Improvements to Nitrogen Production in a Model Bacterium for Biofertilizer Application

A large percentage of the nitrogen needed by society to sustain agricultural processes is accomplished through the industrial Haber-Bosch process. This industrial process is accomplished at a significant cost in energy. In the natural ecosystem, organisms have also had to evolve to overcome the limits of reduced nitrogen compound availability in the environment, even though a large pool of nitrogen is readily available in our atmosphere. Nature has solved this limitation through a select group of organisms known as diazotrophs, which evolved the ability to transform atmospheric nitrogen gas into fixed ammonia through the action of nitrogenase. Other species have evolved to work together with diazotrophs by evolving intricate symbiotic relationships based primarily on the exchange of nitrogen for energy rich compounds. As we look to the future for solutions and alternatives to energy demanding processes such as Haber-Bosch, it is important to understand the methods employed by biological systems that produce the same commodity chemicals, and look at examples where these systems can be altered to enhance nitrogen production in an effort to generate effective biofertilizer strains.

Our laboratory has been focusing on methods to induce and then screen for improved nitrogen production. In relation to this work, we reconstructed a strain which is a prolific ammonium producer. In this specific strain, the genes controlling nitrogen fixation are deregulated, through a modification of the nifLA operon. This modification results in a strain that yields extracellular ammonium levels in the 15-35 mM range, depending on the temperature in which it is cultured. Under these conditions, metabolism has been extensively reorganized to yield such high levels of ammonium. Using whole-genome RNA-Sequencing analysis, we have probed how the cell has altered gene regulation at the transcriptional level to achieve these high levels of nitrogen production. These results will be utilized to inform further biosynthetic strategies to construct stable strains that might be suitable for the production of ammonium and other nitrogen compounds. Recent work is aimed at the development of additional tools to probe alternative regulation at the transcriptional level that would result in elevated nitrogen production. These results are revealing an array of additional genes that play an important role in nitrogen regulation.

Our efforts to produce high levels of nitrogen have resulted in the undesirable evolution of cheaters during extended culture. Cheaters are single cells that have evolved within a community to take advantage of the elevated product levels generated within the culture, and then quit contributing toward the production of the desired product. Based on this evolution, these cells proliferate rapidly to become a dominant percentage of the culture and lower production yields. Our current efforts to direct fixed-nitrogen species toward a terminal product would help to eliminate the role that cheaters are able to play by making it exceedingly difficult to evolve a means to cheat. Our findings related to these efforts will be discussed.