In petroleum-contaminated aquifers, iron (III) serves as an electron acceptor, enabling microbial degradation of organic matter. While previous studies have focused on iron reduction and organic matter degradation under laboratory conditions, research on iron-associated microorganisms in petroleum-contaminated aquifers is limited. To explore the diversity and distribution of such microorganisms in natural settings, this study used metagenomic analysis of an iron-rich, petroleum-contaminated aquifer. Sixteen groundwater samples from both pollution source and background areas were collected for species annotation and functional gene identification. Results show more than 7000 species were identified as iron-reducing microorganisms (IRMs), including several previously well-characterized iron-reducing species (e.g., Geobacter luticola and Geobacter sulfurreducens). However, the majority of IRMs were not found in existing iron-reducing microbial databases. Some of them, such as Sulfurospirillum sp. and Extensimonas perlucida, could be taxonomically classified at the species level, while most were only annotated as unclassified bacteria. In the contamination source zone, these microorganisms proliferated extensively, which led to hydrocarbon degradation predominantly driven by iron reduction in the aquifer. This study enhances our understanding of hydrocarbon-degrading microorganisms and supports the management of petroleum-contaminated sites.

Enhanced Natural Attenuation (ENA) can accelerate pollutant degradation by adding electron acceptors or nutrients. However, its impact on carbon-fixing microorganisms, which are widely found in the natural attenuation process, remains unclear. In this study, four types of ENA materials were added in batch experiments. Chemical analysis and metagenomic sequencing were employed to analyze the degradation kinetics of petroleum hydrocarbons, the consumption pattern of nitrate, as well as the functional genes and population evolution characteristics of carbon-fixing microorganisms. Results showed that nitrate-based enhancement materials significantly improved the petroleum hydrocarbon degradation rate but suppressed the expression of some carbon fixation genes, such as those involved in the Calvin–Benson–Bassham cycle. Nevertheless, the overall abundance of carbon fixation genes did not show a notable decline. Dominant bacterial genera such as Pseudomonas and Achromobacter possessed both hydrocarbon degradation and carbon fixation capabilities. Although the calcium peroxide treatment group only achieved a 40% petroleum hydrocarbon degradation rate, it significantly promoted the abundance of carbon fixation genes involved in the reductive tricarboxylic acid cycle pathway. Therefore, ENA alters carbon fixation pathways but does not diminish carbon fixation potential, indicating its potential for synergistically achieving pollution remediation and carbon fixation.

The mineralization of benzene, toluene, ethylbenzene, and xylene (BTEX) into inorganic substances by microorganisms may affect the water–rock interaction. However, few studies have quantitatively analyzed the processes. To quantitatively reveal this mechanism, in this study, nitrate and toluene were taken as the typical electron acceptor and BTEX, respectively. Based on hydro-geochemical theory, the mechanism and mathematical model were established. In addition, the model was verified with a toluene mineralization experiment. The mechanism model demonstrated that H⁺ was the main factor in the dissolution or precipitation of CaCO₃. The mathematical model derived the equations quantitatively between the amount of toluene mineralization, CaCO₃, and some biogeochemical indicators, including temperature, microbial consumption, and other major ions in groundwater. According to the model, the amount of dissolved CaCO₃ increased with the increasing proportion of completely reduced nitrate. For a complete reaction, the greater the microorganisms’ consumption of toluene was, the smaller the precipitation of CaCO₃. CaCO₃ dissolution was a nonmonotonic function that varied with temperature and the milligram equivalent of other ions. Furthermore, the validation experiments agreed well with the mathematical model, indicating its practicality. The established model provides a tool for assessing the biodegradation of toluene by monitoring the concentration of groundwater ions.