MULTISPECIES MICROBIAL CELL-FACTORIES COMPRISING OIL-RICH EUKARYOTIC MICROALGAE AND ENGINEERED AMMONIUM-EXCRETING BACTERIA

JUAN CÉSAR FEDERICO ORTIZ MARQUEZ; MAURO DO NASCIMENTO; RAFAEL AMBROSIO; LEONARDO CURATTI

Mar del Plata

Congreso; X Congreso Argentino de la Sociedad Argentina de Microbiología General; 2014

The current and projected increases in human population and general welfare poses a serious concern on the trilemma food, energy and environment, since these interests are often conflicting. A key aspect of this is the additional role imposed to agriculture towards biofuels production that entails dramatic changes in the use of arable land and an increase in the demand of agrochemicals, especially N-fertilizer. The use of non-conventional crops such as aquatic microalgae that may thrive in otherwise non-productive land represents one of the most attractive alternatives. However, large-scale cultivation of microalgae might increase the demand of N-fertilizer up to unsustainable levels. Conversely to current agricultural practices, the use of biological nitrogen fixation to partially substitute for synthetic N-fertilizers in eukaryotic algae culture is just starting to be explored. In this study we modified the capacity of ammonia excretion of the free-living diazotrophic bacterium Azotobacter vinelandii by metabolic engineering. For this purpose, regulatory pathways of nitrogen-fixation (ammonium production) and nitrogen assimilation (ammonium consumption) were modified. The first set of mutant strains bear an in-frame deletion of the general nif-genes expression anti-activator nifL. As expected, these strains were unable to sense intracellular ammonium sufficiency, and as a consequence were impaired in nif-genes expression switch-off, produced an excess of ammonium that is released to the medium. In A. vinelandii ammonium assimilation occurs only by the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway, making GS mutants lethal. Then, to modify ammonium assimilation, we first made use of the GS inhibitor L-methionine sulfoximine (MSX) to partially inhibit GlnA what allowed us to better understand the relationship between GS activity, cell growth and ammonium excretion. These results encourage us to isolate a set of mutant strains bearing point mutations at the active site of GS. This single mutation, especially D49S, produced strains with slow diazotrophic growth and ammonium excretion properties. Double mutant strains, show a dramatic increase in the initial rate of ammonium release into the medium but failed to sustain the production. We further observed that that was mostly due to a severe impairment of nif genes expression in the double mutant strains. D49S strains were more efficient ammonium producers under carbon/energy limiting conditions, as would be expected when growing at the expense of microalgae C-exudates in synthetic microbial consortia. This has been experimentally confirm in co-culturing experiments that resulted in the accumulation of oleaginous biomass using air as the sole source of C and N. Ammonium delivery by the different strains had implications for the cell size distribution of microalga. This study represents a step forward towards sustainable microalgae biotechnology.