Bicarbonate, not just carbon dioxide, is the key to growing gut fungi

Anaerobic gut fungi are among the most powerful plant-digesting machines in the herbivore gut, yet growing them alone in the laboratory has always been frustratingly unreliable. A new study published in Fungal Biology and Biotechnology has untangled a long-standing experimental knot, showing that these fungi need bicarbonate dissolved in their medium for reasons that go far beyond simple pH buffering. By systematically separating the gas in the headspace, the buffer chemistry, the pH and the nutrient supply, the researchers found that removing bicarbonate collapses fungal metabolism even when the external pH is held perfectly steady, and that only tiny amounts of the compound are needed to bring growth roaring back.

These organisms are primary colonizers of lignocellulosic plant material in the rumen, penetrating tough cell walls with invasive rhizoidal growth and secreting a broad arsenal of carbohydrate-active enzymes, often organized into fungal cellulosome-like complexes. Their ecological importance has made them attractive targets for biotechnology, particularly lignocellulose valorization and biological hydrogen production, which flows from hydrogenosomes, mitochondria-derived organelles that couple pyruvate oxidation to hydrogen release via reduced ferredoxin-dependent hydrogenases.

The problem with conventional cultivation is that everything is coupled at once. The classical Hungate serum bottle technique relies on a carbon dioxide headspace combined with bicarbonate buffering, so atmospheric composition, carbonate equilibria, dissolved gas concentrations and medium pH all shift together during a batch run. As the fungi ferment, they produce hydrogen and carbon dioxide, raising headspace pressure, altering gas solubility and pushing fermentation equilibria around, while the carbon dioxide and bicarbonate equilibrium simultaneously governs buffering capacity. Earlier studies had shown that modified gas atmospheres change fermentation patterns in rumen fungi, but none had cleanly separated the headspace from the carbonate buffer, leaving the independent contribution of each variable unresolved.

The first experiment did exactly that. Across all six strains, hydrogen formation was consistently higher under nitrogen, and total metabolite output was higher under nitrogen in every strain except Caecomyces churrovis. The effect was most pronounced in Aestipascuomyces dupliciliberans, which delivered the highest hydrogen yield under the nitrogen atmosphere. Because the medium and pH were identical, the difference could be attributed specifically to the gas phase.

The authors propose that this is not merely a matter of hydrogen partial pressure. Elevated dissolved carbon dioxide could, through mass action, push the reversible reactions of malic enzyme and pyruvate:ferredoxin oxidoreductase backward, opposing the oxidative, reductant-generating direction that normally supplies NAD(P)H and reduced ferredoxin to the hydrogenases. Consistent with this, pyruvate:ferredoxin oxidoreductase synthesis was previously reported to be suppressed in Neocallimastix patriciarum grown under a carbon dioxide atmosphere. The authors also note that in axenic batch culture with no hydrogen-consuming partner such as a methanogen, hydrogen-dependent redox constraints may be particularly pronounced, so the observed atmospheric response reflects monoculture constraints rather than the full rumen situation.

The model strain Neocallimastix cameroonii showed limited metabolic activity in the first transfer cycle, likely buoyed by carbonate carried over in the inoculum, but after serial transfer into fresh medium no measurable growth, hydrogen production or sustained metabolite formation remained in any bicarbonate-free buffer. Stable external pH alone, in other words, could not keep the fungi alive.

Without bicarbonate, hydrogen production and total metabolite formation were negligible across all four filamentous strains tested. As bicarbonate was added, hydrogen formation returned in a concentration-dependent, threshold-like manner: N. cameroonii and Orpinomyces joyonii recovered reproducible activity at 0.25 grams per liter or more, A. dupliciliberans needed at least 0.5 grams per liter, and Pecoramyces ruminantium recovered with reduced overall output. Beyond the threshold, hydrogen production plateaued rather than rising proportionally, pointing to a minimum requirement rather than a simple dose-response stimulation. A sodium chloride control matched for sodium content failed to restore activity, ruling out the sodium ion as the active ingredient.

Was the requirement specific to bicarbonate, or would any carboxylate do? None restored the bicarbonate-supported hydrogen production phenotype; total metabolite formation stayed well below the bicarbonate control, and end products shifted toward cytosolic redox balancing, dominated by lactate, succinate and malate. Maleic acid was the one exception, producing elevated soluble metabolite accumulation, but without proportional hydrogen, suggesting a redistribution of carbon flux rather than restored hydrogenosomal metabolism, possibly through weak-acid stress or interactions with enzymes such as fumarase and pyruvate kinase. The authors stress these mechanisms remain speculative for anaerobic gut fungi.

Across initial pH values from 5.8 to 7.0, hydrogen production and total metabolite formation peaked around pH 6.4 in most of the four filamentous strains, although P. ruminantium responded only weakly across the range. That optimum sits neatly within the roughly 6.0 to 6.8 pH window reported for rumen fermentation in vivo, and because the buffer held pH independent of carbonate equilibria, the result reflects a physiological preference rather than a buffering artefact. The authors caution that the two buffers have overlapping pKa values and that medium phosphate contributes some buffering, so the components are not fully independent, and the optimum may shift under higher inorganic carbon supply.

Applied to a nitrogen-variation experiment with the bulbous strain C. churrovis grown on glucose, ammonium sulfate concentrations from 0.3 to 4 grams per litre produced no consistent trend in biomass, total metabolites or hydrogen production, indicating that nitrogen was not limiting within the tested window rather than that nitrogen is unimportant. Taken together, the study shows that headspace composition and inorganic carbon availability are the dominant determinants of anaerobic gut fungal cultivation performance, that bicarbonate plays a physiological role beyond pH stabilization that organic carboxylates cannot replace, and that decoupling gas phase, buffering chemistry and pH control finally gives researchers a reproducible framework for studying and exploiting these remarkable fungi.

Subject of Research: A cultivation study decoupling headspace gas, buffering and pH to show bicarbonate is required for anaerobic gut fungal growth beyond pH buffering.

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

Article References: Schulz, K. E., Pfau, A., Tarhan, T., van Almsick, C., Scholz, D., Sikirić, A. R., Neumann, A., & Ochsenreither, K. (2026). Beyond carbon dioxide: decoupling headspace and buffering reveals a bicarbonate requirement for anaerobic gut fungi growth. Fungal Biology and Biotechnology, 13(1), Article 18. https://doi.org/10.1186/s40694-026-00224-0

Image Credits: AI Generated

DOI: 10.1186/s40694-026-00224-0

Keywords: anaerobic gut fungi, Neocallimastigomycota, bicarbonate, hydrogen production, hydrogenosomes, pH buffering, nitrogen headspace, rumen fungi, cultivation methods, biomass quantification, carboxylic acids, lignocellulose degradation