Evidence for interfacial uptake in hexadecane degradation by Rhodococcus equi: the importance of cell flocculation

The kinetics of hexadecane degradation were studied in four strains of Rhodococcus equi that did not produce biosurfactants. The aim was to analyse the characteristics of alkane uptake and their relevance to a mechanism of interfacial uptake. The kinetic studies involved continuous determination of degradation by electrolytic respirometry in a diphasic system where the hydrophobic phase was hexadecane or a solution of hexadecane in a non-toxic, non-biodegradable solvent, either 2,2,4,4,6,8,8-heptamethylnonane or silicone oil. The technique allowed large variations in interfacial area between the aqueous and hydrophobic phases. For the four strains, the kinetics obtained were reproducible and showed, in almost all cases, an initial short phase of exponential growth, followed by a long phase of linear growth. Specific growth rates during exponential growth varied amongst the strains from 0.11 to 0.20 h(-1) and were independent of interfacial area, in accordance with the very strong adsorption of bacterial cells at the interface of solvent and aqueous media. The degradation rates during linear growth did not increase with interfacial area but increased with efficiency of stirring. These characteristics can be explained by the formation of cellular flocs due to the hydrophobicity of the strains. These flocs were observed during growth on hexadecane in almost all conditions. In one case, with a non-flocculating culture, a kinetic pattern with a longer exponential phase, closer to that expected for simple interfacial uptake, was observed. The results show that strictly interfacial uptake, limited by floc formation (occurring at moderate and higher cell densities, and controlled by stirring efficiency) is a common pattern for growth on long-chain alkanes of micro-organisms that do not produce biosurfactants.