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Human gut microbiota species which are next-generation probiotics (NGPs) candidates are of high interest as they have shown the potential to treat intestinal inflammation and other diseases. Unfortunately, these species are often not robust enough for large-scale cultivation, especially in maintaining diversity in co-culture production. In this study, we describe interactions between human gut microbiota species in the cultivation process with unique substrates. We also demonstrated that it is possible to change the species ratio in co-culture by changing the ratio of carbon sources. We screened 25 different bacterial species based on their metabolic capabilities. After evaluating unique substrate possibilities, we chose Anaerostipes caccae (A. caccae), Bacteroides thetaiotaomicron (B. thetaiotaomicron), and Bacteroides vulgatus (B. vulgatus) as subjects for further study. D-sorbitol, D-xylose, and D-galacturonic acid were selected as substrates for A. caccae , B. thetaiotaomicron , and B. vulgatus respectively. All three species were cultivated as both monocultures and in co-cultures in serial batch fermentations in an isothermal microcalorimeter. Positive interactions were detected between the species in both co-cultures (A. caccae + B. thetaiotaomicron ; A. caccae + B. vulgatus) resulting in higher heat production compared to the sum of the monocultures. The same positive cross-feeding interactions took place in larger-scale cultivation experiments. We confirmed acetate and lactate cross-feeding between A. caccae and B. thetaiotaomicron with flux balance analysis (FBA). Changing the ratio of the selected carbon sources in the medium changed the species ratio accordingly. Such robustness is the basis for developing more efficient industrial co-culture processes including the production of NGPs. • The growth of 25 bacterial species was evaluated in chemically defined medium. • A. caccae , B. thetaiotaomicron , and B. vulgatus were chosen as model organisms. • Stable co-culture was achieved throughout three consecutive batches. • Trophic interactions of A. caccae and Bacteroides spp. boost species' growth rates. • Unique substrates help to control biomass concentrations in co-culture production. [ABSTRACT FROM AUTHOR]