Unraveling and resolving inefficient glucolipid biosurfactants production through quantitative multiomics analyses of Starmerella bombicola strains.

Glucolipids (GLs) are glycolipid biosurfactants with promising properties. These GLs are composed of glucose attached to a hydroxy fatty acid through a ω and/or ω-1 glycosidic linkage. Up until today these interesting molecules could only be produced using an engineered Starmerella bombicola strain (∆ugtB1::URA3 G9) producing GLs instead of sophorolipids, albeit with a very low average productivity (0.01 g·L-1 ·h-1 ). In this study, we investigated the reason(s) for this via reverse-transcription quantitative polymerase chain reaction and Liquid chromatography-multireaction monitoring-mass spectrometry. We found that all glycolipid biosynthetic genes and enzymes were downregulated in the ∆ugtB1 G9 strain in comparison to the wild type. The underlying reason for this downregulation was further investigated by performing quantitative metabolome comparison of the ∆ugtB1 G9 strain with the wild type and two other engineered strains also tinkered in their glycolipid biosynthetic gene cluster. This analysis revealed a clear distortion of the entire metabolism of the ∆ugtB1 G9 strain compared to all the other strains. Because the parental strain of the former was a spontaneous ∆ura3 mutant potentially containing other "hidden" mutations, a new GL production strain was generated based on a rationally engineered ∆ura3 mutant (PT36). Indeed, a 50-fold GL productivity increase (0.51 g·L-1 ·h-1 ) was obtained with the new ∆ugtB1::URA3 PT36 strain compared with the G9-based strain (0.01 g·L-1 ·h-1 ) in a 10 L bioreactor experiment, yielding 118 g/L GLs instead of 8.39 g/L. Purification was investigated and basic properties of the purified GLs were determined. This study forms the base for further development and optimization of S. bombicola as a production platform strain for (new) biochemicals.

A conductive hydrogel based on alginate and carbon nanotubes for probing microbial electroactivity.

Some bacteria can act as catalysts to oxidize (or reduce) organic or inorganic matter with the potential of generating electrical current. Despite their high value for sustainable energy, organic compound production and bioremediation, a tool to probe the natural biodiversity and to select most efficient microbes is still lacking. Compartmentalized cell culture is an ideal strategy for achieving such a goal but the appropriate compartment allowing cell growth and electron exchange must be tailored. Here, we develop a conductive composite hydrogel made of a double network of alginate and carbon nanotubes. Homogeneous mixing of carbon nanotubes within the polyelectrolyte is obtained by a surfactant assisted dispersion followed by a desorption step for triggering electrical conductivity. Dripping the mixture in a gelling bath through simple extrusion or a double one allows the formation of either plain hydrogel beads or liquid core hydrogel capsules. The process is shown to be compatible with the bacterial culture (Geobacter sulfurreducens). Bacteria can indeed colonize the outer wall of plain beads or the inner wall of the conductive capsules' shell that function as an anode from which electrons produced by the cells are collected.

Multiscale electrochemistry of hydrogels embedding conductive nanotubes.

The local functionalities of biocompatible objects can be characterized under conditions similar to the operating ones, using scanning electrochemical microscopy (SECM). In the case of alginate beads entrapping carbon nanotubes (CNTs), SECM allows evidencing of the local conductivity, organization, and communication between the CNTs. It shows that the CNT network is active enough to allow long range charge evacuation, enabling the use of alginate/CNT beads as soft 3D electrodes. Direct connection or local interrogation by a microelectrode allows visualization of their communication as a network and eventually the study of them individually at the nanoscale.