Metabolomics Analysis of Ammonia Secretion during the Fermentation of Klebsiella variicola GN02 with Highly Efficient Endophytic Nitrogen-Fixing Bacteria

The ammonia-secreting performance is one of the key factors for determining nitrogen-fixing capacity of the cell. Klebsiella variicola GN02 is an endophytic nitrogen-fixing bacterium isolated from the roots of mature Pennisetum giganteum z.x.lin. In order to reveal the key factor which influences the ammonia secretion in fermentation process of K. variicola, GC-TOF-MS metabolomics was used. The changes of OD 600 , ammonia nitrogen content and intracellular metabolites of bacteria were measured simultaneously during fermentation, and a total of 172 intracellular metabolites were detected. Then some methods were used to analysis these high-throughput data. Principal component analysis (PCA) showed that the substances with more contribution to major component exhibited an order as mannitol, glycerol, glucose-1-phosphate, 2‑hydroxypyridine, glutamic acid, fructose-6-phosphate, aniline, lyxose and norleucine. Orthogonal projection to latent structures (OPLS) results revealed that the most important substances for the secretion of ammonia from K. variicola GN02 were mannitol, Gly-Pro, fructose-6-phosphate, and other 12 substances including glycerol, Glu and diacylglycerol having a significant impact with variable importance plot (VIP) values higher than 1, which can be used as the key metabolites for the secretion of ammonia from bacteria. Heatmap clustering analysis showed that mannitol, Gly-Pro and fructose-6-phosphate were strongly expressed at different fermentation stages as the major metabolites. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that fructose-mannose metabolism and amino acid metabolism are both metabolic pathways with the greatest impact on the secretion of ammonia in bacteria. Mannitol, fructose-6-phosphate and Glu are at the core hub of the metabolic process in bacteria, and Gly-Pro is the major nitrogen-related metabolite synthesized.