Measuring Temporal Variations of Nucleotide Pools in Microbial Granular Biofilm Performing Enhanced Biological Phosphorous Removal

Microbial communities often face environmental fluctuations that occur on timescales much shorter than their growth rate or proteome turnover. In such cases, cellular responses are likely driven by rapid changes in metabolite pools, particularly energy nucleotides including ATP, ADP, and AMP. However, robust methods to quantify these metabolites in biofilm-forming microbial communities are lacking. Here, we developed and systematically evaluated a metabolomics workflow for a granular biofilm enrichment, which performs Enhanced Biological Phosphorous Removal (EBPR). We combined fast quenching in liquid nitrogen and a boiling water extraction, followed by high resolution mass spectrometry, using porous graphitic carbon chromatography and ¹³ C-labeled internal reference standards. Among tested procedures, a boiling water extraction was most suitable for extraction of nucleotides, as indicated by stable adenylate energy charge (AEC) and isotopic ratios. Applied to an anaerobic–aerobic cycle of a lab scale EBPR system, the method revealed dynamic changes in AEC and uridylate energy charge (UEC) during acetate uptake and polyphosphate degradation. These results demonstrate that energy pool imbalances underlie rapid metabolic switching observed in EBPR systems. Moreover, the established method provides a foundation for performing metabolomic studies of microbial biofilms in general.