Quantifying Bacterial Spores Across Ancient Permafrost Using Lanthanide Luminescence

• Main Author: Lalla, Sidharth
• Other Authors: Mogul, Rakesh, Department of Chemistry and Biochemistry, Liu, Yan, McCulloch, Kathryn
• Format: Thesis
• Language: English
• Published: California State Polytechnic University, Pomona 2020
• Subjects: chemistry; luminescence; quantification; terbium; lanthanide; spore; permafrost
• Online Access: http://hdl.handle.net/10211.3/216717

With the onset of global warming, the thawing of ancient permafrost may lead to the release of age-old dormant microorganisms, or spores; thus, enhancing the potential for a new age of superbugs. The endeavors of this thesis, therefore, were to (A) develop a simple procedure to extract spores in soils with high organic matter, and (B) to apply this method towards quantifying spore abundances in permafrost across a chronosequence of 19 to 33 ky from present. For this method, a tandem filtration device, assembled by combining 3.0 and 0.2 ??m syringe filters, was used to sequentially remove large soil particles and trap bacterial spores, respectively. Permafrost samples were repeatedly (10x) extracted via vortexing and filtering through the tandem system using 0.1 M sodium acetate (pH 5.5). Upon completion, the 0.2 ??m filter was removed, washed (7x), autoclaved, and the microbial contents eluted using buffer. Eluates, containing dipicolonic acid (DPA), were then mixed with Tb(EDTA) (the lanthanide probe) and analyzed using temporally-resolved luminescence spectroscopy. Through this procedure, we respectively obtained 0.121 ?? 0.021 nmol DPA/dry g (or 1.7x105 ?? 0.3x105 spores per dry gram) for the 19 ky sample, and 0.258 ?? 0.036 nmol DPA/dry g (or 3.7x105 ?? 0.5x105 spores per dry gram) for the 33 ky sample. This amounted to an ~2.2-fold increase (p = 0.0072) in spores across the chronosequence. Together, these results (1) corroborate molecular genetic studies showing increases in sporulation in older permafrost, and (2) reveal a current limit of detection of 243 fg/??L of DPA (1.46 nM), or 3.3x104 spores gdw-1.