Heterotrophic nitrification in soils: Approaches and mechanisms

Studies on nitrification, a crucial process of biogeochemical N cycling, have traditionally focused on autotrophic microorganisms. Recent discoveries, however, highlight the importance of heterotrophic nitrification as a key to N cycling, particularly in acidic soils. While molecular approaches have advanced our understanding of the key players in autotrophic nitrification, the biochemical mechanisms and corresponding genes of heterotrophic nitrification are nearly unknown. First, we reviewed the advantages and limitations of existing approaches to analyze heterotrophic nitrification in soils. ¹⁵N labeling of organic compounds (e.g. amino acids) allows to determine solely the nitrification of organic N. Because many bacteria have similar autotrophic nitrification enzymes that oxidize inorganic N, it is necessary to inhibit autotrophic nitrification to determine the heterotrophic N nitrification activity by ¹⁵N techniques. The use of existing inhibitors, however, can mislead the conclusions because not all inhibitors stop autotrophic nitrification completely, and some can decrease heterotrophic nitrification. Their effects strongly depend on the composition of the nitrifier community and soil properties. The use of modern molecular approaches is limited by suitable genetic biomarkers. Second, we propose the following methods to investigate heterotrophic nitrification processes: i) isolation and purification of heterotrophic nitrification enzymes, followed by determination of the amino acid sequence of proteins to design genetic markers; ii) use of DNA-based stable isotopes (¹³C, ¹⁵N); iii) combining fluorescence in situ hybridization with microautoradiography (¹⁴C) to determine the composition of heterotrophic nitrifier communities; and iv) scheme to select autotrophic nitrification inhibitors. We suggest to improve the existing approaches to shed new light on the processes of heterotrophic nitrification, which can reach 99% of total nitrification in forest soils and strongly affect N stocks and fluxes in terrestrial ecosystems.