Cultivable microbial diversity in speleothems using MALDI-TOF spectrometry and DNA sequencing from Krem Soitan, Krem Lawbah, Krem Mawpun, Khasi Hills, Meghalaya, India.

The microbial diversity in the Indian caves is inadequately characterized. This study reports on the culturable microbial communities in caves from the Indian sub-continent. This study aims to expand the current understanding of bacterial diversity in the speleothems and wall deposits from Krem Soitan, Krem Lawbah, Krem Mawpun in Khasi Hills, Meghalaya, India. A culture-dependent approach was employed for elucidating the community structure in the caves using MALDI-TOF spectrometry and 16S rRNA gene sequencing. A high bacterial diversity and a greater bacterial taxonomic diversity is reported using MALDI-TOF spectrometry and 16S rRNA gene sequencing. High microbial enumerations were observed on dilute nutrient agar (5.3 × 103 to 8.8 × 105) followed by M9 minimal medium (4 × 104 to 1.7 × 105) and R2A medium (1.0 × 104 to 5.7 × 105). A total of 826 bacterial isolates were selected and preserved for the study. 295 bacterial isolates were identified using MALDI-TOF spectrometry and the isolates which showed no reliable peaks were further identified by 16S rRNA gene sequencing. A total 91% of the bacterial diversity was dominated by Proteobacteria (61%) and Actinobacteria (30%). In addition, bacterial phyla include Firmicutes (7.45%), Deinococcus-Thermus (0.33%) and Bacteroidetes (0.67%) were found in the samples. At the genus level, Pseudomonas (55%) and Arthrobacter (23%) were ubiquitous followed by Acinetobacter, Bacillus, Brevundimonas, Deinococcus, Flavobacterium, Paenibacillus, Pseudarthrobacter. Multivariate statistical analysis indicated that the bacterial genera formed separate clusters depending on the geochemical constituents in the spring waters suitable for their growth and metabolism. To the best of our knowledge, there are no previous geomicrobiological investigations in these caves and this study is a pioneering culture dependent study of the microbial community with many cultured isolates.

Microbially induced iron precipitation associated with a neutrophilic spring at Borra Caves, Vishakhapatnam, India.

The present investigation uncovers various pieces of evidence for the possible biologically induced mineralization in iron mats associated with a pH-neutral spring in the Borra caves, Vishakhapatnam, India. Electron microscopy [scanning electron microscopy (SEM) and transmission electron microscopy (TEM)] demonstrated large numbers of (i) hollow tubes (diameter ∼1 µm) resembling sheaths of the iron-oxidizing bacteria Leptothrix, (ii) thin (diameter <<1 µm) solid fibers of uncertain origin, (iii) nanoscale subspherical to irregularly shaped particles encrusting tubes and fibers, and (iv) aggregates of broken and partially disintegrated sheaths, fibers, and particles embedded in extracellular polymeric substances (EPS) occasionally including microbial cells. X-ray microanalyses by energy dispersive spectroscopy (EDS) revealed that the mat accumulated largely Fe but also smaller amounts of Si and traces of P and Ca. Particles rich in Si and Al (possibly kaolinite) and Ca (carbonate) were also observed. High-resolution TEM/EDS of unstained ultrathin sections suggests that microbial sheaths were highly mineralized by amorphous to cryptocrystalline Fe-rich phases and less frequently by other fine-grained and fibrous authigenic claylike minerals. Total number of microorganisms in the iron mats was 5.8×10(5) cells, g sed(-1) (wet weight). Analysis of the 16S rRNA gene diversity revealed microorganisms assigned to eight different phyla [Proteobacteria (62%), Chloroflexi (8%), Bacteroidetes (7%), Planctomycetes (1%), Actinobacteria (5%), Acidobacteria (6%), Nitrospira (1%), Firmicutes (5%)]. Within the Proteobacteria, Betaproteobacteria was the predominant class, which accounted for 28% of the sequences. Within this class some obvious similarities between the obtained sequences and sequences from other cave systems could be seen, especially sequences affiliated with Leptothrix, Siderooxidans, Crenothrix, Comamonadaceae, Dechloromonas, and many uncultured Betaproteobacteria. Four (4%) of the sequences could not be assigned to phylum level but were affiliating with the candidate division TM7 (2%), candidate division OP11 (1%), and candidate division WWE3 (1%). The results allow us to infer a possible relationship of microbial sheaths, EPS, and the iron precipitates to microbial community diversity in the Borra cave springs. Understanding biogenic iron oxides in caves has important astrobiological applications as it provides a potential tool for the detection of extraterrestrial life.