Abstract
Hydrothermal liquefaction (HTL) is an emerging method for thermochemical conversion of wet organic waste and biomass into renewable biocrude. HTL also produces an aqueous phase (HTL-AP) side stream containing 2–4% light organic compounds that require treatment. Although anaerobic digestion (AD) of HTL-AP has shown promise, lengthy time periods were required for AD microbial communities to adapt to metabolic inhibitors in HTL-AP. An alternative for HTL-AP valorization was recently demonstrated using two engineered strains of Yarrowia lipolytica, E26 and Diploid TAL, for the overproduction of lipids and the polyketide triacetic acid lactone (TAL) respectively. These strains tolerated up to 10% HTL-AP (v/v) in defined media and up to 25% (v/v) HTL-AP in rich media. In this work, adaptive laboratory evolution (ALE) of these strains increased the bulk population tolerance for HTL-AP to up to 30% (v/v) in defined media and up to 35% (v/v) for individual isolates in rich media. The predominate organic acids within HTL-AP (acetic, butyric, and propionic) were rapidly consumed by the evolved Y. lipolytica strains. A TAL-producing isolate (strain 144-3) achieved a nearly 3-fold increase in TAL titer over the parent strain while simultaneously reducing the chemical oxygen demand (COD) of HTL-AP containing media. Fermentation with HTL-AP as the sole nutrient source demonstrated direct conversion of waste into TAL at 10% theoretical yield. Potential genetic mutations of evolved TAL production strains that could be imparting tolerance were explored. This work advances the potential of Y. lipolytica to biologically treat and simultaneously extract value from HTL wastewater.
Key points
• Adaptive evolution of two Y. lipolytica strains enhanced their tolerance to waste.
• Y. lipolytica reduces chemical oxygen demand in media containing waste.
• Y. lipolytica can produce triacetic acid lactone directly from wastewater.
Graphical Abstract
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
We thank the Genomic Sequencing and Analysis Facility (GSAF, Facility RRID: SCR_021713) at The University of Texas at Austin for providing the sequencing data. SMC thanks Dr. Daniel Deatherage (University of Texas at Austin) and Dr. Edward Marcotte (University of Texas at Austin) for their thoughtful consultation on the genomic variant analysis. The authors acknowledge the Texas Advanced Computing Center (TACC) at The University of Texas at Austin for providing computing resources that have contributed to the research results reported within this paper. URL: https://www.tacc.utexas.edu.
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The code used for the variant analysis is described in detail in the Methods section and the cited reference, https://wikis.utexas.edu/display/bioiteam/Genome+Variant+Analysis+Course+2020. MATLAB code identifying the location of the variant relative to genomic functional annotations is available from the corresponding author on reasonable request.
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LTC and HSA conceived and designed research. LTC, BCL, SAA, SMC and ER conducted experiments. SMC, LTC, JRC, and HSA analyzed data and wrote the manuscript. All authors read and approved the manuscript.
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Coleman, S.M., Cordova, L.T., Lad, B.C. et al. Evolving tolerance of Yarrowia lipolytica to hydrothermal liquefaction aqueous phase waste. Appl Microbiol Biotechnol 107, 2011–2025 (2023). https://doi.org/10.1007/s00253-023-12393-8
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DOI: https://doi.org/10.1007/s00253-023-12393-8