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Multi-strain probiotics are widely regarded as effective products for improving gut microbiota stability and host health, providing advantages over single-strain probiotics. However, in general, it is unclear to what extent different strains would cooperate or compete for resources, and how the establishment of a common biofilm microenvironment could influence their interactions. In this work, we develop an integrative experimental and computational approach to comprehensively assess the metabolic functionality and interactions of probiotics across growth conditions. Our approach combines co-culture assays with genome-scale modelling of metabolism and multivariate data analysis, thus exploiting complementary data- and knowledge-driven systems biology techniques. To show the advantages of the proposed approach, we apply it to the study of the interactions between two widely used probiotic strains of Lactobacillus reuteri and Saccharomyces boulardii , characterising their production potential for compounds that can be beneficial to human health. Our results show that these strains can establish a mixed cooperative-antagonistic interaction best explained by competition for shared resources, with an increased individual exchange but an often decreased net production of amino acids and short-chain fatty acids. Overall, our work provides a strategy that can be used to explore microbial metabolic fingerprints of biotechnological interest, capable of capturing multifaceted equilibria even in simple microbial consortia. • We propose an approach to predict interactions of probiotics across growth conditions. • Metabolism of L. reuteri and S. boulardii is studied in vitro and in silico. • Multi-strain probiotics can establish a mixed cooperative-antagonistic interaction. • Inter-species interaction are characterised in planktonic and biofilm growth. • In silico models well predict amino acid production across cultures. [ABSTRACT FROM AUTHOR]

Genetic heterogeneity is a common trait in microbial populations, caused by de novo mutations and changes in variant frequencies over time. Microbes can thus differ genetically within the same species and acquire different phenotypes. For instance, performance and stability of anaerobic reactors are linked to the composition of the microbiome involved in the digestion process and to the environmental parameters imposing selective pressure on the metagenome, shaping its evolution. Changes at the strain level have the potential to determine variations in microbial functions, and their characterization could provide new insight into ecological and evolutionary processes driving anaerobic digestion. In this work, single nucleotide variant dynamics were studied in two time-course biogas upgrading experiments, testing alternative carbon sources and the response to exogenous hydrogen addition. A cumulative total of 76,229 and 64,289 high-confidence single nucleotide variants were discerned in the experiments related to carbon substrate availability and hydrogen addition, respectively. By combining complementary bioinformatic approaches, the study reconstructed the precise strain count—two for both hydrogenotrophic archaea—and tracked their abundance over time, while also characterizing tens of genes under strong selection. Results in the dominant archaea revealed the presence of nearly 100 variants within genes encoding enzymes involved in hydrogenotrophic methanogenesis. In the bacterial counterparts, 119 mutations were identified across 23 genes associated with the Wood-Ljungdahl pathway, suggesting a possible impact on the syntrophic acetate-oxidation process. Strain replacement events took place in both experiments, confirming the trends suggested by the variants trajectories and providing a comprehensive understanding of the biogas upgrading microbiome at the strain level. Overall, this resolution level allowed us to reveal fine-scale evolutionary mechanisms, functional dynamics, and strain-level metabolic variation that could contribute to the selection of key species actively involved in the carbon dioxide fixation process. [Display omitted] • This is the first use of strain-resolved metagenomics in in-situ biogas upgrading. • A total of 97 high-quality metagenome assembled genomes were reconstructed. • The variants calling approach retrieved 140,518 variants in high abundance genomes. • Two strains were found in M. wolfeii and M. thermophilus at distinct time points. • Eight hydrogenotrophic methanogenesis genes were under selective pressure. [ABSTRACT FROM AUTHOR]