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Abstract Aseptic technique has historically served as a fundamental practice in microbiology, helping to maintain culture purity and integrity. This technique has been widely encouraged and employed for use with cultures of heterotrophic bacteria as well as freshwater and marine algae. Yet, recent observations have suggested that these approaches may bring their own influences. We observed variations in growth among replicate experimental cyanobacterial cultures upon flaming of the culture tube opening during sample transfer and collection. Investigation revealed the pH of culture media had decreased from the initial pH established during media preparation. Flaming of sterile culture media alone confirmed a significant decrease, by as much as 1.7 pH units, and correlated with increased flaming events over time. We hypothesized that the causative factor was the introduction of carbon dioxide (CO₂) into the media. To test this hypothesis, qualitative and quantitative analyses were used to determine the primary driver of pH decline. We further assessed the direct effects of flaming and subsequent pH changes onMicrocystis aeruginosacultures, showing flame‐driven pH changes and/or the introduction of CO₂influenced experimental results. Our observations provide a cautionary tale of how classic and well‐accepted approaches may have unintended consequences, suggesting new approaches may be necessary in research areas assessing pH or carbon‐related effects on microbial communities. 

Summary The over‐enrichment of nitrogen (N) in the environment has contributed to severe and recurring harmful cyanobacterial blooms, especially by the non‐N₂‐fixingMicrocystisspp. N chemical speciation influences cyanobacterial growth, persistence and the production of the hepatotoxin microcystin, but the physiological mechanisms to explain these observations remain unresolved. Stable‐labelled isotopes and metabolomics were employed to address the influence of nitrate, ammonium, and urea on cellular physiology and production of microcystins inMicrocystis aeruginosaNIES‐843. Global metabolic changes were driven by both N speciation and diel cycling. Tracing¹⁵N‐labelled nitrate, ammonium, and urea through the metabolome revealed N uptake, regardless of species, was linked to C assimilation. The production of amino acids, like arginine, and other N‐rich compounds corresponded with greater turnover of microcystins in cells grown on urea compared to nitrate and ammonium. However,¹⁵N was incorporated into microcystins from all N sources. The differences in N flux were attributed to the energetic efficiency of growth on each N source. While N in general plays an important role in sustaining biomass, these data show that N‐speciation induces physiological changes that culminate in differences in global metabolism, cellular microcystin quotas and congener composition.