Inoculum optimization of Clostridium beijerinckii for reproducible growth

Spore-forming solventogenic Clostridium spp. are receiving renewed attention due to their butanol production abilities. However, there is an absence of literature describing the preparation of dense, vigorous and homogeneous seed cultures of Clostridium spp., guaranteeing reproducibility during fermentation. Therefore, we performed a series of growth experiments of Clostridium beijerinckii NCIMB 8052 and its offspring SA-1 to evaluate the influence of inoculum age (harvest time) on the subsequent population's maximum specific growth rate, as a signal of population homogeneity. The organisms were cultivated in Reinforced Clostridial Medium and supplemented sweet sorghum juice. The best inoculum ages coincided with the late-exponential growth phase: between 9 and 11 h in the conditions tested. Additionally, the harvest time was delayed up to 4 h by pre-adapting the seed culture with 0.75 g L(-1) butyric acid. These findings were validated by performing a series of bench-top batch fermentations showcasing reproducibility in growth kinetics with 95% confidence limits. Overall, these experiments allowed us to understand the transient nature of seed cultures of C. beijerinckii NCIMB 8052 and SA-1, while enabling reproducibility and consistent culture performance.