Habitat specificity and network analysis of nitrogen-fixing bacteria in the Heshang Cave, Hubei Province

Abstract:

Karst caves are important karst landforms, serving as natural laboratories to investigate deep biosphere. Despite the diverse microorganisms, little is known about microorganisms capable of fixing nitrogen in the permanent darkness and oligotrophic karst caves. [Objective] To reveal the composition of nitrogen-fixers and their correlation with biotic and abiotic factors in karst caves. [Methods] We collected samples from overlying soils, loose sediments, and weathered rock inside the Heshang Cave, Hubei Province, and conducted high-throughput sequencing of nifH responsible for nitrogen fixation. [Results] The nitrogen-fixing bacteria had the highest alpha diversity in the overlying soils and the lowest α diversity in the sediments. K⁺, NO₂^-, and NO₃^- were the main factors impacting the nitrogen-fixing bacteria, which showed high specificity for habitats. Bradyrhizobium widely distributed in all habitats with high relative abundance. Geobacter dominated in the overlaying soil, whereas Azotobacter was dominant in the sediments. Geobacter, Azotobacter, and Halorhodospira had similar relative abundance in weathered rock samples. The co-occurrence network analysis showed that the nitrogen fixers had closely positive correlations, suggesting their cooperative survival strategy under the oligotrophic conditions. It was noted that a large proportion of nitrogen fixers cannot be classified, which indicated rich novel nitrogen fixers in the caves, hot spots for studying microbial dark matter. [Conclusion] Our study revealed the composition and structure of the nitrogen-fixing microbial community and their correlations with environmental variables in the underground dark oligotrophic Heshang Cave. The findings are of great significance for the in-depth understanding of the nitrogen cycle in deep biosphere.