Nutrients and light effects on stream biofilms: a combined assessment with CLSM, structural and functional parameters

Abstract

Nutrients and light are the most determinant factors for microbial benthic assemblages in oligotrophic forested streams. We investigated the importance of nutrients and light availability on the structure and the function of epilithic biofilms in a Mediterranean forested stream (Fuirosos, Spain). Biofilms grew on artificial substrata in both enriched and unenriched reaches where shade conditions were simulated. Four different treatments were generated: higher light unenriched, lower light unenriched, higher light enriched (HL-E) and lower light enriched. Chlorophyll a, bacterial density, extracellular polymeric substances (EPS), extracellular leucine aminopeptidase (LAmP) and alkaline phosphatase (APase) activities were analysed during the colonisation at days 4, 9, 16, 22 and 52. At day 52, confocal laser scanning microscopy (CLSM) was used to determine differences in biofilm architecture. CLSM evidenced differences in thickness and structural complexity of biofilms grown in different conditions. Biofilms in HL-E were the thickest and had the most complex structure. The CLSM highlighted that the EPS was agglomerated in the upper layer of enriched-grown biofilms, but evenly distributed through the biofilm in unenriched biofilms. CLSM 3D images suggested that cyanobacteria increased under higher nutrient conditions. Nutrient enrichment caused the decrease of APase activity. Interaction between the two factors affected LAmP activity. HL-E had the highest LAmP and the lowest APase activities, an indication that biofilm responses to nutrients mostly occurred with high-light availability. Our results revealed that the conjoint availability of light and nutrients caused the highest changes in biofilm spatial organisation, microbial structure and functioning in oligotrophic forested streams.