The benefits of an electron-transfer mechanism for petroleum biodegrading have been widely acknowledged, but few have studied the spatial pattern of microbial community diversity in groundwater fields, and few discuss the bacterial community’s diversity in relation to electron donors-acceptors distribution, which is largely determined by groundwater flow. Eleven samples in different groundwater fields are collected at a petroleum-contaminated site, and the microbial communities are investigated using 16S rRNA gene sequences with multivariate statistics. These are mainly linked to the chemical composition analysis of electron donor indexes COD, BTEX and electron acceptor indexes DO, NO₃⁻, Fe²⁺, Mn²⁺, and SO₄²⁻, HCO₃⁻. The spatial pattern of the bacterial community’s diversity is characterized and the effect of the electron redox reaction on bacterial community formation in different groundwater field zones is elucidated. It is found that a considerable percentage (>65%) of the bacterial communities related to petroleum degrading suggest that petroleum biodegrading is occurring in groundwater. The communities are subject to the redox reaction in different groundwater field zones: The side plume zone and the upstream of the source zone are under aerobic redox or denitrification redox, and the corresponding bacteria are Rhodoferax, Novosphingobium, Hydrogenophaga, and Comamonas; the source zone and downstream of the source zone are under Fe³⁺, Mn⁴⁺, and SO₄²⁻ reduction redox, and the corresponding bacteria are Rhodoferax, Treponema, Desulfosporosinus, Hydrogenophaga, and Acidovorax. These results imply that groundwater flow plays a definitive role in the bacterial community’s diversity spatial pattern formation by influencing the distribution of electron donor and acceptor.

In petroleum-contaminated aquifers, biodegradation is always associated with various types of microbial metabolism. It can be classified as autotrophic (such as methanogenic and other carbon fixation) and heterotrophic (such as nitrate/sulfate reduction and hydrocarbon consumption) metabolism. For each metabolic type, there are several key genes encoding the reaction enzymes, which can be identified by metagenomics analysis. Based on this principle, in an abnormally low dissolved inorganic carbon (DIC) petroleum-contaminated aquifer in North China, nine groundwater samples were collected along the groundwater flow, and metagenomics analysis was used to discover biodegradation related metabolism by key genes. The major new finding is that autotrophic metabolism was revealed, and, more usefully, we attempt to explain the reasons for abnormally low DIC. The results show that the methanogenesis gene, Mcr, was undetected but more carbon fixation genes than nitrate reduction and sulfate genes were found. This suggests that there may be a considerable number of autotrophic microorganisms that cause the phenomenon of low concentration of dissolved inorganic carbon in contaminated areas. The metagenomics data also revealed that most heterotrophic, sulfate, and nitrate reduction genes in the aquifer were assimilatory sulfate and dissimilatory nitrate reduction genes. Although there was limited dissolved oxygen, aerobic degrading genes AlkB and Cdo were more abundant than anaerobic degrading genes AssA and BssA. The metagenomics information can enrich our microorganic knowledge about petroleum-contaminated aquifers and provide basic data for further bioremediation.

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.

Groundwater at petroleum-contaminated sites typically exhibits elevated dissolved inorganic carbon (DIC) levels due to hydrocarbon biodegradation; however, our prior field investigations revealed an enigmatic DIC depletion anomaly that starkly contradicts this global pattern and points to an unrecognized carbon sink. In a breakthrough demonstration, this study provides the first experimental confirmation that sulfur-oxidizing bacteria (SOB) drive substantial carbon sequestration via a coupled sulfur oxidation autotrophic assimilation process. Through integrated hydrochemical monitoring and 16S rRNA sequencing in an enrichment culture system, we captured the complete DIC transformation trajectory: heterotrophic acetate degradation initially increased DIC to 370 mg/L, but subsequent autotrophic assimilation by SOB dramatically reduced DIC to 270 mg/L, yielding a net consumption of 85 mg/L. The distinctive pH dynamics (initial alkalization followed by acidification) further corroborated microbial regulation of carbon cycling. Critically, Pseudomonas stutzeri and P. alcaliphila were identified as the dominant carbon-fixing agents. These findings definitively establish that chemolithoautotrophic SOB convert DIC into organic carbon through a “sulfur oxidation-carbon fixation” coupling mechanism, overturning the conventional paradigm of petroleum-contaminated sites as perpetual carbon sources. The study fundamentally redefines natural attenuation frameworks by introducing microbial carbon sink potential as an essential assessment metric for environmental sustainability.