Fe (II)/Fe (III) regulated adaptive biofilm responses and microbial metabolic mechanisms for enhanced cycloalkane biodegradation

• Fe (II) added improved cycloalkane removal efficiency by 1.78 to 3.15 times. • The structure of the biofilm was greatly altered with 7.5 μM Fe (II) added. • Iron cycling and cyclohexane oxidizing bacteria played key roles in higher iron levels. • More Fe (II) increased ETSA by 393.7% and ATPase activity by744.3%. • Fe (II)-mediated evolution of microbial composition and regulation of gene functions. Oxidation of organic contaminants by iron-cycling microorganisms is an effective remediation strategy for contaminated environments. However, studies on the removal of cycloalkane using this method have been limited, and little is known about how microbes adapt to iron concentrations and how the latter stimulates contaminant degradation. Here, we investigated the biodegradation of cyclohexane (CyH) in three sequencing batch bioreactors (SBRs) with different iron concentrations (SBRs are named: Low 2.5 μM, Suit 7.5 μM, High 75 μM). The Suit reactor showed a more efficient CyH removal performance (average 98.61 ± 0.62 %) than the Low reactor (average 55.75 ± 3.38 %), which was associated with more extracellular polymeric substances (EPS) release and improved biofilm structure. The High reactor exhibited a gradual adaptation process due to the apparent accumulation of reactive oxygen species (ROS) under iron stress, with an average removal efficiency of (86.83 ± 18.81 %). However, the High reactor had a higher microbial activity (1.08-fold increase in ATPase activity) and electron transport system activity (1.18-fold increase) to facilitate direct electron transfer in the presence of insoluble iron compared to the Suit one, largely due to the dominant genus Novosphingobium . Metagenomics and metatranscriptomics analyses revealed lactone (a key intermediate) formation as a potential degradation pathway across three SBRs in our study under iron regulation. Notably, there were differences in the key functional genes expressed under different iron concentrations: genes related to anti-oxidative stress and iron-driven pathways, biofilm regulation-related pathways, and encoding protein complexes III and IV were found in the High, Suit, and Low reactors, respectively. This study enhances understanding of microbiota adaptation mechanisms to iron-disturbed conditions and provides a new insight into iron-cycling mediated biofilm formation and inhibition.