Four ways of implementing robustness quantification in strain characterisation.

ABSTRACT

BACKGROUND: In industrial bioprocesses, microorganisms are generally selected based on performance, whereas robustness, i.e., the ability of a system to maintain a stable performance, has been overlooked due to the challenges in its quantification and implementation into routine experimental procedures. This work presents four ways of implementing robustness quantification during strain characterisation. One Saccharomyces cerevisiae laboratory strain (CEN.PK113-7D) and two industrial strains (Ethanol Red and PE2) grown in seven different lignocellulosic hydrolysates were assessed for growth-related functions (specific growth rate, product yields, etc.) and eight intracellular parameters (using fluorescent biosensors). RESULTS: Using flasks and high-throughput experimental setups, robustness was quantified in relation to (i) stability of growth functions in response to the seven hydrolysates; (ii) stability of growth functions across different strains to establish the impact of perturbations on yeast metabolism; (iii) stability of intracellular parameters over time; (iv) stability of intracellular parameters within a cell population to indirectly quantify population heterogeneity. Ethanol Red was the best-performing strain under all tested conditions, achieving the highest growth function robustness. PE2 displayed the highest population heterogeneity. Moreover, the intracellular environment varied in response to non-woody or woody lignocellulosic hydrolysates, manifesting increased oxidative stress and unfolded protein response, respectively. CONCLUSIONS: Robustness quantification is a powerful tool for strain characterisation as it offers novel information on physiological and biochemical parameters. Owing to the flexibility of the robustness quantification method, its implementation was successfully validated at single-cell as well as high-throughput levels, showcasing its versatility and potential for several applications.