Microbial Diversity and Mineralogical-Mechanical Properties of Calcitic Cave Speleothems in Natural and in Vitro Biomineralization Conditions

Abstract

Natural mineral formations are a window into important processes leading to carbon storage and mineralized carbonate structures formed through abiotic and biotic processes. In the current study, we made an attempt to undertake a comprehensive approach to characterize the mineralogical, mechanical, and microbial properties of different kinds of speleothems from karstic caves; with an aim to understand the bio-geo-chemical processes in speleothem structures and their impact on nanomechanical properties. We also investigated the biomineralization abilities of speleothem surface associated microbial communities in vitro. Mineralogical profiling using techniques such as X-ray powder Diffraction (XRD) and Tescan Integrated Mineral Analyzer (TIMA) demonstrated that calcite was the dominant mineral in the majority of speleothems with Energy Dispersive X-ray Analysis (EDS) indicating a few variations in the elemental components. Differing proportions of polymorphs of calcium carbonate such as aragonite and vaterite were also recorded. Significant variations in trace metal content were recorded through Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Scanning Electron Microscopy (SEM) analysis revealed differences in morphological features of the crystals which varied from triangular prismatic shapes to etched spiky forms. Microbial imprints and associations were seen in a few sections. Analysis of the associated microbial diversity showed significant differences between various speleothems at Phylum level; although Proteobacteria and Actinobacteria were found to be the predominant groups. Genus level microbial associations showed a relationship with the geochemistry, mineralogical composition, and metal content of the speleothems. The assessment of nanomechanical properties measured by Nanoindentation revealed that the speleothems with a dominance of calcite were stronger than the speleothems with mixed calcium carbonate polymorphs and silica content. The in vitro metabolic activity of the microbial communities associated with the surfaces of the speleothems resulted in calcium carbonate crystal precipitation. Firmicutes and Proteobacteria dominated these populations, in contrast to the populations seen in natural systems. The precipitation of calcium carbonate crystals in vitro indicated that microbial metabolic activity may also play an important role in the synthesis and dissociation of biominerals in the natural environment. Our study provides novel evidence of the close relationship between mineralogy, microbial ecology, geochemistry, and nanomechanical properties of natural formations.

High-Throughput Sequencing Analysis of the Actinobacterial Spatial Diversity in Moonmilk Deposits

“…Previous investigations on the moonmilk microbiome revealed a very diverse microbial community in these deposits [ 9 , 13 , 14 , 15 , 16 , 17 , 19 ]. The high-throughput sequencing approach used in this work complemented previous findings by providing an in-depth picture of the bacterial population, together with a detailed taxonomic fingerprint of the phylum Actinobacteria.…”

“…The origins of various moonmilk crystalline habits, including monocrystalline rods, polycrystalline chains, and nanofibers, are tentatively attributed to the moonmilk indigenous microbial population [ 11 ]. Among a moonmilk microbiome comprising Archaea, Fungi, and Bacteria [ 9 , 10 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ], the indigenous filamentous Fungi [ 20 ] and Actinobacteria [ 11 , 21 ] are believed to mediate moonmilk genesis with cell surfaces promoting CaCO 3 deposition [ 11 , 20 , 21 ]. Actinobacteria were additionally reported to be metabolically capable of inducing favorable conditions for CaCO 3 precipitation, or even directly precipitating carbonate minerals [ 12 , 21 ].…”

“…Actinobacteria were additionally reported to be metabolically capable of inducing favorable conditions for CaCO 3 precipitation, or even directly precipitating carbonate minerals [ 12 , 21 ]. Members of the phylum Actinobacteria are routinely found in this speleothem [ 9 , 10 , 12 , 13 , 14 , 18 , 19 ], as well as in the other subterranean deposits within limestone caves [ 3 , 8 , 22 , 23 ], volcanic caves [ 24 , 25 , 26 ], and ice caves [ 27 ]. The broad distribution of Actinobacteria in the subsurface systems stimulates investigation in order to understand the factors driving their existence in mainly inorganic and highly oligotrophic environments, and the processes that enable them to mediate speleogenesis.…”

“…Instead, in our attempt to isolate moonmilk-dwelling Actinobacteria for assessing their potential for participating in the genesis of these speleothems [ 21 ] and producing antimicrobial compounds [ 10 ], we only recovered members of the genus Streptomyces . Such a dominance of streptomycetes was rather unexpected, according to other moonmilk microbial diversity studies performed through culture-dependent [ 10 , 12 , 13 , 18 ] and culture-independent approaches using clone libraries [ 9 ], denaturing gradient gel electrophoresis (DGGE) fingerprinting [ 14 , 16 , 17 ], automated ribosomal intergenic spacer analysis (ARISA) [ 13 ], and, more recently, high-throughput sequencing (HTS) [ 19 ]. The actinobacterial genera identified in those studies included Rhodococcus , Pseudonocardia , Propionibacterium , Nocardia , Amycolatopsis , Saccharothrix , Geodermatophilus , Mycobacterium , Aeromicrobium , Kribella , Nocardioides , Actinomycetospora , Nonomuraea , Euzebya , Rubrobacter , and Arthrobacter , in addition to Streptomyces .…”

Analysis and optimization of process parameters for in vitro biomineralization of CaCO3 by Klebsiella pneumoniae, isolated from a stalactite from the Sahastradhara cave

“…3). The percentage of carbon was found to be quite high as compared to that reported in stalactite of Lake cave (4.9 AE 1.7%) and Mammoth cave (5.4 AE 1.5%) as reported by Dhami et al 40 They reported the presence of many other elements like N, Si, K, Na, S, Mg, Al, Fe and P which were not detected in stalactite of Sahastradhara cave except magnesium.…”