Development of a Cultivation System for Producing Pure Mycelium Materials from Distillers' Spent Grains

Stillage is a byproduct of distilleries which is rich in organic matter, minerals, and acidic components. It is commonly used as animal feed and has high potential for use as an alternative substrate for microorganisms. Filamentous fungi are saprophytes that can utilize stillage solids to grow as threadlike mycelium. The structure and composition of the filamentous mycelium has shown promise to produce pure mycelium materials (PMM), which might have many potential applications, for instance as a leather-like material or alternative protein food ingredient. Basidiomycota fungi (including true mushrooms) species are presently used in industry to produce PMM due to the ease of suppressing sporulation and encouraging vegetative growth. The production of PMM is a relatively new area and sufficient quantitative data is lacking on the effective cultivation and processing steps required to optimize the materials for different potential applications. In this study, a series of iterative prototypes were designed using Pleurotus ostreatus to identify environmental parameters that favored aerial mycelium growth while suppressing fruiting body formation. Prototypes varied in their methods of moisture retention and air exposure and enabled homogenous PMM growth using solid-state fermentation (SSF). Production of PMM by Mucoromycota fungus Rhizopus microsporus var. oligosporus was also designed and tested. The influence of important operational parameters on the aerial mycelium growth characteristics was studied including 1) substrate packing density, 2) external support geometry, 3) substrate carbon-to-nitrogen (C:N) ratio, and 4) aerial delivery of additives. The results showed that stillage was a favorable substrate to produce PMM and that the studied operational parameters allowed for effective control of the mycelium fiber length, density, and moisture content. R. oligosporus displayed rapid growth, enhanced 3 to 4 times compared to Basidiomycota fungus Pleurotus ostreatus (oyster mushroom). Increasing substrate packing density and the length of external supports was found to encourage development of longer aerial mycelium fibers while aerial delivery of additives was found to have limited effects on fiber length but significantly influenced mycelium density and moisture content. It was also found that the use of unprocessed stillage solids was effective at delaying the sporulation of this Mucoromycota fungus and promoting development of aerial mycelium, which was hypothesized to be related to its natively low C:N ratio. Together, these results indicate promise for the efficient production of tunable PMM from inexpensive organic substrates.