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Microbial degradation of fluorinated compounds raised significant attention because of their widespread distribution and potential environmental impacts. Here, we report a bacterial isolate, Rhodococcus sp. NJF-7 capable of defluorinating monofluorinated medium-chain length alkanes. This isolate consumed 2.29 ± 0.13 mmol L− 1 of 1-fluorodecane (FD) during a 52 h incubation period, resulting in a significant release of inorganic fluoride amounting to 2.16 ± 0.03 mmol L− 1. The defluorination process was strongly affected by the initial FD concentration and pH conditions, with lower pH increasing fluoride toxicity to bacterial cells and inhibiting enzymatic defluorination activity. Stoichiometric conversion of FD to fluoride was observed at neutral pH with resting cells, while defluorination was significantly lower at reduced pH (6.5). The discovery of the metabolites decanoic acid and methyl decanoate suggests that the initial attack by monooxygenases may be responsible for the biological defluorination of FD. The findings here provide new insights into microbial defluorination processes, specifically aiding in understanding the environmental fate of organic semi-fluorinated alkane chemicals. [ABSTRACT FROM AUTHOR]