Library of Azotobacter Vinelandii Identifies a Strain with Biofertilizer Potential

Nitrogen is the most expensive nutrient input for photosynthesis-dependent crops. Nitrogen fertilizers that are generated through current industrial methods are both economically and environmentally costly due to the large amounts of fossil fuels consumed during production and transportation. This research aims to develop a novel means to make nitrogen fertilizers by engineering bacteria to extracellularly produce excess nitrogen to support the growth of algae or other photosynthetic species. Azotobacter vinelandii naturally fixes atmospheric nitrogen and grows under aerobic conditions, making it an ideal bacterium for use as a renewable biofertilizer to support crop growth.

Transposon mutagenesis was used to generate random insertion mutants of A. vinelandii to identify genes associated with a nitrogen-producing phenotype. Strains of interest were selected by growing them on agar plates containing no nitrogen along with a nitrogen biosensor strain that is only able to grow in the presence of an exogenous nitrogen source. Using these high-throughput methods to screen the library of A. vinelandii, six mutants were identified that enable the biosensor strain to grow. Each of these mutants had a transposon insertion at various locations within a specific gene. Site-directed deletion of this gene in A. vinelandii also resulted in a strain that supports the growth of the biosensor strain, confirming that disruption of this specific gene is responsible for the nitrogen-producing phenotype. When grown in liquid culture or on agar media where no other nitrogen source is present, this gene deletion strain, AZBB109, also enables the growth of various algae strains that lack the ability to fix nitrogen on their own.

Efforts are underway to determine the nitrogen compound responsible for the nitrogen production phenotype in this gene disruption. Regardless, strain AZBB109 has potential as a biofertilizer to sustain algal cultures, and prospective applications in other important agricultural crops. This research shows that these screening methods can successfully identified an A. vinelandii strain capable of producing nitrogen at levels that allow various strains of algae to grow under conditions that they otherwise could not. Future experiments will focus on discovering additional gene manipulations in A. vinelandii that further enhance its level of extracellular nitrogen production.