An internal recycling mechanism between ammonia/ammonium and nitrate driven by ammonia-oxidizing archaea and bacteria (AOA, AOB, and Comammox) and DNRA on Angkor sandstone monuments

The ancient Angkorian sandstone monuments in Cambodia are well known for biodeterioration of the sandstone under tropic climate. This study examined ammonia oxidizing bacteria (AOB and Comammox) and archaea (AOA) in stone-dwelling microbiome from three important Angkor monuments, namely Angor Wat, Bayon of Angkor Thom, and Preah Vihear, by DNA-based metagenomics analysis, RNA-based functional gene reverse-transcriptional (RT)-qPCR quantification, and N-15 isotope analysis. Our metagenomics datasets on these three monuments reveal a rich microbiome and abundant microbial nitrogen transforming reactions of ammonia oxidation and nitrite oxidation, Comammox and dissimilatory nitrate reduction to ammonium (DNRA), and these results support an internal recycling mechanism between ammonia/ammonium and nitrate by AOA, AOB, Comammox and DNRA on the stone monuments to support the microbial community. Though a wide range of AOB and AOA lineages together with Comammox were retrieved from these metagenomes as the potential ammonia-oxidizers, only AOA and Comammox were most abundant, likely to contribute to the biochemical processes of ammonia oxidation. The RNA-based qPCR quantification of the functional gene amoA in this study showed that AOA were the more active ammonia oxidizers over AOB in the microbiome of these Angkor sandstone monuments. In addition, a rich population of nitrite/nitrate producing AOA and Comammox drove the sequestration of CO₂ onto the stone and this process was in turn supported by DNRA to provide the substrate ammonia/ammonium for a further cyclic reaction to take place continuously between ammonia/ammonium and nitrate. This model is further supported by the high enrichment of stable isotope ¹⁵N signature of NO₃⁻ in sandstone surface biofilms. The findings of this study are insightful for elucidation of nitrate accumulation by an internal N cycling mechanism proposed, and are important for understanding the sustainable microbial community and protection management of stone cultural heritage.