Media optimization to improve growth and polyhydroxyalkanoate (PHA) production from lignocellulosic side streams in a halophilic archae-on Haloferax mediterranei

Demand for biobased and biodegradable plastics has increased significantly in recent years due to the environmental pollution associated with petroleum-based plastics. Among bioplastics, polyhydroxyalkanoates (PHAs) are the most promising, as they are completely renewable and biodegradable in all environments; however, their high production cost limits widespread commercial use. Utilizing inexpensive carbon feedstocks, such as lignocellulosic side streams, could significantly reduce production costs. The aim of this study was to evaluate the feasibility of using a lignocellulosic side stream as a carbon feedstock and to optimize media for growth and PHA production. Lignocellulosic hydrolysate is a cheap and abundant resource; however, it contains many inhibitory compounds that can hinder microbial growth. Therefore, the halophilic archaeon Haloferax mediterranei was selected for this study, as it is a well-established PHA producer known to tolerate a wide range of inhibitory compounds, and its performance was compared to the halotolerant bacterium Halomonas halophila. Strains were cultivated in Bioscreen assays and shake flask with varying carbon and nitrogen sources to determine optimal conditions for growth and PHA production. Growth was measured spectrophotometrically, and PHA content of the cell pellets was determined indirectly via acid hydrolysis and quantification of crotonic acid. In the Bioscreen assays, Haloferax mediterranei had good tolerance to the hydrolysate, showing the most growth at 10 % concentration (OD₆₀₀ = 0.90, µ = 0.1296 d⁻¹). Conversely, Halomonas halophila had lower growth at 10 % concentration (OD₆₀₀ = 0.55, µ = 0.0384 d⁻¹), with its highest growth achieved at 1 % concentration (OD₆₀₀ = 1.08, µ = 0.096 h⁻¹). However, accurate analysis of the hydrolysate media was hindered by background colour, high salt concentrations, and particle content, which is why research focus switched to the dynamics and kinetics of cell growth in defined single and mixed sugar media. Halomonas halophila showed superior performance in mixed-sugar media mimicking hydrolysate composition without inhibitors, achieving the highest growth and PHA content at 10 % concentration in media recommended by Obruca et al. (2014) (OD₆₀₀ = 8.10, µ = 0.7392 d⁻¹, PHA = 39.1 %). In comparison, Haloferax mediterranei cultivated at 10 % concentration in media recommended by Sato et al. (2021) reached lower values (OD₆₀₀ = 4.39, µ = 0.3216 d⁻¹, PHA = 27.1 %). Future studies should focus on identifying filtration techniques to prevent background colour and particles from obscuring measurements.