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N2 gas is an effective fertilizer for bioethanol production by Zymomonas mobilis
Edited by Lonnie O. Ingram, University of Florida, Gainesville, FL, and approved December 31, 2014 (received for review October 28, 2014)

Significance
Recently there has been a surge in ethanol biofuel production from cellulosic feedstocks, offering more environmental benefits than conventional ethanol production from food crops. However, cellulosic feedstocks are low in nitrogen, requiring that millions of dollars be spent on nitrogen supplements to grow the ethanol-producing microbes. Zymomonas mobilis is a bacterium that has long been viewed as a potential rival to Baker’s yeast as an ethanol producer. Contrary to published remarks, we discovered that Z. mobilis can use the most abundant gas in the atmosphere, N2, as a nitrogen source and does so without detriment to the high ethanol yield. Using N2-utilizing Z. mobilis could offset much of the monetary and environmental costs of current industrial nitrogen supplements.
Abstract
A nascent cellulosic ethanol industry is struggling to become cost-competitive against corn ethanol and gasoline. Millions of dollars are spent on nitrogen supplements to make up for the low nitrogen content of the cellulosic feedstock. Here we show for the first time to our knowledge that the ethanol-producing bacterium, Zymomonas mobilis, can use N2 gas in lieu of traditional nitrogen supplements. Despite being an electron-intensive process, N2 fixation by Z. mobilis did not divert electrons away from ethanol production, as the ethanol yield was greater than 97% of the theoretical maximum. In a defined medium, Z. mobilis produced ethanol 50% faster per cell and generated half the unwanted biomass when supplied N2 instead of ammonium. In a cellulosic feedstock-derived medium, Z. mobilis achieved a similar cell density and a slightly higher ethanol yield when supplied N2 instead of the industrial nitrogen supplement, corn steep liquor. We estimate that N2-utilizing Z. mobilis could save a cellulosic ethanol production facility more than $1 million/y.
Footnotes
↵1T.A.K. and B.L. contributed equally to this work.
- ↵2To whom correspondence should be addressed. Email: jmckinla{at}indiana.edu.
Author contributions: T.A.K., B.L., and J.B.M. designed research; T.A.K., B.L., A.L.P., and J.B.M. performed research; T.A.K., B.L., A.L.P., and J.B.M. analyzed data; and T.A.K., B.L., and J.B.M. wrote the paper.
Conflict of interest statement: The authors have a patent pending related to this material.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1420663112/-/DCSupplemental.