| Summary: | To fulfil the growing interest in investigating microbial interactions in co-cultures, a novel two-compartment bioreactor system was developed, characterised, and implemented. The system allowed for the exchange of amino acids and peptides via a polyethersulfone membrane that retained biomass. Further system characterisation revealed a Bodenstein number of 18, which hints at backmixing. Together with other physical settings, the existence of unwanted inner-compartment substrate gradients could be ruled out. Furthermore, the study of Damkoehler numbers indicated that a proper metabolite supply between compartments was enabled. Implementing the two-compartment system (2cs) for growing <i>Streptococcus thermophilus</i> and <i>Lactobacillus delbrueckii</i> subs. <i>bulgaricus</i>, which are microorganisms commonly used in yogurt starter cultures, revealed only a small variance between the one-compartment and two-compartment approaches. The 2cs enabled the quantification of the strain-specific production and consumption rates of amino acids in an interacting <i>S. thermophilus</i>–<i>L. bulgaricus</i> co-culture. Therefore, comparisons between mono- and co-culture performance could be achieved. Both species produce and release amino acids. Only alanine was produced <i>de novo</i> from glucose through potential transaminase activity by <i>L. bulgaricus</i> and consumed by <i>S. thermophilus</i>. Arginine availability in peptides was limited to <i>S. thermophilus’</i> growth, indicating active biosynthesis and dependency on the proteolytic activity of <i>L. bulgaricus</i>. The application of the 2cs not only opens the door for the quantification of exchange fluxes between microbes but also enables continuous production modes, for example, for targeted evolution studies.
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