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, 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.

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.