Serial cultures in invert emulsion and monophase systems for microbial community shaping and propagation.

BACKGROUND: Microbial communities harbor important biotechnological potential in diverse domains, however, the engineering and propagation of such communities still face both knowledge and know-how gaps. More specifically, culturing tools are needed to propagate and shape microbial communities, to obtain desired properties, and to exploit them. Previous work suggested that micro-confinement and segregation of microorganisms using invert (water-in-oil, w/o) emulsion broth can shape communities during propagation, by alleviating biotic interactions and inducing physiological changes in cultured bacteria. The present work aimed at evaluating invert emulsion and simple broth monophasic cultures for the propagation and shaping of bacterial communities derived from raw milk in a serial propagation design. RESULTS: The monophasic setup resulted in stable community structures during serial propagation, whereas the invert emulsion system resulted in only transiently stable structures. In addition, different communities with different taxonomic compositions could be obtained from a single inoculum. Furthermore, the implementation of invert emulsion systems has allowed for the enrichment of less abundant microorganisms and consequently facilitated their isolation on culture agar plates. CONCLUSIONS: The monophasic system enables communities to be propagated in a stable manner, whereas the invert emulsion system allowed for the isolation of less abundant microorganisms and the generation of diverse taxonomic compositions from a single inoculum.

Invert emulsions alleviate biotic interactions in bacterial mixed culture.

The large application potential of microbiomes has led to a great need for mixed culture methods. However, microbial interactions can compromise the maintenance of biodiversity during cultivation in a reactor. In particular, competition among species can lead to a strong disequilibrium in favor of the fittest microorganism. In this study, an invert emulsion system was designed by dispersing culture medium in a mixture of sunflower oil and the surfactant PGPR. Confocal laser scanning microscopy revealed that this system allowed to segregate microorganisms in independent droplets. Granulomorphometric analysis showed that the invert emulsion remains stable during at least 24 h, and that the introduction of bacteria did not have a significant impact on the structure of the invert emulsion. A two-strain antagonistic model demonstrated that this invert emulsion system allows the propagation of two strains without the exclusion of the less-fit bacterium. The monitoring of single-strain cultures of bacteria representative of a cheese microbiota revealed that all but Brevibacterium linens were able to grow. A consortium consisting of Lactococcus lactis subsp. lactis biovar diacetylactis, Streptococcus thermophilus, Leuconostoc mesenteroides, Staphylococcus xylosus, Lactiplantibacillus plantarum and Carnobacterium maltaromaticum was successfully cultivated without detectable biotic interactions. Metabarcoding analysis revealed that the system allowed a better maintenance of alpha diversity and produced a propagated bacterial consortium characterized by a structure closer to the initial state compared to non-emulsified medium. This culture system could be an important tool in the field of microbial community engineering.

A selection process based on the robustness of anti-Listeria monocytogenes activity reveals two strains of Carnobacterium maltaromaticum with biopreservation properties in cheese.

Biopreservation is an approach consisting of using microorganisms as protective cultures and/or their metabolites to optimize the microbiological quality and shelf life of food by ensuring safety or reducing food waste. Biopreservation strain selection pipelines mainly focus on inhibition strength to identify strains of interest. However, in addition to inhibition strength, inhibition activity must be able to be expressed despite significant variations in food matrix properties. In this study, the anti-Listeria monocytogenes EGDelux properties of a collection of 77 Carnobacterium maltaromaticum strains were investigated by high throughput competition assays under varying conditions of co-culture inoculation level, time interval between inoculation with C. maltaromaticum and L. monocytogenes, pH, and NaCl, resulting in 1309 different combinations of C. maltaromaticum strains and culture conditions. This screening led to the selection of two candidate strains with potent and robust anti-L. monocytogenes activities. Deferred growth inhibition assays followed by halo measurements, and liquid co-culture followed by colony counting, revealed that these two strains exhibit a wide anti-Listeria spectrum. Challenge tests in Camembert and Saint-Nectaire cheese revealed both strains were able to inhibit a cocktail of five strains of L. monocytogenes with high potency and high reproducibility. These results highlight the importance of including the robustness criterion in addition to potency when designing a strain selection process for biopreservation applications.

Mining Biosynthetic Gene Clusters in Carnobacterium maltaromaticum by Interference Competition Network and Genome Analysis.

Carnobacterium maltaromaticum is a non-starter lactic acid bacterium (LAB) of interest in the dairy industry for biopreservation. This study investigated the interference competition network and the specialized metabolites biosynthetic gene clusters (BGCs) content in this LAB in order to explore the relationship between the antimicrobial properties and the genome content. Network analysis revealed that the potency of inhibition tended to increase when the inhibition spectrum broadened, but also that several strains exhibited a high potency and narrow spectrum of inhibition. The C. maltaromaticum strains with potent anti-L. monocytogenes were characterized by high potency and a wide intraspecific spectrum. Genome mining of 29 strains revealed the presence of 12 bacteriocin BGCs: four of class I and eight of class II, among which seven belong to class IIa and one to class IIc. Overall, eight bacteriocins and one nonribosomal peptide synthetase and polyketide synthase (NRPS-PKS) BGCs were newly described. The comparison of the antimicrobial properties resulting from the analysis of the network and the BGC genome content allowed us to delineate candidate BGCs responsible for anti-L. monocytogenes and anti-C. maltaromaticum activity. However, it also highlighted that genome analysis is not suitable in the current state of the databases for the prediction of genes involved in the antimicrobial activity of strains with a narrow anti-C. maltaromaticum activity.