Effects of an organic carbon source, sodium citrate, on the structure and microbial community composition of an early-stage multispecies biofilm model. EFFECTS OF SODIUM CITRATE ON SIMULATED FRESHWATER BIOFILMS

In recent years, most biofilm studies have focused on fundamental investigations using multispecies biofilm models developed preferentially in simulated naturally occurring low-nutrient medium than in artificial nutrient-rich medium. However, because biofilm development under low-nutrient growth media is slow, natural media are often supplemented with an additional carbon source to increase the rate of biofilm formation. Although carbon source supplementation may indeed accelerate biofilm formation and biomass accumulation, there are knowledge gaps in interpreting the effects of such supplementation on the resulting biofilm in terms of structure and microbial community composition—changes that can influence the outcome of the intended simulated multispecies biofilm model. We investigated the effects of supplementation of a simulated fresh water medium with sodium citrate on the resulting structure, bacterial community composition, and microbial network interactions of an early-stage multispecies biofilm model. Qualitative and quantitative analyses of acquired confocal laser scanning microscopy data confirmed that sodium citrate supplementation distinctly increased biofilm biomass. Notably, the analysis of 16S rRNA gene sequencing data revealed that the microbial community structure of biofilms grown in sodium citrate-supplemented conditions was characterized with increased relative abundance and dominance of Proteobacteria compared with that of biofilms grown in sodium citrate-free conditions. Further, network analysis revealed that sodium citrate supplementation promoted distinct network interconnections between the most abundant phyla, characterized by higher number of co-exclusion interactions and fewer co-inclusion interactions. Our findings suggest that the supplementation of a low-nutrient medium with a carbon source for generating multispecies biofilms may alter their structure and microbial community composition such that the biofilm phenotype differs from that which was originally meant to be investigated. Therefore, the possibility of such changes in the biofilm phenotype should be considered when supplementing experimentally simulated naturally occurring media.