湖北和尚洞固氮微生物群落的生境特异性及网络分析

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

摘要:

岩溶洞穴是喀斯特地貌的重要组成部分，由于其长期黑暗、寡营养等极端条件，使其成为研究陆地深部生物圈的天然实验室。尽管近年来对洞穴微生物多样性的研究不断深入，但对洞穴中固氮微生物的认识却相对薄弱。【目的】查明洞穴中固氮微生物群落的特征以及与生物、非生物因子之间的关系。【方法】本文以湖北和尚洞为例，通过对固氮菌功能基因nifH进行高通量测序和多元统计分析，系统研究了洞穴内外3种生境(洞穴上覆土壤、洞内松散沉积物以及风化岩壁)固氮微生物群落的空间分布特征及其与环境因子之间的关系。【结果】结果表明和尚洞不同生境间固氮菌群落α多样性存在显著差异(P<0.05)，以洞穴上覆土壤α多样性最高，洞穴内沉积物α多样性最低。K⁺、NO₂^-和NO₃^-是驱动和尚洞固氮微生物群落组成的重要因子。和尚洞内固氮菌群落组成和分布具有生境特异性，除慢生根瘤菌属(Bradyrhizobium)在3种生境中相对丰度均较高外，上覆土壤以地杆菌属(Geobacter)占主导，洞穴沉积物以固氮菌属(Azotobacter)占主导，而风化岩壁中地杆菌属(Geobacter)、固氮菌属(Azotobacter)以及需盐红螺菌属(Halorhodospira)等主要类群占比相当。共现性网络分析结果显示洞穴固氮菌之间形成了紧密的正相关关系，暗示着固氮菌在洞穴寡营养的极端环境中以协作为主的生存策略。值得注意的是，洞穴固氮菌中有很大一部分未分类的类群，暗示着洞穴中可能蕴含着大量新的固氮类群，是挖掘微生物暗物质的热点区域。【结论】该研究结果揭示了地下黑暗寡营养的和尚洞固氮微生物群落的组成，对深入了解地下黑暗生物圈的氮循环过程、固氮菌与环境之间以及固氮菌种群之间的互作关系具有重要意义。

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.