figure 2.

To characterize the structure of microbial communities in riverbed sediments and aquifer media during river water recharge to groundwater, a total of 115 sediment microbial samples were collected simultaneously from the shallow seepage path within 17.0 m of the nearshore zone of the Liaohe River. The samples were cut and dispensed into sterilized self-sealing bags in a nitrogen-filled anaerobic operating chamber by applying a Beeker portable sediment sampler. The cutting knives were sterilized using anhydrous alcohol and dried before each cut. Approximately 20.0 g of samples were collected per stratum position at each point. The samples were kept in a mobile refrigerator and brought back indoors, and kept frozen in a −20 °C refrigerator. Three parallel samples were taken for each point according to biostatistical requirements.

Microbial samples were collected from each groundwater observation hole in the deep seepage path, totaling 20 samples. Then, 10 L water samples were collected from each observation hole and pumped through 0.22 μm filter membranes; then, membranes with microorganisms were cut with sterile scissors, sealed in 5 mL sterile tubes, grouped into sterilized self-sealing bags, and frozen at −20 °C for storage.

2.1.3. Collection of Microbiological Samples from River Water and Groundwater

Prior to sampling, the wells undergo pumping and flushing. A total of 20 microbial samples were collected in this study, covering river water and various groundwater observation wells. The specific sampling and processing procedures are as follows: 10 L of water sample was collected from each sampling site, including river water (RS1, RS2, RS3, RS4), groundwater from stratified monitoring wells in the monitoring profile (GS1, GS2, GS3, PW5 production well), and groundwater from stratified monitoring wells in the nearshore zone (RB well group); subsequently, the water sample was filtered through a 0.22 μm filter membrane, the filter membrane with intercepted microorganisms was cut into pieces using sterile scissors, and then placed into a 5 mL sterile tube and sealed. Finally, the tubes were grouped into sterilized self-sealing bags and stored under freezing conditions at −20 °C [15].

2.2. Determination of Microbial Community Structure

The structure of microbial communities in riverbed sediments and aquifer media was analyzed by 16SrRNA, combined with PCR amplification (Polymerase Chain Reaction)—high-throughput sequencing results.

The FastDNA Spin Kit for Soil (Soil Genomic DNA Extraction Kit: MP Biomedicals, Santa Ana, CA, USA) was utilized in an ultra-clean bench, in conjunction with a water bath and an MP Fast Prep instrument, to extract total DNA from sediment samples in the study area. The extracted genomic DNA was subjected to agarose electrophoresis to assess its integrity and concentration. Genomic DNA was precisely quantified using a micro-UV spectrophotometer (NanoDrop ND-1000UV-Vis: Thermo Fisher Scientific, Waltham, MA, USA) to ascertain the appropriate amount of DNA to be included in the PCR reaction (50 ng). Subsequently, PCR amplification was conducted using a PCR amplifier model 2700 (GeneAmp PCR System 2700: Applied Biosystems, Foster City, CA, USA). The PCR products were monitored via 1% agarose gel electrophoresis and visualized using a Tanon-1600 gel imager. The successfully amplified PCR products were stored in a −20 °C refrigerator for future experiments [16].

The microbial diversity within the samples was evaluated by targeting the V3-V4 region of the 16SrRNA gene using the Miseq platform. PCR amplification was performed with the primersV3-V4-F:5′-GTACTCCTACGGGGAGGCAGCA-3 and V3-V4-R:5′-GTGGACTACHVGGGGTWTCTAAT-3′. The reaction mixture was 50 μL, containing 0.3 μL of Pyrobest DNA Polymerase (2.5 U/μL), 5 μL of 10× Pyrobest Buffer, 10 ng of DNA sample (1 μL), 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), and 4 μL of dNTPs (2.5 mM), with the remaining volume made up with ddH₂O. The reaction conditions were as follows: 95 °C for 5 min, 25 cycles of 95 °C for 30 s, 56 °C for 30 s, 72 °C for 40 s, followed by a 10 min extension at 72 °C, and storage at ℃. To minimize PCR bias, three independent PCR reactions were conducted for each sample, followed by purification and sequencing on the Ion Torrent platform [17].

The PCR products were quantified by qubit inclinometer (nucleic acid and protein quantification instrument—Life tech qubit model 2.0) and sequenced using the illumina MISEQ instrument to obtain the sequences. Using a DELL server, low-quality data were filtered out by Mothur (v1.48.0) software with the following control conditions: removal of low-quality reads (Q < 30), removal of lengths less than 200 bp reads, and truncation of low-quality data to improve the correctness of subsequent analyses. Chimera removal was performed using QIIEM (v1.8.0) software at 0.97 similarity, clustered into OTU (Operational Taxonomic Units), used for species classification, and sequence classification information was compared to the Silva database.

After clustering the OTU sequences used for species classification, their taxonomic information was compared to the Silva database to characterize the microbial species composition at the genus level in riverbed sediments, shallow infiltration paths with increasing distance from the river, and deep infiltration paths. The abundance information in each OTU of individual samples was counted, and the abundance of OTU was used as a preliminary indication of the species richness of the samples. Based on the total OTU composition of each sample, individual sample Alpha diversity indices and inter-sample Beta diversity indices were calculated, and species composition was characterized. Statistical analyses between samples, as well as the analysis with environmental factors in addition to trend correspondence analysis (DCA), were plotted using the R language.

3. Results and Analysis

3.1. Microbial Species Composition in Riverbed Sediments and Aquatic Media

During the study, significant spatial inhomogeneity of species composition in streambed sediments and media with different infiltration paths in the study area was revealed (Figure 3).

The dominant indigenous microorganisms present in the river bed sediments and aquatic media of the study area were Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and Acidobacteria. Proteobacteria were involved in the cycling of C, N, S, and metal elements in the material cycle of the ecosystem, and were one of the major microbial species in the environment of the research area, with a wide range of metabolic species, which were mostly parthenogenetic or exclusively anaerobic and heterotrophic, and Gram-negative staining.

The high content of Proteobacteria detected in the research area includes the following:

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Alphaproteobacteria

Sphingomonas, which was mainly used in the environment to degrade aromatic compounds, such as phenol, phenanthrene, and naphthalene [18], was mainly found in aquifers with shallow infiltration pathways. The genus Rhodobacter had denitrifying denitrification [19] and was mainly found in shallow aquatic media. Novosphingobium degraded aromatic organic compounds, such as phenanthrene, nitrobenzene, and phenol, in the environment [18,20].

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Betaproteobacteria

Dechloromonas of the family Oxalobacteraceae and Rhodotoraceae, Vogesella of the order Neisseriales, Ferribacterium, Aqabacterium, Gallionella, Albidiferax, Limnohabitans and Hydrogenophaga of the family Comamonadaceae, Polaromonas, Sideroxydans, Polynucleatobacter, and others [21,22]. Among them, Gallionella, Hydrogenophaga, and Albidiferax were typical iron-reducing bacteria that reduced iron and most arsenite, and chemically convert manganese to other forms of oxides. Polaromonas had the ability to degrade polycyclic aromatic warps, and Sideroxydans was a typical divalent iron oxidizing bacterium. Polynucleobacter was a bacterium necessary for the survival of plankton in freshwater systems.

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Gammaproteobacteria

Perlucidibaca in the order Bdellovibrionales, Aeromonas, and Aeromicrobium was a pathogenic bacterium. Flavobacterium were freshwater fish pathogens detected in HS2 and in sediments from deep infiltration pathways. Flavobacterium activated intracellular enzymes in the presence of Mn²⁺, prompting valence Flavobacterium to degrade PAHs. Shewanella could stimulate catalytic electron donor–electron acceptor reactions under anaerobic conditions. Pseudomonas could efficiently degrade PAHs and participated in denitrification, Fe and Al/Ca cation exchange in phosphate, while competitive adsorption of phosphate affected the release and fixation of arsenic (As) [23].

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Deltaproteobacteria

Most of the δ—Proteobacteria were strictly anaerobic bacteria, including sulfate-reducing bacteria, sulfur-reducing bacteria, and others. They were extensively involved in biological cycles, such as sulfate reduction and iron (III) reduction, in the environment. Geobacter degraded organic pollutants by using Fe(III), MnO₄, and AsO₃⁻ as electron acceptors. Desulfobacca and Desulfatiferula were involved in sulfate and sulfur reduction [24].

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Epsilonproteobacteria

Arcobacter and Sulfuribacter were facultative anaerobic sulfur-oxidizing bacteria. In addition to proteobacteria, the dominant species endemic to riverbed sediment HS2 was Synechococcus, a species of Cyanobacteria, a unicellular organism with primitive anisotropic cytoplasm, and a photoenergetic inorganic autotrophic bacterium. Synechococcus could utilize nitrate and ammonia as the only source of nitrogen for its own growth and metabolism, and its growth activities utilized inorganic compounds such as hydrogen sulfide and sodium thiosulfate to induce C-fixation reduction, accompanied by elemental sulfur release. Synechococcus was also a major component of micro-phytoplankton and was widely involved in micro-food chains in ecosystems [25].

Arthrobacter of the phylum Actinobacteria, which was widely involved in denitrification in the process of organic matter degradation, and was mainly distributed in the sediments near the riverbed in the shallow and deep infiltration paths. Bacillus, Desulfosporosinus of the phylum Firmicutes, and Desulfosporosinus were strictly anaerobic sulfate-reducing bacterium, which can degrade xylene and other substances [24]. Candidatus Brocadia of the phylum Planctomycetes was an autotrophic bacterium, distributed close to the production wells, had the ability to oxidize nitrogen in nitrite, which produced hydrazine, which was mostly used for the reduction or removal of ammonia and nitrogen from wastewater [26]. Geothrix of the phylum Acidobacteria, a chemoenergetic heterotrophic bacterium widely found in aquifer sediments, could utilize trivalent iron and other highly valent metal elements and continued to participate in the biogeochemical cycling of metal elements in the environment by using metabolic substrates as electron donors. The dominant species in HS2 and deep water-bearing media was the species of Planctomycetia, the dominant species in sediments of shallow infiltration paths was Thermoleophilia, and the dominant species in deep infiltration paths was Spirochaetes and Oscillatoriophycideae in the prokaryotic cyanobacteria kingdom. Therefore, during the infiltration process of river water, abundant and diverse proteobacteria were widely present in the sediment of the aquifer, actively participating in the biogeochemical cycles of C, N, S, and metal elements. Under anaerobic conditions, proteobacteria stimulated the catalytic electron donor–electron acceptor reaction involved in denitrification, reduction in Mn, Fe, SO₄²⁻, and most of arsenite, and the degradation of organic and other pollutants.

3.2. Characterization of Microbial Response to Redox Subzone in Hyporheic Zones

The evolution of groundwater chemistry during river water infiltration was inextricably linked to microbial community structure and function. Microorganisms in aqueous media obtain nutrients and energy for their growth and metabolism from the groundwater environment, which contributed to changes in the structure and function of the microbial community to a certain extent. It also directly or indirectly influenced the evolution of groundwater hydrochemistry. Therefore, microorganisms in the environment have a certain response pattern to the redox subzone.

3.2.1. Characterization of Spatial Distribution of Microbial Communities in Riverbed Sediments and Hyporheic Zones

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Distribution characteristics of microbial dominant bacteria in riverbed sediments

From the function and content of dominant bacteria in the infiltration flow path of the river water (

Table 1. Characterization of the function and content of dominant bacteria in the redox subzone.

Infiltration Path	Redox Subzone	Dominant Bacterium	Functionality	Abundance
Riverbed	Aerobic zone	Flavobacterium Synechococcus	In the presence of Mn2+, it can activate intracellular enzymes to promote the degradation of polycyclic aromatic hydrocarbons (PAHs) by Flavobacterium, which is mainly used in the environment for the degradation of phenol, phenanthrene, naphthalene, and other aromatic compounds [25].	18.24%
Shallow flow infiltration paths	O2/NO3−-reducing zone	Arthrobacter	Participation in denitrification in the degradation of organic matter [26].	11.26%
	Mn(IV)-reducing zone	Geobacter	Degradation of organic pollutants using Fe(III), MnO4, and AsO3− as electron acceptors.	15.91%
	Fe(III)-reducing zone	Pseudomonas Geothrix	Efficient degradation of polycyclic aromatic hydrocarbons (PAHs), participation in denitrification, and exchange of Fe with Al/Ca cations in phosphates, in conjunction with competitive adsorption of phosphates affecting As release and immobilization [23].	27.28%
	Sulfate-reducing zone	Arcobacter Desulfobacca Desulfatiferula Desulfosporosinus	Sulfate and sulfur reduction [24].	13.41%
Deep water infiltration paths	O2/NO3−-reducing zone	Arthrobacter	Participation in denitrification in the degradation of organic matter [26].	17.69%
	Mn(IV)-reducing zone	Geobacter Albidiferax Flavobacterium	Degradation of organic pollutants using Fe(III), MnO4, and AsO3− as electron acceptors, typical of iron-reducing bacteria, reducing iron and most arsenite and chemically converting manganese to other forms of oxides.	12.91%
	Fe(III)-reducing zone	Pseudomonas Geothrix Hydrogenophaga	Efficient degradation of polycyclic aromatic hydrocarbons (PAHs), participation in denitrification, and exchange of Fe with Al/Ca cations in phosphates, in conjunction with competitive adsorption of phosphates affecting As release and immobilization [23].	14.07%
	Sulfate-reducing zone	Arcobacter Desulfobacca Desulfatiferula Desulfosporosinus	Sulfate and sulfur reduction [24].	12.23%

), it can be seen that the river bed is an aerobic region, and the main dominant bacteria are Flavobacterium and Synechococcus, with an abundance of 18.24%, which mainly catalyzes the degradation process of organic matter [27].

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Characterization of the spatial distribution of microbial communities in the hyporheic zone

As shown in Figure 4, the content and spatial distribution characteristics of dominant bacteria in aqueous media had a good correlation with the redox subzone in groundwater. Based on the diversified reactions of functional bacteria, there were both synergistic and antagonistic effects between biogeochemical reaction processes.

The total abundance of dominant bacteria in the redox subzone of the shallow water flow infiltration path was 67.86%. Among them, the main dominant bacteria in the O₂/NO₃⁻-reducing zone were Arthorbacter, mainly involved in denitrification during organic matter degradation, with an abundance of 11.26%. The main dominant bacterium in the Mn(IV) reduction zone was Geobacter, which can utilize Fe(III), MnO₄, AsO₃⁻, and others as electron acceptors to participate in the degradation process of organic matter [28], with 15.91% abundance. The main dominant bacteria in the Fe(III) reduction zone were Pseudomonas and Geothrix, with a higher abundance of 27.28%. The main eubacteria in the sulfate reduction zone were Arthorbacter, Desulfobacca, Desulfatiferula, and Desulfosporosinus, with an abundance of 13.41%. The higher abundance of Fe(III)-reducing bacteria compared to Mn(IV)-reducing bacteria in the shallow water infiltration pathway media was consistent with the relatively high Fe²⁺ content of shallow groundwater.

The total abundance of dominant bacteria in the redox subzone of the deep water flow infiltration path was 56.90%. The abundance of the main dominant bacteria in the O₂/NO₃⁻-reducing band was 11.26%. The main dominant bacteria in the Mn(IV) reduction zone were Geobacter, Albidiferax, and Flavobacterium, which could reduce iron and most arsenite with an abundance of 12.91%. The abundance of the main dominant bacteria in the Fe(III) reduction zone was 14.07%. The abundance of the main dominant bacteria in the sulfate reducing zone was 12.23%. Among them, the main dominant bacteria in the O2/NO₃⁻-reducing zone, Fe(III)-reducing zone, and sulfate-reducing zone were similar to those along the shallow water flow path. The abundance of Mn (IV)-reducing bacteria and Fe (III)-reducing bacteria in the medium of deep water infiltration path was comparable. As analyzed earlier, the effective form content of manganese in the deep water-containing medium was lower than that of iron, so the Fe²⁺ content in deep groundwater was relatively high.

3.2.2. Characterization of the Synergistic Evolution of Microorganisms and Groundwater Hydrochemistry in Aqueous Media

In the RDA (Redundancy Analysis) of dominant bacteria and water chemistry (Figure 5), Q1–Q5 were shallow water flow path monitoring points: HB1-6, HB2-6, HB3-6, HB4-6, and HJ1-1, respectively; S1–S5 were deep water flow path monitoring points: HJ1-4, HJ2-4, HJ3-4, and HJ4-4, respectively.

The results from the RDA correspondence analysis (

Table 2. RDA analysis of dominant bacteria and water chemistry indicators.

Parameters	Axis I	Axis II
DOC (mg/L)	0.1936	−0.3633
pH	−0.2480	−0.3636
DO (mg/L)	−0.0909	−0.2840
Mn2− (mg/L)	−0.0984	0.2950
SO42− (mg/L)	0.3190	−0.4916
Fe2+ (mg/kg)	−0.1560	0.4831
Eh (mV)	0.1544	−0.1462
As (ng/L)	−0.2404	0.5177
Eigenvalues	0.894	0.080
Species environment correlations	0.995	0.921
* CPV of species data	89.4	97.5
* CPV of species environment relation	91.6	94.8

Note: * CPV: cumulative percentage of variance.

and Figure 5) showed that the first and second distribution axes explained 89.4–97.5% of the variation in microbial composition. The cumulative values of the biocomposition and environmental correlation between the first and second distribution axes amounted to 91.6–94.8%. From the relationship between the angle between the dominant species and the sampling points, it can be seen that the angle between the shallow groundwater and the species is mostly acute, which characterizes the higher abundance of species in the shallow aquifer medium and the richer diversity of populations. The environmental factors (DO—dissolved oxygen, Eh—oxidation reduction potential, and pH) had short lines to the origin, characterizing them as important environmental factors controlling groundwater hydrochemical indicators. By the relatively small angle between the Fe and Mn arrows and each other, it was shown that they have a good correlation with each other, and there is a certain synergy and symbiosis in the process of hydrochemical evolution.

As shown by the length of the arrows between the dominant bacteria and the origin, Albidiferax, Pseudomonas, Geothrix, Desulfobacca, Desulfatiferula, and Desulfosporosinus had shorter arrows, and had a greater impact on the environment with respect to groundwater water chemistry. The environmental factor Fe made a plumb line to Albidiferax, Pseudomonas, Geothrix, Desulfobacca, Desulfatiferula, Desulfosporosinus, and other dominant bacterial species in the medium, which fell in the positive direction of the Fe and Mn arrows and was closer to the origin. This shows that the dominant bacteria in this part of the population have a good positive correlation with Fe and Mn, characterizing a good positive correlation between the dominant bacteria and Fe and Mn. Similarly, there was a good correlation between Sulfuritalea and the environmental factor SO₄²⁻. Consequently, the function and activity of the dominant species in the environment were characterized by a synergistic evolution with the water chemistry.

4. Conclusions

This study took the Huangjia water source area in the Shenyang section of the main stream of the Liaohe River as the research object to explore the synergistic evolution relationship between hyporheic zone microorganisms and groundwater hydrochemistry under the influence of riverbank filtration. The results showed that the dominant bacterial phyla in the study area were Proteobacteria, Actinobacteria, etc. Proteobacteria contained a variety of functional bacteria, which were involved in the cycles of C, N, S, Fe, and Mn. The spatial distribution of microbial communities was heterogeneous and related to redox zonation, and there were differences in the abundance and types of dominant bacteria between shallow and deep seepage paths. RDA (Redundancy Analysis) showed that the dominant bacteria had a good correlation with environmental factors, such as Fe, Mn, DO (dissolved oxygen), and Eh (oxidation-reduction potential). Their functions and activities evolved synergistically with the hydrochemical environment. This not only provides a microbiological theoretical basis for redox zonation but also offers a reference for understanding the migration and transformation of Fe and Mn in the hyporheic zone.