Development of a feeding strategy for high cell and PHA density fed-batch fermentation of Ralstonia eutropha H16 from organic acids and their salts

• A fed batch fermentation strategy was designed for Ralstonia eutropha using organic acids as feedstock.
• Organic acids were fed by pH-stat, and organic acid salts were fed using pO2-dependent feed control.
• A fed batch strategy using three successive carbon/nitrogen ratios (10, 90, to infinite) was employed to maximize PHA productivity.
• Biomass yield of 112.0 g/L, PHA yield of 93.5 g/L, and a PHA productivity of 2.13 g/L/h was achieved using the strategy outlined in this work.

The utilization of inexpensive carbon sources, including waste streams, for production of value added products has been explored for over two decades. Palm oil mill effluent (POME), a waste water stream from the palm oil industry, can be used to produce organic acids and their salts by anaerobic digestion. These organic acids are suitable as the main carbon source for growth and production of polyhydroxyalkanoates (PHAs) by organisms like Ralstonia eutropha. The presence of propionate/propionic acid results in the biosynthesis of the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)], which is desired for its favourable thermal and mechanical properties compared to the polyhydroxybutyrate (PHB) homopolymer. A specially-designed fed-batch strategy is necessary to decrease the toxic effects of organic acids in cultures of R. eutropha. A pH-stat fed-batch culture technique, using organic acids as the main carbon source, was adopted in combination with an additional pO2-dependent feed for delivering organic acid salts. The developed strategy is highly reproducible and shows a high productivity of >2 g PHA/L/h. After 44 h of fermentation, 112.4 ± 2.3 g/L cell dry weight (CDW) with 83.3 ± 1.1% P(HB-co-HV)/CDW containing 5.6 ± 0.4 mol% 3-hydroxyvalerate (3HV) were achieved. The described productivity, CDW and P(HB-co-HV)/CDW values are by far the highest reported results for R. eutropha cultivated on mixed organic acids as the main carbon source.