Evidences for microbial precipitation of calcite in speleothems from Krem Syndai in Jaintia Hills, Meghalaya, India

Speleothems from Krem Syndai, Meghalaya in Northeast India were studied for their microbial diversity using 16S rDNA-based phylogenetic approach and conventional microbiological techniques along with geochemistry, mineralogy and in vitro experiments to understand participation of microorganisms in CaCO3 precipitation. Speleothems imaged by scanning electron microscopy showed round coccoid-like, sporangia-like and spinose calcified structures, numerous broken cocci shells with spotted interiors inside a calcite crystal, honeycomb long reticulate, smooth, flat, twisted, ribbon-like, tubular, beaded, microbe-mineralized filaments and extracellular polymeric substances (EPS). Fourier spectroscopy indicated the presence of various organic compounds. d13C and d18O isotopic ratios of speleothems ranged from -4.65 to -7.34% and -3.06 to -6.80%, respectively. Total number of microbial cells using SYBR Gold was high. Fluorescence in situ hybridization (FISH) indicated approximately 3 * 105 to 5 * 105 cells g sed-1 in the speleothems out of which the number of microbes belonging to Eubacteria ranged from 1.8 * 105 to 3.6 * 105 cells, g sed-1. FISH showed ~45% active microbial cells of the total cell number in samples. DNA-based high-throughput amplicon sequencing revealed 19 bacterial phyla in the speleothem. Approximately 42% of the sequences were similar to Proteobacteria (Alphaproteobacteria: 22.4%, Betaproteobacteria: 8.9%, Gammaproteobacteria: 8.6%). Sequences similar to Nitrospiraceae (22.8%) had the highest proportion of sequences belonging to a single family. Bacterial strains isolated from the speleothems raised alkalinity and precipitated calcite in the laboratory cultures which was confirmed by X-ray diffraction (XRD) analyses. These isolates belonged to Bacillus spp., Actinomycetes spp., Streptomyces spp., Pseudomonas spp., Micrococcus spp., Staphylococcus spp., Xanthobacter spp. and Arthrobacter spp. Overall, the results showed unequivocal evidence of bacterial fingerprints during CaCO3 precipitation in the cave.