Development of screening procedure for fungi emulating concrete environment for application in self-healing concrete and spore production

Concrete is strong but susceptible to cracking. Self-healing concrete (SHC) can be a way to solve this without manual intervention. Microorganisms’ ability to biomineralize can be leveraged to find a way to develop SHC. However, the concrete environment is tough for microbes. Choosing the right microorganisms for application in concrete environments requires the development of an effective hierarchy mimicking concrete-relevant pH and temperature. A hierarchy is developed and 18 alkalotolerant strains were screened using it, and Scopulariopsis brevicaulis was identified as the best candidate for the intended application. The spore-damaging effects of high pH and temperature were also investigated separately and combinedly. Results indicated the critical value of locally lower pH near spores, at least slightly (from 12.9 to 12), and to limit temperature to below 53^oC for ensuring survivability of spores in concrete. Due to the high potential of S. brevicaulis utilization in the development of SHC, it requires effective production of S. brevicaulis spores. Suitability of soy-based substrates: soy molasses (SM), soy hull (SH), and soy flour (SF) were first assessed, and SH was identified as suitable. Spore production was next compared between solid-state fermentation (SSF) and submerged fermentation (SmF) and SSF produced spores earlier and with a more than 4.5-fold higher rate using SH. Spore production by SSF was studied further for different factors: initial spore inoculum, moisture, SH particle size, sugar supplementation, N-source supplementation, pH, salt addition, light (vs. dark) condition, and occasional mixing/shaking plus water addition. High initial medium pH of 10 and 11 or addition of 15 g/L NaCl significantly improved the spore yield and 60%-67% moisture was identified as optimal. Higher spore yields were also obtained by using larger ( >850 µm) SH particles. This work significantly improved the technical and economic feasibility of producing S. brevicaulis spores for industrial SHC development.