Micromolar concentrations of metabolites enable coexistence of bacterial species in chemostats

Competition for a single limiting resource is expected to lead to competitive exclusion, yet diverse microbial communities persist even in nutrient-poor environments. Cross-feeding of essential metabolites is one mechanism that can promote coexistence between species, but its contribution is difficult to pinpoint experimentally. Here, we studied a prototroph-auxotroph pair growing on a single carbon source in chemostats. In minimal medium, the prototroph Comamonas testosteroni (Ct) supplied thiamine to the thiamine-auxotroph Ochrobactrum anthropi (Oa), allowing stable coexistence in agreement with consumer-resource theory. Contrary to our expectation that supplying thiamine would remove the dependency and lead to exclusion of Ct, coexistence persisted even when thiamine was supplemented. Our theoretical anlaysis showed that coexistence between competitors can be maintained by trace concentrations of an additional metabolite if it is taken up at sufficiently high affinity by the weaker competitor. Consistent with this prediction, targeted metabolomics and spent-medium assays identified growth-enhancing compounds at micromolar concentrations in Oa spent medium and as residues in fresh medium. Model analysis further showed that such weak positive effects can qualitatively change coexistence outcomes in chemostats while remaining undetected in standard batch interaction assays. Together, our results show that trace metabolites and subtle positive effects can reshape coexistence outcomes and should be incorporated into ecological models and interaction measurements.