Kinetic phenotyping of Fusarium venenatum A3/5 across waste-relevant carbon substrates

This study presents a high throughput phenotyping framework to systematically evaluate Fusarium venenatum A3/5 kinetics across waste-relevant carbon substrates and a real heterogeneous beverage-industry feedstock. The workflow combines microlitre cultivation, condition-specific OD 600 -cell dry weight (CDW) calibration, time-series UHPLC quantification of extracellular substrates and byproducts, single/multi-phase modified Gompertz modelling and process-metric extraction to resolve complex growth regimes, carbon allocation strategies, substrate uptake efficiencies, and byproduct formation/reassimilation dynamics. Process performance was strongly influenced by carbon identity in defined single substrate conditions. Glucose supported the highest uptake rate of 0.538 g·L -1 ·h -1 , while glucose and sucrose supported rapid growth, with μ m values of 0.13 h -1 and 0.16 h -1 respectively, but low apparent biomass yields of 0.15 g·g -1 and substantial ethanol accumulation at 4.64 ±0.49 g·L -1 and 6.62 ±0.79 g·L -1 , respectively. In contrast, fructose supported slower uptake of 0.186 g·L -1 ·h -1 and growth progression with T 90 = 173.6 h, but achieved the highest biomass yield of 0.49 g·g -1 , whereas galactose demonstrated a similar growth and uptake phenotype to fructose but supported a much lower biomass yield of 0.18 g·g -1 . Lactose represented a kinetically distinct regime of slow linear growth with only 34% substrate consumed over 234.5 h. In dual-substrate systems, glucose was preferentially utilised, and secondary xylose or galactose utilisation produced low-yield diauxic growth demonstrating that mixed-substrate performance was non-additive and conditioned by prior glucose metabolism. Expired functional drink supported the best process performance of tested conditions, achieving 18.37 ± 0.64 g·L -1 total biomass and μ m = 0.33 h -1 compared with 12.49 ± 1.21 g·L -1 and μ m = 0.10 h -1 for a compositionally matched synthetic control. Overall, our developed framework simultaneously achieves high throughput and enhanced system observability for scale-down bioprocess kinetics phenotyping. Our findings reveal distinct, process-relevant growth and carbon allocation behaviours in Fusarium venenatum A3/5 cultures across alternative carbon substrates and identify beverage-sector waste streams as promising new feedstocks for carbon-efficient bioprocesses relevant to future mycoprotein production.