Sequence of inoculation influences the nature of extracellular polymeric substances and biofilm formation in Azotobacter chroococcum and Trichoderma viride
&NA; Extracellular polymeric substances (EPS) are important structural components of biofilms. In the present study, the EPS in biofilms developed using two agriculturally beneficial organisms—Azotobacter chroococcum (Az) and Trichoderma viride (Tv) were quantified and characterised. Time course experiments were undertaken to optimise the EPS yield of biofilm samples resulting from coculture and staggered inoculation. The EPS produced during biofilm formation was found to differ quantitatively and qualitatively in individual cultures (Az alone, Tv alone), and in treatments differing in the sequence of inoculation of bacterium and fungus (Az + Tv coculture, staggered inoculation of Az followed by Tv i.e. Az ‐ Tv, or Tv followed by Az i.e. Tv ‐ Az). Significant enhancement in terms of growth and biofilm formation, as compared to individual inoculation was recorded, with Tv ‐ Az exhibiting higher values of these attributes. The EPS from biofilms showed significantly higher concentrations of protein, acetyl, and uronic acids, while planktonic EPS recorded higher total carbohydrates. Fourier transform infrared spectroscopy analyses illustrated the significant influence on chemical and structural aspects of EPS (planktonic and biofilm). This represents a first report correlating EPS production, cell aggregation and biofilm formation during bacterial‐fungal biofilm development, which can have implications in the colonisation of soil and plants.