Fractionation of nitrogen isotopes in the enzymatic deamination of alanine: an insight into the quantitative evaluation of anaerobic metabolism in ecosystems

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Nitrogen isotope ratios of amino acids have been employed as a tool for illustrating the resource utilization of organisms and the trophic transfer between organisms in food webs. This tool is proposed based on the empirical observation that a universal trophic enrichment in 15 N is found in glutamic acid from diet to consumer species. Since glutamic acid is deaminated and incorporated into the tricarboxylic acid cycle (aerobic metabolism), the isotope ratios of glutamic acid have been used for evaluating life energy production through aerobic metabolism. However, little is known about the change in the isotope ratio associated with ‘anaerobic’ metabolism such as fermentation in organisms. Therefore, we determined the isotopic fractionation factor ( α ) for the deamination of alanine, a representative amino acid related to anaerobic metabolism, and conducted laboratory-cultured experiments to see the change in the nitrogen isotope ratio through anaerobic metabolism. Moreover, we compared the trophic enrichment in 15 N between alanine and glutamic acid in natural samples to visualize the contribution of anaerobic metabolism in food webs. In the in vitro enzymatic deamination of alanine, the isotope ratios of alanine are gradually increased, with the α of 0.9923. In the culture of the eukaryote Saccharomyces cerevisiae and the bacterium Lactococcus lactis with organic substrates under hypoxic conditions, the enrichment in 15 N is negligibly small for glutamic acid (by ~ 0‰), but considerably large for alanine (by ~ 5‰) in the culture. These results demonstrate that the activity of anaerobic metabolism is recorded in the isotope ratios of alanine but not glutamic acid in organisms. Moreover, we visualize the contribution of anaerobic metabolism in natural samples: (1) hypoxia within pollen provision for the diet of bee larvae induces a large enrichment in 15 N of alanine; and (2) this ‘anaerobic’ enrichment in 15 N can be identified in the cross-plot of the isotope ratios for alanine and glutamic acid in marine and terrestrial species, dissolved organic nitrogen, and the bees that potentially record of hypoxia within their diets. Thus, we suggest that the comparison of the isotope ratios between alanine and glutamic acid can be useful for evaluating the experience of hypoxia and the activity of anaerobic metabolism in biological and environmental samples.