Abstract <p>We investigated the mechanism underlying the coexistence of the phenol-degrading bacteria <i>Cupriavidus</i> sp. strain P-10 and <i>Comamonas thiooxydans</i> strain R2 using a chemostat culture supplemented with phenol as the sole carbon and energy source. Quantitative PCR (qPCR) targeting the genes encoding the large subunit of phenol hydroxylase <i>dmpN</i> and <i>aphN</i> for strains P-10 and R2, respectively, revealed the coexistence of the two strains even under phenol-limiting conditions. According to the reverse transcription qPCR targeting <i>dmpN</i>, <i>aphN</i>, catechol 2,3-dioxygenase genes (<i>dmpB</i> and <i>aphB</i>), and catechol 1,2-dioxygenase gene (<i>catA</i>) of strain P-10, both strains transcribed these genes, suggesting that phenol was metabolized individually. However, mathematical simulations demonstrated that the two strains never coexisted because competitive exclusion occurred at any phenol sharing ratio. The simulation revealed that strain P-10 outcompeted strain R2 when it occupied more than 64% of the phenol. These results suggest that phenol sharing, with fluctuations in the ratio within an appropriate range, is indispensable for coexistence under resource-limiting conditions. The phenol concentration in the chemostat remained spatially heterogeneous for 15&#xa0;s. If coexistence was accomplished through the phenol-sharing, either strain P-10 or R2 could invade another single-strain chemostat culture using phenol, even when inoculated at a significantly lower cell density than the resident strain. Strains P-10 and R2 successfully invaded and coexisted with other strains in the chemostat culture. These results suggest that fluctuation of the limiting substrate sharing ratio enabled stable coexistence of bacterial strains under competitive conditions.</p> Key point <p>• <i>Phenol concentration was spatiotemporally heterogeneous in the chemostat culture</i></p>