A novel strategy for extraction of intracellular poly(3-hydroxybutyrate) from engineered Pseudomonas putida using deep eutectic solvents: Comparison with traditional biobased organic solvents

Polyhydroxyalkanoates (PHAs) represent a category of microbial polyesters that offer both biodegradability and biocompatibility, if produced in sufficient quantities, they could serve as an alternative to many conventional plastics in use today. However, these microbial polymers are intracellularly stored, necessitating a more complex downstream extraction and purification process. Downstream processes often constitute the most financially burdensome stage in biomolecule production. One significant drawback of many existing extraction processes is their reliance on harsh organic solvents, such as chloroform, and high temperatures. This study presents and compares two novel downstream processes for the extraction and purification of poly(3-hydroxybutyrate) (PHB), a type of short-chain-length PHA, utilizing bio-based green solvents and natural deep eutectic solvents (NADES), respectively. The soil bacterium Pseudomonas putida, engineered to produce PHB from sugars, was adopted as a model for testing these extraction procedures. Initially, biomass was disrupted using a hypotonic buffer containing lysozyme to enhance the extraction efficiency in the downstream process. After extensive screening, the bio-based solvent ethyl acetate was selected for PHB extraction from P. putida biomass, yielding ∼ 95 wt% of the homo-polymer with a purity of ∼ 97 wt%, results comparable to those achieved with the traditional benchmark solvent, chloroform. Furthermore, a hydrophobic natural deep eutectic solvent (hydrophobic NADES) was synthesized, comprising L-menthol and acetic acid in a 1:3 M ratio, and employed as the extraction solvent in combination with methanol as the anti-solvent. The optimized extraction process resulted in a homo-polymer yield of ∼ 66 wt% with a high purity of ∼ 85 wt%. These results are promising considering the benefits associated with the use of NADES, they are less toxic and much easier to handle than ethyl acetate and have the potential to be recycled. Therefore, it represents a promising avenue for a more sustainable PHB extraction process, devoid of harmful organic solvents.