Microbes have been isolated from soil and water samples after an enrichment culture process with kerosene and tested for biosurfactant production by measuring the surface and interfacial tensions and emulsification power of culture broths. The isolation and characterization of extracellular surface‐active agents from the culture broth of Pseudomonas aeruginosa 44T1 strain have been made. Preliminary structure identification with specific TLC reagents of the CHCl3MeOH (2:1) extracts showed two spots with a glycolipidic structure and Rf values of 0.70 and 0.45, respectively, using the solvent system CHCl3:MeOH:H2O (65:25:4). Separation of surface active agents by Chromatographic absorption column in Florisil or silica gel and further spectroscopic study (IR, 1H‐NMR, 13C‐NMR) and chemical degradation techniques (acid and alkaline hydrolysis) gave a structure of β[β(2‐0‐α‐L‐rhamnopyranosyloxy)decanoyl] decanoic acid and β[β (2‐0‐α‐L‐rhamnopyranosyl‐α‐L‐rhamnopyranosyloxy) decanoyloxy] decanoic acid (glycolipid A and glycolipid B, respectively) for the two glycolipids detected. A new method of mass spectrometry, fast atom bombardment mass spectrometry (FABMS), was used to probe molecular structure. The mass spectra obtained contain molecular weight recognition and sequence information signals, and they are in agreement with the proposed structures. Physicochemical evaluations of the two isolated glycolipids were made. The minimum surface tension obtained was 25 mN/m in water solutions. At pH 7 the CMC value was 11 ppm. In both cases, at pH 3 the CMC was displaced to lower and at pH 9 it was displaced to higher concentration values. Glycolipid B showed a lower value of interfacial tension (0.2 mN/m) than glycolipid A (1.0 mN/m). Glycolipid A showed a lower CMC value at alkaline pH, whereas glycolipid B had a lower CMC at acidic pH than at alkaline pH.
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