Untargeted GC–MS reveals differential regulation of metabolic pathways in cyanobacterium Anabaena and its biofilms with Trichoderma viride and Providencia sp.

Cyanobacteria and their biofilms are used as biofertilizing options to improve plant growth, soil fertility, and grain quality in various crops, however, the nature of metabolites involved in such interactions is less explored. The present investigation compared the metabolite profiles of cyanobacterial biofilms: Anabaena torulosa- Trichoderma viride (An-Tr) and A. torulosa- Providencia sp. (An-PW5) against the individual culture of A. torulosa (An) using untargeted gas chromatography-mass spectroscopy. Metabolites were identified using the NIST mass spectral library and the relative peak area of cultures analysed, after normalization with an internal standard, ribitol. An-Tr biofilm recorded approximately 66.85% sugars, with increased quantity and numbers of sugars and their conjugates, which included maltose, lactose, and d-mannitol, but decreased amino acids concentrations, attributable to the effect of Tr as partner. Heat map and cluster analysis illustrated that An-Tr biofilm possessed a unique cluster of metabolites. Partial least square-discriminate analysis (PLS-DA) and pathway analyses showed distinct modulation in terms of metabolites and underlying biochemical routes in the biofilms, with both the partners- PW5 and Tr eliciting a marked influence on the metabolite profiles of An, leading to novel cyanobacterial biofilms. In the An-PW5 biofilm, the ratios of sugars, lactose, mannitol, maltose, mannose, and amino acids serine, ornithine, leucine and 5‑hydroxy indole acetic acid were significantly higher than An culture. Such metabolites are known to play an important role as chemoattractants, facilitating robust plant -microbe interactions. This represents a first-time study on the metabolite profiles of cyanobacterial biofilms, which provides valuable information related to their significance as inoculants in agriculture.