Mycelium as Manifold (And the Peril of Biomass Proxies)
Fungal physical plasticity is a source of developmental opportunity. The independence of specific growth rate and growth velocity offers a tunable design space, where plasticity itself sits at the foundation of effective work with filamentous fungi. It is the basis for extracting diverse functional outcomes from the same organism, and for navigating between exploration, consolidation, and stabilization in mycelial systems. But that same plasticity can just as easily be confused. What appears at first to be a clean and useful relationship can unravel under the weight of context, becoming messy, conditional, or even outwardly misleading. Inputs that seem tightly coupled at one scale or under one set of assumptions may decouple entirely under another. A cleanly resolved linear relationship in isolation can collapse when embedded in real substrates, real geometries, and real temporal dynamics. Just as fundamental as the design opportunities of physical plasticity are the ways it complicates how we perceive and measure it.
Few places make this tension more visible than the problem of fungal biomass quantification. It is a fascinating and humbling corner of fungal biotechnology because it sits precisely at the intersection of a seemingly fundamental question—How much mycelium do I have?—and the realities of fungal physical plasticity. In practice, the question is rarely singular. “How much mycelium” collapses multiple, often competing ideas into a single phrase: how much living tissue is present, how much metabolically active biomass exists, how much structural material has been deposited, or how much future growth potential is embodied in the current state. A diffuse, rapidly expanding network and a dense, slow-growing mat may carry similar dry mass while expressing radically different biological intent, and as such compositional distributions. A system rich in senescent interior tissue may report very differently from one dominated by actively extending margins, even when both are labeled, operationally, as “biomass.”
It is in this gap between what we think we’re asking, what we’re actually asking, and what the organism is actually expressing, that the mycelium engineer can easily find their ego bruised. Proxies that appear robust can behave unpredictably, practically and quietly degrading into over-simplified metrics. And conclusions drawn too confidently from simplified metrics can expose hidden assumptions about what biomass ought to mean, rather than what it does mean in a given biological and physical context.
The example of ergosterol sits squarely in this space. It offers a powerful, fungus-specific signal, and with it the promise of clarity. But it is also an active component of the full vocabulary of fungal plasticity, being context dependent and, as such, carries interpretive risk.
The Potential of Ergosterol
Ergosterol is the primary sterol component of fungal cell membranes, serving many of the same functions that cholesterol serves in animal cells. It helps maintain membrane structure and fluidity, ensuring appropriate rigidity, permeability, and activity of membrane-bound proteins. Because ergosterol is largely specific to fungi and absent in animal cells (and most other organisms), it provides a uniquely fungal signal in complex or mixed biological systems. The promise of ergosterol, then, is straightforward and deeply appealing: a practical, fungus-specific proxy for estimating living fungal biomass.
The appeal here is as psychological as technical. Ergosterol offers a single, chemically defined molecule that appears to cut cleanly through the morphological ambiguity of filamentous growth. Unlike visual estimates, colony diameter, or indirect metabolic readouts, ergosterol feels beautifully concrete: extract, quantify, plot. It’s definable in clean units, around which error can be defined and thresholded, conferring a sense of analytical authority. In systems where mycelium resists tidy measurement, ergosterol offers a number that feels stabilizing. This is particularly powerful in solid-state fermentation systems (SSF), which offers tremendous economic and process advantages, yet fungal biomass is dispersed through complex biological matrices that make direct quantification exceptionally difficult, and where the ability to estimate the fungal biomass fraction across operational and parameter space carries immense learning and practical value.
The root of ergosterol concentration variation in fungal biomass, however, lies in the very property that makes filamentous fungi so powerful to work with: physical plasticity. Fungi modulate membrane composition in response to environmental and metabolic conditions, and ergosterol synthesis is significantly influenced by factors such as growth phase, nutrient availability, oxygen levels, and the physical mode of growth (pellet morphology, liquid versus solid substrate, surface-associated versus aerial growth). As fungi adapt to changes in aeration, substrate composition, mechanical constraint, and developmental stage, they adjust the proportion of ergosterol within their cells to maintain membrane integrity and function.
What ergosterol faithfully reports, then, is not biomass in the abstract, but membrane state as it reflects a particular physiological and developmental context. This distinction is easy to overlook because the signal is real, reproducible, and fungus-specific. Yet even within a single genetic individual, ergosterol content can vary by more than an order of magnitude depending on surrounding conditions and the morphological or metabolic state of the mycelium. A rapidly expanding hyphal front, a dense transport-oriented interior, and a stressed or oxygen-limited region may all carry very different sterol profiles while belonging to the same organism.
The Difficulty with Physical Plasticity
Accurate use of ergosterol as a fungal biomass proxy depends on calibration under the specific growth conditions of interest. Ergosterol content per unit of biomass is not fixed, and any attempt to convert sterol concentration into biomass necessarily rests on a calibration curve that is contingent on context. A rigorous calibration would need to span different biomass levels and different physiological states (exponential growth, stationary phase, stressed conditions) to capture how ergosterol-to-biomass ratios shift across the organism's developmental trajectory. What ergosterol reports is always inseparable from how, where, and when the fungus was grown, and from the physiological state through which that growth unfolded. This dependency becomes especially pronounced in SSF that are inherently defined by eccentricity; moisture, oxygen, and nutrient availability are distributed unevenly through space and time. Actively extending margins, consolidated interior regions, stressed zones, and senescent tissue can coexist within the same body, each expressing different membrane compositions and sterol demands. Under these conditions, ergosterol concentration may vary substantially within a single sample as a necessary and faithful reflection of heterogeneous biological states when navigating eccentric environments. Without spatially resolved measurement or calibration that explicitly acknowledges this heterogeneity, biomass estimates can drift far from the notion of reality generalized in the calibration curve.
This difficulty is compounded by the fact that fungal biomass itself is not a singular quantity. The term quietly bundles together living tissue, metabolically active regions, structural mass, and future growth potential, all of which can decouple from one another as development proceeds. Ergosterol may track some of these facets more closely than others depending on growth phase and environmental history, but its relationship to any single definition of biomass remains conditional. Physical plasticity ensures that membrane composition, metabolic state, and structural investment do not scale uniformly. Temporal and environmental sensitivity further complicate interpretation. Ergosterol can persist after cell death in dark or anaerobic environments, while degrading rapidly under UV exposure. In systems where growth, differentiation, and decay occur simultaneously across space, these effects overlap in ways that resist clean correction. Taken together, this makes ergosterol a context-dependent biomass proxy whose reliability emerges only through careful calibration and sustained validation within each specific application.
Where this complexity becomes most consequential is in how measurements are used. In practice, ergosterol-derived biomass estimates often inform process decisions, guide comparisons across runs, or serve as evidence of progress or stability. When calibration is loose or when plasticity is underappreciated, shifts in ergosterol signal may be read as changes in biomass rather than as changes in physiological state, spatial distribution, or membrane composition. Over time, these interpretations can accumulate into conclusions that feel coherent yet rest on misaligned assumptions, with real consequences for time, capital, and technical direction.
Biomass as a Physiological Manifold
One way to understand where ergosterol becomes misleading is to step back from the molecule itself and consider the physicality it is being asked to represent. Ergosterol is a single biochemical feature, measured along a single axis. Biomass, as it is operationally invoked in fungal systems, is an emergent property of a high-dimensional physiological state space shaped by growth phase, metabolism, architecture, history, and environment. From this perspective, fungal biomass is better understood not as a scalar quantity, but as a manifold; an organizing metaphor for a structured physiological surface embedded in higher-dimensional state space. Different regions of this surface correspond to different expressions of fungal life (i.e. exploratory growth, consolidation, transport, stress response, senescence), each with its own internal relationships between membrane composition, metabolism, and structure. Ergosterol occupies one coordinate within this space, but its position relative to “biomass” shifts as the organism moves across that surface.
Physical plasticity is what gives this manifold its curvature. As physiological state changes, the local relationship between ergosterol concentration and any particular notion of biomass can remain coherent, yet that relationship need not hold globally. A calibration curve derived in one region of the manifold may fail quietly in another, because the organism has moved to a different neighborhood of its physiological state space. Biomass does not live on a single axis, and no single biochemical feature can track it uniformly across the full expressive range of a filamentous fungus.
Appreciating this effectively places ergosterol as a biotechnological tool. Interpreted as a coordinate on a physiological surface rather than as a scalar endpoint, the measurement regains meaning without overreach. In this sense, ergosterol becomes particularly instructive; its utility and limitations reflect the broader reality of working with filamentous fungi, where measurements remain informative only when they are situated within the organism’s physical, developmental, and historical logic. Taking ergosterol seriously as a biomass marker, therefore, requires a parallel seriousness about plasticity itself as a defining feature that shapes what any proxy can reasonably claim. Cultivating this restraint in interpretation prevents technical error and cultivates a way of seeing, equipping the mycelium engineer to navigate other measurement problems where there may, or may not be, a promise of clarity.
References
Charcosset, J.-Y., & Chauvet, E. (2001). Effect of culture conditions on ergosterol as an indicator of biomass in the aquatic hyphomycetes. Applied and Environmental Microbiology, 67(5), 2051–2055. https://doi.org/10.1128/AEM.67.5.2051-2055.
Nout, M. J. R., Bonants-van Laarhoven, T. M. G., de Jongh, P., & Rombouts, F. M. (1987). Ergosterol content of Rhizopus oligosporus NRRL 5905 grown in liquid and solid substrates. Applied Microbiology and Biotechnology, 26(5), 456–461. https://doi.org/10.1007/BF00253532
Eliaš D, Tóth Hervay N, Gbelská Y. Ergosterol Biosynthesis and Regulation Impact the Antifungal Resistance and Virulence of Candida spp. Stresses. 2024; 4(4):641-662. https://doi.org/10.3390/stresses4040041
Mille-Lindblom, C., von Wachenfeldt, E., & Tranvik, L. J. (2004). Ergosterol as a measure of living fungal biomass: Persistence in environmental samples after fungal death. Journal of Microbiological Methods, 59(2), 253–262. https://doi.org/10.1016/j.mimet.2004.07.010