MICROBES ON MOONMILK: A SPELEOLOGICAL EXPLORATION

Moonmilk is a secondary, microcrystalline cave deposit that forms on ceilings, walls, and other speleothems in association with condensing or dripping water. Recent studies show that the metabolic activity of the cave microbiome plays a pivotal role in cave biogenesis by inducing changes in the environment that can lead to cave corrosion or mineral precipitation. We set out to characterize the crystalline and microbial community structures of moonmilk collected from three limestone caves in southwestern Virginia using eDNA metabarcoding and scanning electron microscopy (SEM).

Caves are protected by state law and it is illegal to remove speleothems, biota, or anything else from caves except for scientific research purposes. To obtain the samples used in this research we worked with the Appalachian Cave Conservancy and the Virginia Department of Conservation and Recreation’s Natural Heritage Division, and acquired written permission from the landowners. Very small samples (~ 4 cm²) of damp moonmilk in the form of chips of crust or surface swabs were collected in order to minimize disturbance at each site. Control samples were collected as surface swabs of the walls around the sample sites.

SEM images indicated that the moonmilk was composed of rhombohedral calcite minerals with the presence of spherical bacteria (cocci) on the surface. Solitary cocci measured approximately 0.5 µm in diameter and larger aggregates of cocci exhibited spore chain morphology indicative of growth and reproduction. Elemental analysis of these structures with Energy Dispersive X-ray spectroscopy (EDS) detected Ca, C, Al and Si oxides, with maximum carbon oxides of 51.50-54.00 % by weight in the spore chain samples. Calcium content was greatest in the solitary spheres at up to 36.56 % by weight, and several also contained silica and aluminum up to 4.58 % and 2.57 % by weight, respectively. Beta-diversity PCoA analyses of the eDNA results revealed that the microbial community structures between the moonmilk samples were more variable than those collected from nearby control samples. The differences between the moonmilk and control samples, however, were not statistically significant. The eDNA results support the premise that the microbiome and character of moonmilk may vary between caves or regions due to external environmental inputs.