Extracellular particles of polymeric material formed in n-hexadecane fermentation by Pseudomonas aeruginosa

In n-hexadecane fermentation by Pseudomonas aeruginosa, the production of rhamnolipids (biosurfactants) caused by nitrogen source limitation was observed during the stationary phase. The rhamnolipids caused severe foaming in the process, particularly at higher pH (ca. > 6.8). To reduce the foaming, several runs were made at a lower pH, i.e. 6.5 +/- 0.1. Some, however, behaved anomalously different. The rhamnolipid synthesis was very low. Instead, a large quantity of waxy particles with sizes up to 3-5 mm were formed. The waxy material was practically insoluble in all of the organic solvents tested, suggesting the cross-linked polymeric structure. A white, brittle solid was obtained after the material was thoroughly washed (with 0.05 M sodium bicarbonate, distilled water, and hexane) and vacuum-dried. Characterization of the washed material was made by Fourier-transformed infrared (FTIR) spectroscopy, thin-layer chromatography (TLC), and wet chemical analyses. It was found to contain less than 2.5% (w/w) proteins and less than 9% carbohydrates, of which only one sugar component was observed and tentatively identified as glucose by the TLC analysis. The material was, therefore, predominantly hydrocarbon-based, with oxidized functional groups such as esters, alcohols, and carboxylic acids being identified by FTIR. The lipase-catalyzed esterification of fatty acids and alcohols, within the oil droplets or at the oil/water interface of low local water activity, is postulated to play an important role in the waxy particle formation.