Natural isolates of the soil‐dwelling bacterium Bacillus subtilis form robust biofilms under laboratory conditions and colonize plant roots. B. subtilis biofilm gene expression displays phenotypic heterogeneity that is influenced by a family of Rap‐Phr regulatory systems. Most Rap‐Phr systems in B. subtilis have been studied independently, in different genetic backgrounds and under distinct conditions, hampering true comparison of the Rap‐Phr systems’ impact on bacterial cell differentiation. Here, we investigated each of the 12 Rap‐Phr systems of B.subtilis NCIB 3610 for their effect on biofilm formation. By studying single ∆ rap ‐ phr mutants, we show that despite redundancy between the cell–cell communication systems, deletion of each of the 12 Rap‐Phr systems influences matrix gene expression. These Rap‐Phr systems therefore enable fine‐tuning of the timing and level of matrix production in response to specific conditions. Furthermore, some of the ∆ rap ‐ phr mutants demonstrated altered biofilm formation in vitro and colonization of Arabidopsis thaliana roots, but not necessarily similarly in both processes, indicating that the pathways regulating matrix gene expression and other factors important for biofilm formation may be differently regulated under these distinct conditions.

Natural isolates of the soil-dwelling bacterium Bacillus subtilis form robust biofilms under laboratory conditions and colonize plant roots. B. subtilis biofilm gene expression displays phenotypic heterogeneity that is influenced by a family of Rap-Phr regulatory systems. Most Rap-Phr systems have been studied independently, in different genetic backgrounds and under distinct conditions, hampering true comparison of the Rap-Phr systems impact on bacterial differentiation. Here, we investigated each of the 12 Rap-Phr systems of B. subtilis NCIB 3610 for their role in biofilm formation. While Δ11 rap-phr mutants displayed increased matrix gene expression under biofilm inducing conditions, only some of the mutants demonstrated altered biofilm formation and colonization of Arabidopsis thaliana roots. Therefore, matrix gene expression does not directly correlate with biofilm formation in vitro and on the root. Our results suggest that each of the 12 Rap-Phr systems influences matrix gene expression, thereby allowing fine-tuning of the timing and level of matrix production in response to specific conditions, but additional factors also contribute to biofilm architecture and root colonization.

To make biofuel production feasible from an economic point of view, several studies have investigated the main associated bottlenecks of the whole production process through approaches such as the “cradle to grave” approach or the Life Cycle Assessment (LCA) analysis, being the main constrains the feedstock collection and transport. Whilst several feedstocks are interesting because of their high sugar content, very few of them are available all year around and moreover do not require high transportation’ costs. This work aims to investigate if the “zero miles” concept could bring advantages to biofuel production by decreasing all the associated transport costs on a locally established production platform. In particular, a specific case study applied to the Technical University of Denmark (DTU) campus is used as example to investigate the advantages and feasibility of using the spent coffee grounds generated at the main cafeteria for the production of bioethanol on site, which can be subsequently used to (partially) cover the campus’ energy demands.