Beyond carbon dioxide: decoupling headspace and buffering reveals a bicarbonate requirement for anaerobic gut fungi growth

Abstract Background Anaerobic gut fungi ( Neocallimastigomycota ) are major lignocellulose degraders in herbivore gastrointestinal tracts, but reproducible axenic cultivation remains difficult because conventional serum-bottle systems intrinsically couple CO 2 headspace, bicarbonate buffering, dissolved inorganic carbon availability, and pH control. This study aimed to decouple these variables and determine whether bicarbonate is required beyond its buffering function. Results Comparison of CO 2 - and N 2 -based headspaces in otherwise identical bicarbonate-buffered cultures showed consistently higher hydrogen production under N 2 . Screening of chemically defined non-carbonate buffers demonstrated that stable external pH alone was insufficient to sustain activity, as bicarbonate-free cultures lost metabolic performance after serial transfer. Reintroduction of sodium hydrogen carbonate under N 2 restored activity in a strain-dependent manner, revealing low bicarbonate thresholds that supported reproducible growth-associated metabolism in filamentous taxa. To test whether this effect reflected a general carboxylate requirement, twenty structurally diverse carboxylic acids were screened using Aestipascuomyces dupliciliberans ; none restored the bicarbonate-supported hydrogen production phenotype, although maleic acid increased soluble metabolite accumulation. A dual-buffer strategy combining Bis-Tris with minimal bicarbonate enabled independent pH control and identified maximal metabolic activity at pH 6.4. In addition, an OD 750 -based biomass assay calibrated with activated carbon was established and validated in an ammonium sulfate perturbation experiment, providing a practical framework for biomass estimation and future analyses of nitrogen effects. Conclusions Headspace composition and inorganic carbon availability are key determinants of anaerobic gut fungal cultivation. The results indicate that bicarbonate supports sustained anaerobic gut fungal activity beyond extracellular pH stabilization and cannot be functionally replaced by the tested organic carboxylates. Decoupling gas phase, buffering chemistry, and pH control provides a more interpretable and reproducible cultivation framework for physiological studies and biotechnological application of anaerobic gut fungi.