<b>Low initial metabolite production enhances stability in syntrophic bacterial consortia</b>

Bacteria frequently engage in syntrophic interactions with other microorganisms through metabolite exchange, yet the mechanisms sustaining their stability remain poorly understood. Here, we combine evolutionary experiments and validation assays to investigate how phenotypic heterogeneity in amino acid production stabilizes syntrophic interactions between Escherichia coli lysine- (ΔL) and arginine-auxotroph (ΔA). In a 27-day evolution experiment, growth ceased in 70% of cocultures, with mutual benefit transitioning to amensalism as both auxotrophs reduced partner-essential amino acid production. However, 30% of cocultures maintained stable mutual beneficial relationships and ancestral fitness levels, with amino acid production levels unchanged. Subsequent validation experiments revealed that pre-existing phenotypic heterogeneity - variability in amino acid production among genetically identical strains - determined consortium outcomes. Cocultures pairing initial low-production ΔL and ΔA strains exhibited greater stability than high-production consortia. We showed that higher amino acid production was rather unfavorable to consortium fitness. High amino acid production strains incurred fitness trade-offs to elevated mortality and metabolic redundancy, resulting in the destabilizing of mutually beneficial interactions. Our findings demonstrate that pre-existing phenotypic heterogeneity in initial levels of amino acid production determines variation in the outcomes of inter-strain interactions, offering insights into the stability of microbial communities.