Controlling changes in cell surface hydrophobicity reduces mass transport limitations in Rhodococcus biotransformations

Burning sulfur-containing petroleum and coal contributes to environmental degradation in various ways. Although removal of inorganic sulfur from these fuels may be accomplished by physical, chemical, or biological means, organically bound sulfur is difficult to remove. One possible strategy for reducing the organic sulfur content is to expose these substrates to microorganisms or enzymes that can specifically break carbon-sulfur bonds, thereby releasing the sulfur in a water-soluble, inorganic form. A particularly promising bacterial genus for carrying out this chemistry is Rhodococcus.; Prior to this work, the rate limiting step for Rhodococcus based biodesulfurization has been thought to be saturation of the enzyme mediated reactions in the '4s' metabolic pathway. This is valid if, and only if, the Rhodococcus cells are distributed evenly at the oil:water interface where the biodesulfurization reactions occur. The oil:water interface is the only local environment in the system that allows the cells simultaneous access to both the oil-phase hydrophobic substrate dibenzothiophene (DBT) and the water-phase hydrophilic nutrients (glucose) essential for bioconversion. To test the validity of this assumption, we designed a cryo-confocal microscopy technique to visualize the location of cells in an oil:water emulsion.; Microscopy results revealed that cell aggregation during the biocatalyst [Rhodococcus] growth phase created a critical problem for oil:water hydrophobic/hydrophilic biotransformation systems. These aggregates preferentially dispersed in both the organic and aqueous bulk phases rather than at the oil-water interface, suggesting the invalidity of the presumption that enzyme kinetics governed bioconversion rates in these systems. Rather, this dispersion phenomenon indicated that the biotransformation rate is at least partially limited by the mass transport of the substrates to the biocatalyst. Resolution of this cell dispersion/mass transport issue was initially achieved by manipulating the fermentation media composition. This approach produced cells that efficiently disperse to the oil:water interface in a monolayer, thus increasing the effective biotransformation rate of the system by 2.7x. However, this approach significantly limited production of the cells. An alternative solution to the problem was to genetically alter the organism to control surface properties that cause aggregation, regardless of the fermentation media composition.