Dependency of biological nitrogen fixation on organic carbon in acidic mine tailings under light and dark conditions

Several ecological restoration techniques have been used to enhance the levels of available organic matter in mine tailings. However, the effect of organic carbon on nitrogen fixation remains poorly understood. In this study, nitrogen fixation in two mine tailings treated with glucose was analyzed using 15N isotope labeling, pyrosequencing and DNA-based stable isotope probing (SIP) to identify nitrogen fixers and their activity under organic carbon addition (0.8 mg glucose-C/g soils). Following glucose addition, the total nitrogen content increased significantly (1.6–5.4% of initial N content), and the 15N content increased from 0.89 ± 0.20 mg·kg−1 to 37.08 ± 3.34 mg·kg−1 during a 28-day incubation period. In addition, glucose-stimulated nitrogen fixation in dark conditions was 8.53-fold (Shuimuchong) and 1.22-fold (Yangshanchong) higher than that in light conditions. At the same time, the addition of glucose led to distinctly different phylotypes of the bacterial communities and an increase in the abundance of the nifH gene in the two mine tailings. 13C-glucose SIP showed that the highest copy number of the 16S rRNA and nifH genes in the 13C-glucose-labeled treatment peaked in the heavy fraction, with a buoyant density of 1.737 g·mL−1. However, the pyrosequencing results revealed that the vast majority of the nifH genes were unique and belonged to yet uncultured bacteria, while the potentially known groups were associated with Azotobacter, acidophilic Leptospirillum and Pseudanabaena, accounting for 13.83% of all the total nifH genes. These results indicate that the addition of organic carbon may increase the nitrogen content by stimulating nitrogen fixers, highlighting the vital roles of organic carbon in increasing nitrogen accumulation during the restoration of mine tailings.