Factors affecting production of Scopulariopsis brevicaulis spores for use in self-healing concrete

Abstract

Concrete durability is compromised by its susceptibility to cracking, necessitating innovative solutions like self-healing concrete (SHC). Scopulariopsis brevicaulis is capable of biomineralization and its spores were found to hold high potential for use in SHC. Realizing this potential requires clean and effective production of S. brevicaulis spores, which remains unexplored. Here the factors and processes conducive to high productivity of S. brevicaulis spores were investigated. Suitability of cheap, renewable soy-based substrates: soy molasses (SM), soy hull (SH), and soy flour (SF) were first evaluated, and SH was found suitable. The comparison of SH-based solid-state fermentation (SSF) with submerged fermentation (SmF) revealed SSF's superiority, producing spores earlier and with a more than 4.5-fold higher rate. Further study of SSF parameters, including 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, highlighted conditions that significantly boost spore production. Optimal moisture content (60-67%) and elevated medium pH (10-11) and salt addition (15 g/L NaCl) were key to enhancing yield, the latter likely induced stress-driven sporulation. Using larger SH particles (> 850 µm) also proved beneficial, improving oxygen transfer. Electron microscopy confirmed the effective attachment and penetration of spore chains into SH particles. This work significantly improved the technical and economic feasibility of producing S. brevicaulis spores for industrial SHC development.

Keywords: Scopulariopsis brevicaulis; Solid-state fermentation; Soy hull; Spore.

Fig. 1

Effects of soy-based substrates and culture conditions: pH, NaCl addition, and light (L) vs. darkness (D), on spore production of S. brevicaulis grown on agar plates; systems arranged in the order of increasing average spore productivity from left to right, showing enhanced production in systems with NaCl addition (Group A according to Tukey’s test) and the higher pH 10 (Group B) but no significant effects from the other factors (Group C)

Fig. 2

Comparison of spore production profiles in SSF and SmF using SH as substrate

Fig. 3

Effect of spore inoculation levels (1X = 2.5 × 10⁵ spores/g SH) on SSF spore production yield

Fig. 4

Effect of SH-to-water ratio on SSF spore production yield

Fig. 5

Effect of sugar addition, in wt% of SH, on SSF spore production yield

Fig. 6

Effect of low levels of N-source ((NH₄)₂SO₄) supplementation, in wt% of SH, on SSF spore production yield

Fig. 7

Effect of high levels of (NH₄)₂SO₄ supplementation on SSF spore production yield

Fig. 8

Comparison of SSF spore production profiles for systems having SSF spores as inoculum, with and without SSF extract (in place of water for SSF medium preparation), against the control inoculated with PDA spores

Fig. 9

Effects of SH particle size on spore yields compared in two batches of SSF experiments

Fig. 10

SEM pictures of SSF grown fungal spores on SH particles after 14 days, showing a a SH particle covered with spores (100X), b spores formed in chains (500X), and c spore shape and rough, spiky surface (1760X)

Fig. 11

Effect of pH on SSF spore yield: a time profiles of spore yields obtained in SSF with initial medium pH of 5.5 and 10, showing comparable spore production in up to 8 days but higher production after 14 days in the SSF with high medium pH; b final spore yields after 14 days of SSF with initial medium pH of 5.5, 9, and 11 (three flasks at each pH), showing a clearly higher spore yield at pH 11