Questioning Assumptions & (Inoculum) Potential
The Challenge
If you work in private R&D - outside of patent disclosures - very often the reality is that the bulk of your work is not visible outside of the commercial results. A few years ago I had an opportunity to be a member of a team (lead by the brilliant mycelium engineer Peter Mueller and involving most of Ecovative's R&D team at the time) that plowed new ground in solid-state fermentation systems, meaningfully challenging previous state-of-the-art assumptions, and had the privilege to share it (to at least a reasonable level of detail) in a wonderful review paper The Amazing Potential of Fungi: 50 Ways We Can Exploit Fungi Industrially (see section 41).
This work summarized a mycelium composite cultivation methodology that combined unique cultivation and learning strategies with an actively aerated solid-state bioreactor system. The system addressed the inherent limitations of passively aerated tray-based fermentation by incorporating programmable control over airflow, temperature, and humidity through a modular air pretreatment and distribution infrastructure. The bioreactor enabled uniform colonization of lignocellulosic substrates in 0.7 m³ blocks while significantly reducing the need for aseptic processing while supporting consistent material formation across depth gradients. Engineering features included pressure-rated humidification chambers, custom-designed nozzle arrays for homogeneous gas distribution, and vessel geometries optimized to mitigate airflow bypass events (e.g., “burping”) under increasing back pressure during colonization. Development of the system integrated quantitative image-based analysis of epoxy-embedded thin sections to characterize inter-particle hyphal morphology and correlate structural features with environmental parameters and mechanical outcomes. Additionally, a bi-directional scaling strategy was employed that parallelized a novel scaled-down bioreactor system with the pilot scale system (see my previous article on poly-directional scaling).
Questioning Assumptions from the Previous State-of-the-Art
The goal was a deep bed (>60 cm) static fermentation targeting high inter-particle colonization density and uniform mechanical characteristics along the depth of the bed; a single monolithic block of myceliated material that could meet relevant mechanical performance throughout its volume.
If you’ve ever grown a passively aerated tray of myceliated substrate deeper than about 16cm you can understand how ambitious this goal is. Actively aerated deep bed bioreactors over 60 cm pose significant challenges for producing densely myceliated blocks without mechanical agitation. As mycelium grows and inter-particle porosity decreases, back pressure rises, leading to airflow channeling, oxygen gradients, and uneven colonization. Metabolic heat and CO₂ further exacerbate vertical temperature and gas differentials, while pressurized air can lift sections of the substrate (“burping”), disrupting uniform growth.
There were a tremendous number of reasons to expect that pursuing a deep bed reactor would be untenable, with the known challenges alone enough to warrant caution: airflow channeling, oxygen and temperature gradients, metabolic heat accumulation, physical deformation of the bed, uneven colonization, and the inevitable escalation of complexity with scale. But there was enough unknown opportunity, enough unresolved space relative to the specific material qualities we were selecting for, that it justified trying. Sometimes the greatest argument for experimentation isn’t a calculated confidence in success, but rather an appreciation for how incomplete the prevailing assumptions might be when viewed through the lens of different targets.
Rethinking Mycelium Propagation Conventions
The notion of building monolithic myceliation systems for consumer products that required strict aseptic control was not quite feasible. This, like most mycelium technology scenarios, required creative reconsideration of what the mycelium propagation pipeline might be or could tolerate. This then drives to appreciating the concept of inoculum potential. Admittedly, the definition of this concept can be somewhat nebulous and may vary depending on the reference, but I will try do define it in a functionally relevant way that captures the spirit of the concept:
Inoculum potential refers to the capacity of an introduced fungal propagule to successfully colonize a given substrate or environment, taking into account its biological vigor and the ecological context in which it is deployed. This concept integrates quantity and viability of the inoculum, geometric distribution of the inoculum, compatibility with the substrate, timing and staging of resource availability, and the competitive landscape. Inoculum potential can be considered an ecological problem directed to managing the evolution of priority effect, particularly under non-aseptic conditions. While often treated as a technical or process parameter, it fundamentally reflects multi-dimensional management of ecological dynamics.
As an artist and former teacher I appreciate the flexibility of positive and negative space in visual design; when perception flips the negative space to positive such that, suddenly, you may realize the complexity of the information around the object you initially considered the focal point. Similarly, when it came to rethinking mycelium propagation as a function of re-evaluating the concept of inoculum potential, what I realized was that it was not about management of the spawn or spawning (amount and type of inoculum), but rather about creatively forming the temporal ecological context around the spawning. The details of these strategic tools are largely wrapped up in patent disclosures (see references).
In this case, by operating in concert as a multi-disciplinary team between the cultivation strategy and the physical bioreactor system, we were able to negotiate creative reimagining of what an ecologically managed propagation pipeline might look like in order to minimize expensive physical controls. Ultimately this holistic approach resulted in not only successfully navigating the inherent physical problems of deep bed bioreactors, but was able to achieve operation with minimally processed feedstocks in an open top chamber under operationally realistic conditions for mass produced mycelium composite products.
Cutting to the Chase
If you have competing development targets - in this case maximizing the mechanical strength of mycelium binding between substrate particles vs. allowing for air passage for respiration and heat dissipation - you are best served cutting to the chase and directly observing the tissue binding the particles together (see my previous note on the dangers of indirect biomass detection).
In this project, that meant leveraging quantitative imaging, not as an afterthought but as a central design tool. As detailed in the review, we used epoxy-embedded thin sections coupled with high-resolution image analysis to directly characterize the hyphal network bridging substrate particles. This approach provided far more than a basic measure of colonization, it captured both the quantity and the microstructural organization of inter-particle mycelium, offering a window into the physical architecture that ultimately governs composite integrity.
Critically, this wasn’t just observational. We integrated high-throughput image analysis and predictive modeling to actively link hyphal density and network organization to mechanical performance outcomes. By quantifying both structural features and mechanical properties across environmental conditions, we could model how variations in the complexity of the hyphal network translated into changes in bulk material behavior.
This approach of blending direct observation with predictive modeling allowed us to bypass assumptions and speculation about how process decisions might influence the final product. Instead, we operated with an evolving, data-driven understanding of how fungal network complexity, spatial environmental variation, and mechanical performance were interwoven. In practical terms, it gave us the means to design not just a product, but a propagation strategy and bioreactor system tuned to the mechanically relevant mycelium structure.
The Value of Bi-Directional Scaling
Previously I described bi-directional scaling as the practice of developing both larger and smaller versions of a system in parallel; treating scale as a recursive learning space, not a linear sequence. It’s about creating feedback between formats, so insights at one scale accelerate progress at another.
This project provided a lesson in putting that philosophy to work.
While developing the pilot scale actively aerated bioreactor we faced classic deep-bed challenges: airflow dynamics, metabolic gradients, mechanical variability. Conventional bench systems did not easily simulate these conditions, and direct use of the pilot scale system for complex multi-dimensional characterization and optimization wasn’t a realistic or efficient option.
Instead, we built a tailored bench-scale analogue: an array of >50 individual units at <100mL operation volume per unit, each designed to simulate the gas exchange and temperature dynamics according to substrate depth conditions of the full reactor. This wasn’t a generic lab model, it preserved critical scale-dependent behavior in a controlled and high-throughput format.
We first validated this system using mechanical and hyphal network models developed from the full-scale reactor. Once validated, we rapidly applied structured process optimization (response surface design) at the bench scale to refine operational parameters within the large system. Further, it allowed us to understand and order critical time-dependent sensitivities driving ultimate mechanical performance relative to spatial distribution through the depth of the bed.
The result was true recursive learning: mechanical and biological insights from the pilot scale informed bench-scale design, and rapid bench-scale optimization accelerated full-scale improvement. It turned scale from a hurdle into a design tool.
A Holistic Lesson in Mycelium R&D
In the end this wasn’t just a story about bioreactors or inoculum, but was a reminder that real progress in mycelium R&D comes from multidisciplinary teaming, questioning assumptions, embracing biological complexity, and designing systems that learn with you. Whether it’s propagation pipelines, scale-dependent behavior, or hyphal organization, the opportunity space keeps expanding as long as we’re willing to challenge the edges of what we think we already know.
References
Hyde, K.D., Xu, J., Rapior, S. et al. The amazing potential of fungi: 50 ways we can exploit fungi industrially. Fungal Diversity 97, 1–136 (2019). https://doi.org/10.1007/s13225-019-00430-9
Winiski, J., Van Hook, S., Lucht, M., & McIntyre, G. (2018). Process for solid-state cultivation of mycelium on a lignocellulose substrate. U.S. Patent No. 9,914,906. United States Patent and Trademark Office.
Mueller, P.J., Winiski, J.M., & O'Brien, M.A. (2022). Process and apparatus for producing mycelium biomaterial. U.S. Patent No. 11,343,979. United States Patent and Trademark Office.