N-biofertilization of oil-producing microalgae with an engineered diazothrophic bacterium towards sustainable production of biofuels

ORTIZ MÁRQUEZ JC; AMBROSIO R; CURATTI L

Monterrey

Congreso; 19TH INTERNATIONAL CONGRESS ON NITROGEN FIXATION; 2015

University of California at Davies

N-biofertilization of oil-producing microalgae with an engineered diazothrophic bacterium towards sustainable production of biofuelsJuan C. F. Ortiz Márquez, Ambrosio R & Leonardo CurattiInstituto de Investigaciones en Biodiversidad y Biotecnología (INBIOTEC-CONICET), Mar del Plata, Argentina. Fundación para Investigaciones Biológicas AplicadasConflicting forces towards food and energy security and environment conservation pose a serious concern for the years to come. 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 N2 fixation (BNF) to partially substitute for synthetic N-fertilizers in algae culture has not been explored in detail. We have investigated different alternatives to increase the capacity of ammonia excretion of the free-living diazotrophic bacterium Azotobacter vinelandii by metabolic engineering. To increase BNF we isolated ∆nifL strains that were unable to sense intracellular ammonium sufficiency for nif-genes expression switch-off and produced ammonium in excess that is released into the medium. To modify ammonium assimilation, we isolated mutant strains bearing point mutations at the active site of glutamine synthetase gene glnA. The glnA-D49S mutation produced strains with slow diazotrophic growth that resulted more efficient ammonium producers under carbon/energy limiting conditions than ∆nifL strains. Double mutant strains (∆nifL, glnA D49S), showed a dramatic increase in the initial rate of ammonium release into the medium but failed to sustain the production because of a severe impairment of nif genes expression and diazotrophic growth. A new set of conditional mutant strains containing an exogenously induced copy of glnA allowed us to fine tuning conditions that optimize ammonium excretion properties by an improved balance between growth an ammonium release.Inoculation of ammonium excreting A. vinelandii into microalgae cultures in the absence of sources of C and N other than air, allowed production of microalgae biomass containing nearly 40 % oil, suggesting that BNF can make a positive impact on N-fertilization of microalgae for the production of biofuels and other commodities.