Development of new microbial platforms tolerant to lignocellulosic biomass hydrolysates

Abstract

The conversion of lignocellulosic biomass into value-added bioproducts and energy represents a strategic alternative to mitigate dependence on fossil fuels. However, high production costs and methodological constraints in lignocellulose bioprocessing — specifically regarding yield and scalability — remain significant hurdles. A major challenge lies in efficiently overcoming lignocellulosic recalcitrance without compromising subsequent enzymatic hydrolysis and microbial fermentation. Alternative pretreatment methods, such as those employing amines, aim for the partial removal of lignin, solubilization of hemicellulose, and minimal formation of degradation products (e.g., furaldehydes). Nevertheless, a bottleneck persists regarding the recycling, reuse, and inherent toxicity of these solvents, which can impair bioprocess efficiency. Consequently, despite advancements in pretreatment, the development of microbial platforms tolerant to lignocellulosic hydrolysates remains essential. Given the versatility of pretreatments and the heterogeneity of biomass types, the stress factors encountered during fermentation are highly complex, often limiting the efficacy of rational engineering approaches. Therefore, this doctoral thesis presents both manual and automated Adaptive Laboratory Evolution (ALE) platforms as efficient tools for developing stable microbial strains applicable to lignocellulosic hydrolysates. The first chapter details the development of new Pseudomonas sp. BJa5 and P. putida KT2440 strains tolerant to furfural and 5-hydroxymethylfurfural via manual ALE, including the analysis of key mutations identified in evolved isolates and the detoxification potential of the evolved Pseudomonas sp. BJa5 strains. The second chapter addresses the development of novel P. putida KT2440 and Rhodosporidium toruloides IFO0880 strains tolerant to butylamine using automated ALE platforms. This section includes the screening of best-performing evolved isolates in an agricultural biomass hydrolysate and the identification of convergent and fixed mutations throughout the evolutionary trajectory. The concepts, results, and discussions derived from this thesis contribute to the generation of robust microorganisms for industrial application, the identification of key mutations for reverse engineering to elucidate tolerance mechanisms, and the advancement of novel ALE methodologies and approaches.