Glycerol is generated in large quantities as the major byproduct of biodiesel manufacturing. Developing new processes requiring large volumes of glycerol is essential for a sustainable and profitable biodiesel production. Rhamnolipids are high-value effective biosurfactants that can be produced by Pseudomonas aeruginosa from glycerol. These biosurfactants have been recognized for their immense potential in bioremediation and enhanced oil recovery. Industrial production of rhamnolipids is still a challenge especially under aerobic conditions because of the highly foaming nature of the culture broth and the complex regulatory mechanisms involved. Foaming in aerobic rhamnolipid fermentation appears extremely fast and is too stable to be handled by common foaming control methods. An alternative approach to avoid foaming problems is to use a denitrifying fermentation, taking advantage of the ability of P. aeruginosa to perform nitrate respiration. Isolates of glycerol-utilizing P. aeruginosa were obtained from soil samples of a biodiesel production plant. With an improved methylene blue/cetyl trimethylammonium bromide (CTAB) agar plate’s method, the highest rhamnolipid producers from glycerol were identified. P. aeruginosa E03-40 and P. aeruginosa PAO1 were selected as the production strains for batch denitrifying studies. Successful cell growth and rhamnolipid production were obtained with glycerol as substrate under denitrifying fermentation conditions. Higher cell concentrations, using a more controlled nitrate addition better matching the respiration needs of the culture, were attained from the NAD(P)H fluorescence signal monitoring. The foaming problems associated with commonly used aerobic rhamnolipid fermentations were also avoided using the denitrifying approach in which nitrate (instead of oxygen) was employed as electron acceptor for respiration.
In comparison to the free-cell fermentation, immobilized systems were also evaluated in this study under denitrifying conditions. Immobilized systems relying on oxygen as electron acceptor have been previously investigated but oxygen transfer limitation presents difficulties for continuous rhamnolipid production. The immobilized approach based on a hollow fiber set-up eliminated the transfer limitation problems and was found suitable for a continuous rhamnolipid production using glycerol as carbon source.
Another aspect that was addressed in this study was directed towards the development of a robust analysis for rhamnolipid quantification with the similar aim of large scale production of rhamnolipids. The currently used methods are tedious and laborious. A fast method for rhamnolipid analysis can significantly enhance the strain selection, metabolic engineering, and process development for improved production. A qualitative method was proposed earlier to differentiate rhamnolipid producing and non-producing strains using agar plates containing methylene blue and CTAB. By systematically investigating the complexation of rhamnolipids and methylene blue, with and without the presence of CTAB, a rapid and simple method for rhamnolipid analysis was developed. The method was successfully applied to the batch samples obtained from the denitrifying fermentations used during this project.