Poly(3-Hydroxybutyrate) Production from Glycerol by Zobellella denitrificans MW1 via High-Cell-Density Fed-Batch Fermentation and Simplified Solvent Extraction

Abstract

Industrial production of biodegradable polyesters such as polyhydroxyalkanoates is hampered by high production costs, among which the costs for substrates and for downstream processing represent the main obstacles. Inexpensive fermentable raw materials such as crude glycerol, an abundant by-product of the biodiesel industry, have emerged to be promising carbon sources for industrial fermentations. In this study, Zobellella denitrificans MW1, a recently isolated bacterium, was used for the production of poly(3-hydroxybutyrate) (PHB) from glycerol as the sole carbon source. Pilot-scale fermentations (42-liter scale) were conducted to scale up the high PHB accumulation capability of this strain. By fed-batch cultivation, at first a relatively high cell density (29.9 ± 1.3 g/liter) was obtained during only a short fermentation period (24 h). However, the PHB content was relatively low (31.0% ± 4.2% [wt/wt]). Afterwards, much higher concentrations of PHB (up to 54.3 ± 7.9 g/liter) and higher cell densities (up to 81.2 ± 2.5 g/liter) were obtained by further fed-batch optimization in the presence of 20 g/liter NaCl, with optimized feeding of glycerol and ammonia to support both cell growth and polymer accumulation over a period of 50 h. A high specific growth rate (0.422/h) and a short doubling time (1.64 h) were attained. The maximum PHB content obtained was 66.9% ± 7.6% of cell dry weight, and the maximum polymer productivity and substrate yield coefficient were 1.09 ± 0.16 g/liter/h and 0.25 ± 0.04 g PHB/g glycerol, respectively. Furthermore, a simple organic solvent extraction process was employed for PHB recovery during downstream processing: self-flotation of cell debris after extraction of PHB with chloroform allowed a convenient separation of a clear PHB-solvent solution from the cells. Maximum PHB recovery (85.0% ± 0.10% [wt/wt]) was reached after 72 h of extraction with chloroform at 30°C, with a polymer purity of 98.3% ± 1.3%.