A semi-automated image processing method for analysing the taxonomic composition of algal biofilms

Abstract Benthic phototrophic communities play a crucial role in the functioning of shallow lake ecosystems. The complexity of benthic algal communities poses challenges for exploring their fine structures using traditional methods such as light microscopy. However, confocal laser scanning microscopy (CLSM) offers the potential for microscale-level structural investigation of phototrophic biofilms, thereby contributing to a deeper understanding of the structural and compositional changes within these communities. The primary objective of this study was to develop a relatively rapid, semi-automated, and freely available image processing method to facilitate the tracking of spatio-temporal alterations in intact biofilms. To evaluate our image processing method, we conducted an eight-week long outdoor mesocosm experiment, manipulating temperatures based on two global warming scenarios (assuming intermediate- and high degree of greenhouse gas emission), with increases of 3°C and 5°C, respectively, compared to the reference/unheated mesocosms. Eight limestone cubes were placed on the bottom of each mesocosm to provide 'natural' surfaces for phytobenthos colonization, which was subsequently analysed by confocal microscopy. We hypothesized that increasing water temperature might have a significant impact on the taxonomic composition of algal biofilms, particularly on the abundance of phototrophic prokaryotes and eukaryotes. The designed 'FIJI macro' efficiently analysed the recorded images, including single images, series of Z-stack images, and projection images. Our image processing method effectively discriminated photosynthetic prokaryotes and eukaryotes based on their pigment composition and proved suitable for the high-throughput analysis of structural and compositional changes in algal biofilms. Moreover, this study confirmed that climate-induced warming can lead to an increasing dominance of cyanobacteria in benthic phototrophic communities at the expense of eukaryotic algae.