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Optimization of cultural factors influencing the production of extracellular vesicles and proteinase by Pseudomonas fragi ATCC 4973 Myhara, Robert Michael


Pseudomonas fragi ATCC 4973 was grown in trypticase soy broth (TSB), on a trypticase soy broth + 1.5% agar (TSA) surface, and in a defined citrate broth. The citrate broth contained glutamine as the sole nitrogen source, Pseudomonas fragi grown in TSB started proteinase production at 24 h, during the late logarithmic early stationary growth phase. Pseudomonas fragi grown on TSA surfaces initiated proteinase production at 4 h, 20 hours earlier than in liquid medium. Electron micrographs of P. fragi grown on TSA revealed extracellular vesicles ca. 20 nm in diameter "blebbing" off the surface of the cells. These vesicles were absent from the surface of P. fragi cells grown in TSB, although vesicles could be isolated from the culture supernatant. Isolated extracellular vesicles were ca. 20 nm in diameter and contained a proteinase similar to that found in the supernatant. Electrophoretic analysis showed the vesicles and outer cell membrane of P. fragi to share similarities in their composition. Use of the centroid search technique of Aishima and Nakai, showed the optimum cultural conditions for proteinase production by P. fragi, in defined citrate broth to be: incubation temperature, 12.5 C; incubation time, 38 h; initial pH, 6.8; organic nitrogen concentration, 314 mmole nitrogen/L (glutamine); a gas mixture containing 16.4% oxygen flowing over the medium (7.42 ppm dissolved oxygen). Oxygen was the major factor influencing proteinase production by P. fragi. A comparison of optimization techniques suitable for microbiological experiments showed that the centroid search technique of Aishima and Nakai, the modified super simplex of Nakai and Kaneko and the simplex technique of Morgan and Deming all required similar time and experiment numbers to obtain the optimum point.

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