Isolation and Characterization of Bacteria That Efficiently Eliminate Ammonium Nitrate.

Nitrous oxide (N₂O) has a 200-300-fold-stronger greenhouse effect than carbon dioxide. N₂O is produced in agricultural fields through a denitrification process by microorganisms. In this process, nitrate ion (NO₃^-) is reduced to nitrogen (N₂) through subsequent reductive reactions by denitrifying bacteria under an anaerobic condition. The reduction of N₂O to N₂, the final step of denitrification is, however, strongly suppressed by oxygen. Because agricultural fields are under aerobic conditions, the large amount of N₂O is produced from fertilized NO₃^-, and account for 52% of man-made N₂O. Regarding global climate change, it is important to decrease N₂O emission in agricultural fields. To decrease N₂O emission, we thought that it could be possible by managing microorganisms with an ability to convert NO₃^- into N₂ or into N-containing organic compounds under aerobic conditions in agricultural fields. We, therefore, attempted to isolate bacteria that were suitable for our purpose. Seventy-seven rhizosphere soil samples were collected from vegetable fields.  Enrichment culture was performed in a screening medium containing 0.1% NH₄NO₃ and 2% glucose. Finally, four strains (named N-I, N-II, N-III, and N-IV) were selected because of efficient NH₄NO₃ elimination under an aerobic condition, and identified as Enterobacter cloacae on the basis of morphological, physiological, biochemical, and genetical properties. To identify the products of NH₄NO₃, total nitrogen of couture (supernatant and strain) was analyzed. The four strains did not denitrify but synthesized other nitrogen compounds and accumulated them in the supernatants.  Although the supernatants contained few amino acids, the acid hydrolysates from the supernatants showed some amino acids by HPLC analysis, suggesting that the nitrogen compounds were similar to peptide. Subsequently, we purified peptide-like compounds and are working on structural analyses for the compounds.