Hydrochemical controls on microbial community structure in the Reshuigou hot springs, northeastern Qinghai-Tibet Plateau

Technical Summary: Hydrochemical Controls on Microbial Community Structure in the Reshuigou Hot Springs

Problem Statement
Geothermal hot springs represent complex ecosystems where deep thermal fluids, water-rock interactions, and surface discharge environments converge. While previous research has often emphasized temperature and pH as the primary drivers of microbial community structure, recent evidence suggests that hydrochemical backgrounds—including salinity, sulfate, chloride, redox conditions, and spatial isolation—play critical, non-linear roles in shaping these communities. In the Gonghe-Guide Basin on the northeastern margin of the Qinghai-Tibet Plateau, a region rich in fault-controlled geothermal systems, there is a need to understand how specific hydrochemical evolution processes structure microbial niches. This study addresses the question of how hydrochemical conditions in the Reshuigou hot springs influence microbial community composition, moving beyond simple taxonomic listing to integrate geochemical origins with ecological responses.

Methodology
The study analyzed six water samples (RSG01, RSG02, RSG03, RSG13, RSG14, RSG15) collected from the Reshuigou discharge belt in Guide County, Qinghai Province, in April 2023. The research employed an integrated approach combining hydrogeochemistry and microbial ecology:

• Hydrochemical Analysis: Field parameters (temperature, pH, ORP, DO, EC) were recorded in situ. Major ions (cations and anions) and trace elements (F⁻, As) were quantified. Data were processed using Piper, Gibbs, Schoeller, and Stiff diagrams to classify hydrochemical facies and identify controlling mechanisms. Mineral saturation indices (SI) for calcite, aragonite, dolomite, gypsum, anhydrite, fluorite, and halite were calculated using PHREEQC 3.8.6 with the wateq4f.dat database.
• Microbial Community Profiling: Microbial biomass was concentrated via 0.22 μm filtration. DNA extraction followed, targeting the V4-V5 region of the 16S rRNA gene using 515F/907R primers. Sequencing was performed on the Illumina MiSeq PE300 platform. Data processing involved demultiplexing, denoising with DADA2, and taxonomic annotation against the SILVA database.
• Statistical Integration: Multivariate analyses were conducted to link hydrochemistry and biology. These included Principal Coordinates Analysis (PCoA) based on Bray-Curtis distances, Redundancy Analysis (RDA) with Hellinger-transformed community matrices, and Spearman rank correlation tests. Environmental fitting was used to assess the consistency between hydrochemical variables and ordination structures.

Key Results

• Hydrochemical Characteristics: The Reshuigou waters are characterized by medium-to-high temperatures (46.00–60.70 °C), weak alkalinity (pH 7.93–7.96), reducing conditions (ORP -182.20 to -39.50 mV), and moderate-to-high mineralization (TDS 1452–1587 mg/L). The hydrochemical facies are consistently Na+K/Cl-SO₄ type. Ion-ratio analysis and Gibbs diagrams indicate that the water chemistry is controlled by deep circulation-related water-rock interaction, silicate dissolution, and inputs of sulfate and chloride, rather than simple atmospheric precipitation or evaporation. PHREEQC results suggest calcite and aragonite are near saturation, while fluorite is close to saturation, consistent with elevated F⁻ levels.
• Microbial Community Structure: The communities are dominated by Proteobacteria, Aquificota, Deinococcota, Chloroflexi, and Bacteroidota. At the genus level, Hydrogenobacter, Thermus, Herbaspirillum, and Delftia are prominent.
  • Community Types: The study identified distinct community assemblages:
    • Hydrogenobacter-Aquificota type (RSG14, RSG15): Associated with chemoautotrophic processes (hydrogen/sulfur oxidation).
    • Thermus-Deinococcota type (RSG03): Linked to higher temperatures and thermophilic heterotrophy.
    • Proteobacteria single-dominance type (RSG02): Characterized by Herbaspirillum and Delftia, suggesting a response to oxidative mixing or local discharge conditions.
    • Chloroflexi-enhanced type (RSG01): Indicative of reducing micro-niches.
• Environmental Controls: RDA and correlation analyses revealed that no single factor controls the community. Instead, structure is jointly shaped by temperature, redox conditions (ORP), mineralization (TDS), major ion composition (Ca²⁺, K⁺, Cl⁻), and the carbonate system. For instance, Proteobacteria dominance in RSG02 correlates with higher TDS and Cl⁻, while Chloroflexi shows associations with reducing conditions and HCO₃⁻.

Key Contributions
This study provides a unified framework linking deep hydrothermal hydrochemical backgrounds with shallow discharge conditions to explain microbial niche differentiation in a fault-controlled hot spring system.

1. Integrated Framework: It moves beyond reporting hydrochemical facies or microbial lists separately, demonstrating how mineral saturation states and ion ratios serve as ecological filters alongside temperature.
2. Regional Specificity: It characterizes the specific Na+K/Cl-SO₄ facies of the Reshuigou system and its associated microbial assemblages, contributing to the understanding of geothermal water-microbe coupling on the northeastern Qinghai-Tibet Plateau.
3. Differentiation Mechanisms: The study highlights that even within a single geothermal system with relatively uniform pH and moderate temperature gradients, variations in redox state, mineralization, and local mixing create distinct micro-niches that select for different dominant taxa (e.g., the shift from Hydrogenobacter to Proteobacteria).

Significance and Claims
The authors claim that the microbial community structure in Reshuigou is not determined by a single environmental variable but is shaped by the interplay of local discharge conditions, oxidative mixing processes, and micro-niche differentiation under a shared deep hydrothermal background. The study emphasizes that while temperature and pH are fundamental, ion composition and redox conditions act as sensitive ecological differentiation factors in this weakly alkaline geothermal system. These findings offer a regional case study for biogeochemical research, illustrating how hydrochemical evolution structures microbial niches in fault-controlled hot springs. The paper maintains a modest stance, noting that due to the limited sample size (n=6), statistical results should be interpreted as environmental-response clues and biological plausibility rather than definitive causal evidence, requiring further validation.