Effects of nitrate limitation on the metabolome of Tetraselmis suecica biofilms

Phototrophic biofilms are photosynthetic microbial communities adhered to submerged surfaces. Research has largely focused on multispecies periphyton and benthic diatoms, while Chlorophyte-based, monospecific biofilms remain understudied - despite their increasing industrial relevance, particularly for the production of high value compounds. Here, we investigate the impact of nitrogen limitation on the metabolome of the green microalga <i>Tetraselmis suecica</i> grown in nitrogen-replete and nitrogen-limited monospecific biofilms. A specific culture system was developed to optimise the analysis of both the entire biofilm metabolome and spatial biochemical variations across cell layers. The Droop model was used to determine optimal initial conditions and sampling times. Then, metabolomic analysis by UHPLC-ESI(+)-QToF-HRMS/MS coupled with complementary biochemical analyses was performed on both conditions. Compared to nitrogen-replete biofilms, nitrogen-limited biofilms exhibited elevated C:N ratios (+277.4 %), reduced photosynthetic activity, and decreased pigment content (-18 % for Chl <i>a</i> and <i>b</i>). While total biovolume remained similar between experimental conditions, nitrogen limitation led to a redistribution of cell biomass, with increased surface layer biovolume (+112.3 %) at the expense of deeper layers. Macromolecular ratios of carbohydrates/proteins and lipids/proteins increased two- to three-fold, respectively, under nitrogen-limited conditions. The characterized metabolomic profile was dominated by monogalactosyldiacylglycerols (MGDGs) and digalactosyldiacylglycerols, whose relative abundances were significantly higher in nitrogen-replete condition. Notably, the annotated lipid MGDG(18:3/16:4) previously exhibited nitric oxide inhibitory activity. Given the previously observed role of nitric oxide in bacterial biofilm formation and diatom adhesion, we hypothesize that a feedback loop-like mechanism of adhesion regulation dependent on nitric oxide and nitrogen environmental conditions exists in monospecific phototrophic biofilms.