Process development and environmental impact assessment of sustainable poly(3-hydroxybutyrate) separation and purification via Paraburkholderia sacchari cell lysis using crude enzymes

Abstract

Downstream separation and purification of poly(3-hydroxybutyrate) has been developed via combined crude enzyme bacterial cell lysis followed by solvent extraction and circular utilisation of bacterial lysate as nutrient supplement for PHB production. Enzymatic lysis of Paraburkholderia sacchari cells was initially evaluated using crude enzymes produced via solid state fermentation of Aspergillus oryzae. The optimum protease activity (156 U per g cell dry weight) resulted in 66.9 % lysis of residual cell weight. PHB purification with 1,3-dioxolane, dimethyl carbonate, anisole, ammonium laurate and chloroform were evaluated at different processing parameters (extraction duration, temperature) using wet and dry bacterial cells as well as enzymatically lysed wet bacterial cells. The highest recovery yield (94.5 %) and purity (98.1 %) were obtained with 1,3-dioxolane at 80°C and 6 h processing time using enzymatically disrupted bacterial cells. The bacterial cell lysate was used as nutrient supplement for circular PHB production in fed-batch P. sacchari bioreactor culture leading to the production of 78.7 g_PHB/L, 56.9 % (w/w) PHB content and 2.3 g/(Lꞏh) productivity. Low global warming potential (1.3 CO₂-eq/kg_PHB) and abiotic depletion fossil (14.4 MJ/kg_PHB) were estimated for PHB purification via enzymatic cell lysis and PHB extraction with 1,3-dioxolane demonstrating the development of a sustainable and circular process for PHB production.