Medium chain length alkane solvent-cell transfer rates in two-liquid phase, Pseudomonas oleovorans cultures

The oxidation of medium chain length alkanes and alkenes (C6 to C12) by Pseudomonas oleovorans and related, biocatalytically active recombinant organisms, in two‐liquid phase cultures can be used for the biochemical production of several interesting fine chemicals. The volumetric productivities that can be attained in two‐liquid phase systems can be, in contrast to aqueous fermentations, limited by the transport of substrates from an apolar phase to the cells residing in the aqueous phase and by toxic effects of apolar solvents on microbial cells. We have assessed the impact of these possible limitations on attainable productivities in two‐liquid phase fermentations operated with mcl‐alkanes. Pseudomonas oleovorans grows well in two‐liquid phase media containing a bulk n‐octane phase as the sole carbon source. However, cells are also damaged, typically resulting in a cell lysis rate of about 0.08 to 0.10 h−1. These rates could be lowered by 50 to 70% to 0.03 h−1 and substrate yields increased from 0.55 to 0.85 g g−1 by diluting octane in non‐metabolizable long‐chain hydrocarbon solvents. Transfer rates of medium chain length (mcl) alkanes from the apolar phase to the cells were determined by following growth and the rate at which carbon‐containing metabolites accumulated in the different phases of the cultures. mcl‐Alkane solvent‐cell transfer rates of at least 79, 64, and 18 mmol per liter of aqueous medium per hour were determined for n‐heptane, n‐octane, and n‐decane, respectively. Rates of up to 30 mmol L−1 h−1 were observed under octane‐limiting conditions in systems where the apolar substrate was dissolved to concentrations below 3% (v/v) in hexadecene. Based on low power input experiments, we estimated the maximum obtainable mass transfer rates in large scale processes to be in the range of 13 mmol L−1 h−1 for decane and higher than 45 mmol L−1 h−1 for octane and heptane. The results indicate that high solvent to cell mass transfer rates and minimized cell damage will enable high production rates in two‐liquid phase bioprocesses, justifying ongoing efforts to attain high densities of catalytically, highly active cells in such systems. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 10–23 1998.