Quantification of endospores in ancient permafrost using time-resolved terbium luminescence.

Abstract (100 words) Herein, we detail a simple procedure for quantifying endospore abundances in ancient and organic-rich permafrost. We repeatedly (10x) extracted and fractionated permafrost using a tandem filter assembly composed of 3 and 0.2 μm filters. Then, the 0.2 μm filter was washed (7x), autoclaved, and the contents eluted (including dipicolinic acid). Time-resolved luminescence using Tb(EDTA) yielded a LOD of 1.46 nM DPA (6.55x103 endospores/mL). In summary, DPA/endospore abundances were ∼2.2-fold greater (p=0.007297) in older 33 ky samples (258 ± 36 pmol DPA gdw-1; 1.15x106 ± 0.16x106 spores gdw-1) versus younger 19 ky samples. This suggests that dormancy increases with permafrost age. While ancient permafrost harbors a diverse microbiome, changes in microbial activity and dormancy over geological timescales are not well-characterized. In this report, therefore, we detail a simple procedure for quantifying endospore abundances from Pleistocene-aged permafrost (containing substantial soil organics) using particle filtration, time-resolved lanthanide luminescence, and chelated terbium as a probe (Tb(EDTA)). To separate endospores, we repeatedly (10x) extracted and fractionated permafrost using a tandem filter assembly comprised of sequentially connected 3 and 0.2 μm filters. Then, the 0.2 μm filter was washed (7x), autoclaved, and the contents (including the endospore marker molecule dipicolinic acid (DPA)) eluted using buffer. Time-resolved luminescence of the target complex (Tb(EDTA)(DPA)) eliminated background fluorescence, and yielded a LOD of 1.46 nM DPA, or 6.55x103 endospores/mL. In sum, DPA/endospore abundances in permafrost (formed 19 and 33 ky before present) were greater in the oldest samples (33 ky: 258 ± 36 pmol DPA gdw-1; 1.15x106 ± 0.16x106 spores gdw-1) versus youngest samples (19 ky: 121 ± 21 pmol DPA gdw-1; 5.38x105 ± 0.93x105 spores gdw-1). This ∼2.2-fold increase (p=0.007297) suggests that dormancy may increase with permafrost age. Accordingly, our tandem filtration and luminescence assay serves as a potential cost-effective tool for microbial ecology and biodefense applications.