Species Dynamics and Gene Expression in Algal Polycultures Under Mixotrophic Cultivation in Outdoor Pilot-Scale...

Mixotrophic cultivation of microalgal polycultures can enhance biomass productivity and nutrient recovery from wastewaters compared to autotrophic systems. To better understand non-axenic polycultures under mixotrophic conditions, this study examined species composition and gene expression in a local freshwater green algal polyculture cultivated in outdoor raceway ponds in Southeast Sweden during spring and autumn. Cultures were cultivated under autotrophic conditions in leachate-KH₂PO₄ medium or under mixotrophic conditions with glucose or whey permeate supplementation. Community composition was analyzed using high-throughput sequencing of 18S and 16S rRNA genes, while transcriptomic responses were assessed by differential gene expression analysis. The eukaryotic community remained stable and dominated by green algae (Chlorophyta) under autotrophic conditions, whereas mixotrophic cultivation promoted increases in Ciliophora and Rotifera. Transcriptomic analysis revealed downregulation of photosynthesis-related genes alongside upregulation of nutrient transporters under mixotrophy, consistent with enhanced nutrient removal. Mixotrophic cultivation showed potential for enhanced carbohydrate accumulation and upregulated pathways for oleic acid and amino acid biosynthesis, including arginine, glutamine, glycine, proline, and asparagine. These results demonstrate that mixotrophic conditions drive both microbial community shifts and metabolic reprogramming, underscoring the importance of cultivation strategy and polyculture selection for targeted applications such as nutrient recovery and bioproduct generation.