Geochemistry
Elemental concentrations in spring waters: Analyses of the drip and spring waters showed various trace and major elements (supplementary table 1). The temperature in drip water was 17-19 °C, whereas air temperature inside the cave was 20-23 °C. The pH of the cave samples was neutral (7.1 to 7.7) and the conductivity ranged from 12 to 421 µs with significant variations (p <0.05). The average total carbon content was high (~16.44) and significant differences (p <0.05) were noted among the samples. The inorganic carbon content of the water samples varied significantly (p <0.05). It was higher in LBS3 (29.34 ± 0.2 mg L-1) and lowest in LBS2 (1.26 ± 0.2mg L-1). The bicarbonate contents in drip and spring water also varied and ranged from 36.6 ±0.07 to 287.92 ±0.46 and 129.32 ±0.13 to 424.56 ±0.31 mg L-1 while the value in the pool water was 500.2 ±0.1 mg L-1. The results showed that Ca2+ concentrations significantly differ (p <0.05) in drip and spring water samples of different distances and zones along the length of the cave and ranged from 4.8 ± 0.2 to 30.4± 0.16 mg L-1 and 16.0 ± 0.68 to 43.2 ± 0.14 mg L-1, respectively. Significant (p <0.05) disparities were observed in sulphate (0.01 ± 0.01 to 21.95 ± 0.86 mg L-1) and nitrate (0.06 ± 0.02 to 4.6 ± 0.2 mg L-1) contents. Significant variations (p <0.05) were observed in magnesium (7.77 ± 0.07 to 98.1 ± 0.02 mg L-1), sodium (1.3 ± 0.1 to 6.2 ± 0 mg L-1), chloride (7.1 ± 0.05 to 19.02 ± 0.01 mg L-1), total hardness (44 ± 0.29 to 452 ± 0.1 mg L-1), total dissolved solids (18.9 ± 0.02 to 299 ± 0.12 mg L-1) and salinity (11.3 ± 0.1 to 189 ± 0.69 mg L-1) contents in the drip, pool and spring waters (supplementary table 1).
Microbiology
Isolation and identification of microorganisms: A total of 826 bacterial strains were isolated from all the cave samples on different media. Except for KSSTc6, bacterial colonies were observed in most of the cave samples cultured on dilute Nutrient Agar (NA), M9 and R2A medium. KSSTc1, LBSTc1, LBSTc2. LBWDc1 showed no growth on dilute NA and M9 media (Table 1). Speleothems from all three caves showed high microbial enumerations 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 5.78×105). From the above media, 295 isolates were identified using MALDI-TOF (227 isolates) and 16S rRNA gene (68 isolates) based sequencing.
Taxonomic analysis showed that the bacterial isolates belonged to 16 genera under five phyla. Taxonomic analysis based on relative abundance revealed that Proteobacteria (61%) was the dominant bacterial group followed by Actinobacteria (30%), Firmicutes (7.45%), Bacteroidetes (0.67%) and Deinococcus-Thermus (0.33%; Figure 1a; supplementary table 3).
Proteobacteria and Actinobacteria were the most abundant bacterial community (91%, Figure 1a). The bacterial phyla Proteobacteria (1.1% - 21.5%) was ubiquitously present in all the speleothems samples except KSSTc4, MPSTc1, LBSTc1, LBSTc2, LBWDc1, and LBWDc3 from Krem Soitan and Krem Lawbah. Within the Proteobacteria, Alpha-, Beta- and Gamma-proteobacteria were found in the samples. Gammaproteobacteria were the most abundant class (8 - 96%) detected in most of the samples. Alphaproteobacteria (4.1%) and Betaproteobacteria (2.1%) were found in samples KSSTc3 and KSSMc1, respectively. Actinobacteria (1.1% - 24.7%) was also observed in most of the samples except KSSTc1, KSSTc7, KSSTc8 from Krem Soitan and LBSTc1, LBWDc2 from Krem Lawbah. Bacterial members Deinococcus-Thermus and Bacteroidetes were present as a minor group and were exclusive to the stalactite samples of Krem Soitan. Other significant phyla which constituted >5% of the sequences included the Firmicutes (7.45%). The phylum Firmicutes was present in the stalactites and stalagmites of Krem Soitan and Krem Lawbah (supplementary table 3)
The bacterial genera Acinetobacter, Arthrobacter, Bacillus, Brevundimonas, Deinococcus, Enterobacter, Flavobacterium, Kocuria, Microbacterium, Paenarthrobacter, Paenibacillus, Pseudarthrobacter, Pseudomonas, Staphylococcus, Streptococcus and Variovorax were also identified in the cave samples (Figure 1b; supplementary table 4). A total of 71 bacterial species were identified from all the cave deposits. Pseudomonas (55%) and Arthrobacter (23%) were the most abundant genera (nine out of fifteen samples) in the study (supplementary table 4). The genus Arthrobacter was detected in all three cave samples whereas Pseudomonas was noted in Krem Soitan and Krem Lawbah only. Among the Pseudomonas and Arthrobacter, the following strains were identified. They include: A. oxydans, A. oryzae, Pseudarthrobacter oxydans, P. koreensis, P. chlororaphis, P. granadensis, P. alkylphenolica, and Paenarthrobacter nicotinovorans (Figure 1b; supplementary table 5).
Diversity in Krem Soitan: Maximum numbers of isolates were recovered from stalagmite KSSMc1 (47 strains) followed by stalactite KSSTc5 (37 strains) (supplementary table 4). Among the Proteobacteria, members belonging to Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria were observed. The members noted were Enterobacter, Pseudomonas, Brevundimonas and Variovorax genera. Among the Enterobacter genera, E. asburiae, E. cloacae, E. lugwigii and E. tabaci were identified. Twenty four bacterial strains of Pseudomonas chlororaphis (51%) was observed in the stalagmite (KSSMc1). Among the family Comamonadaceae, Variovorax paradoxus was exclusive to the stalagmite, KSSMc1. One strain of Brevundimonas vesicularis was also identified (KSSTc3) as a relatively minor population (<1%). Among the Actinobacteria, strains belonging to Arthrobacter sp., Pseudoarthrobacter sp., Paenarthrobacter sp., Kocuria sp., Microbacterium sp., were observed. Eighteen strains belonged to genus Arthrobacter in KSSTc5 (48%; Figure 1b). A. aurescens, A. ginsengisoli, A. histidinolovorans, A. ilicis, A. methylotrophus, A. nicotinovorans, A. oxydans, A. pascens, A. polychromogenes, A. sulfonivorans were the main strains identified. Pseudarthrobacter oxydans was also observed in the stalactite and stalagmite. Five strains of Paenarthrobacter nicotinovorans were also identified in the stalactites. It was noted that <1% of the bacterial genera belonged to Deinococcus, Microbacterium and Paenibacillus. Kocuria belonging to Micrococcaceae, were also detected as a minor population (<1%). Within the Acinetobacter genus, four strains of A. johnsonii, three strains of A. woffii were identified in stalactite samples. Among the Bacteroidetes and Firmicutes, Flavobacterium was noted in only one stalactite sample (KSSTc8) and Bacillus genera was observed in two samples, (KSSMc1 and KSSTc7) from Krem Soitan. One strain of Deinococcus ficus was identified from the stalactite (KSSTc4).
Diversity in Krem Mawpun: Among the Actinobacteria, 11 strains belonging to Arthrobacter sp.including seven strains of A. oxydans and two each of A. polychromogenes and A. sulfonivorans were identified from the stalactite (MPSTc1; supplementary table 5).
Diversity in Krem Lawbah: Thirty one strains were identified from the wall deposit LBWDc2 and twenty four strains each from LBWDc4 and LBSTc3. Among the Proteobacteria, all the thirty one strains identified from the wall deposit LBWDc2, belonged to the genus Pseudomonas. The strains P. granadensis, P. koreensis, P. chlororaphis, P. jessenii, were observed from wall deposits LBWDc4 and LBWDc2. Among the genus Arthrobacter, 19 strains were observed (79 %). A. oxydans (five strains), A. pascens (two strains), A. polychromogenes (six strains), Pseudarthrobacter polychromogenes (four strains); A. ginsengisoli, A. sulfonivorans (one strain each)were identified from stalagmite (LBSTc3) (supplementary table 5). Minor percentages of the members belonging to the phylum Firmicutes was present in samples LBSTc1, LBSTc3, LBWDc4. Among the Firmicutes, Bacillus, Staphylococcus and Streptococcus genera were observed. One strain of Staphylococcus hominis was identified from stalactite LBSTc1 (<1%) and 17 strains belonged to Staphylococcus warneri from wall deposits LBWDc4. Streptococcus gallolyticus was noted in LBSTc3 (<1%). Bacillus safensis was present in one sample (LBSTc3) (supplementary table 5)
To identify the relationship among the samples based on species level distribution, the UPGMA (unweighted pair group method with arithmetic mean) cluster analysis was performed. Analysis revealed that most of the samples from the same cave formed a separate cluster (Figure 2). Most of the Krem Soitan samples clustered together except KSSMc1, KSSTc1 and KSSTc4. Similarly, samples from Krem Lawbah grouped together at 40% similarity level except sample LBWDc1 (Figure 2).
Phylogenetic analysis as ascertained by the neighbour-joining tree where the close lineage of bacteria strains was retrieved by doing blast search in Eztaxon database (Fig 3). It was found that bacterial strain KSSMR06 and LBSTR64 showed close lineage with type strain Bacillus altitudinis and Bacillus safensis, respectively (supplementary figure 3). Bacterial strains KSSTR28, KSSTR31, KSSTR25, KSSTR26, LBSTR73 showed high sequence identity among themselves and branched together with type strain Arthrobacter ginsengisoli. Similarly, bacterial strains LBSTR66, LBWDR91, LBWDR90, and LBSTR67 form a separate clade with type strain Pseudarthrobacter oxydans (supplementary figure 4).Most of thebacterial strains belonging to Proteobacteria showed close lineage with type strains Pseudomonas hutmensis and Pseudomonas kribbensis (supplementary figure 5).In addition bacterial strainKSSTM20, KSSTM23formed clade with Enterobacter sichuanesis belonging to Gammaproteobacteria and KSSTR29 formed clade with type strain Deinococcus ficus (supplementary figure 5).
Phylogenetic analysis also indicated other bacterial strains such as LBSTR70, KSSTR47, KSSTR43, KSSMR04, KSSTM46, and KSSTM44. These strains form a separate clade in the phylogenetic tree. It was found from BLASTN search in NCBI database (supplementary table 6) that they showed 92 - 98% similarity with their respective closely related organisms. This may indicate that these are novel strains of the closely related organisms. Hence, they may form separate clades in the phylogenetic tree (Figure 3 and supplementary Figure 3, 4, 5).
Association among the taxa and geochemical parameters: Correlation analysis showed that Enterobacter, Variovorax, Acinetobacter, Paenibacillus, Flavobacterium genera had a positive associationwith Pseudomonas whereas Arthrobacter, Staphylococcus, Deinococcus and Paenarthrobacter genera showed a negative association with Pseudomonas (Figure 4). Association analysis among the bacterial genera and geochemical factors indicated that Bacillus, Enterobacter, Pseudarthrobacter, Brevundimonas and Streptococcus showed a positive association with electrical conductivity, total dissolved solids, and salinity of the samples whereas Staphylococcus and Kocuria showed a negative association with these geochemical factors. Most of the predominant bacterial groups i.e., Arthrobacter, Bacillus, Pseudoarthobacter, Microbacterium, Paenarthobacter, Acinetobacter, Paenibacillus and Streptococcus showed moderate to strong positive association with inorganic carbon and total carbon. In contrast, Staphylococcus and Kocuria showed a negative association. Arthrobacter, Bacillus, Paenarthrobacter and Streptococcus showed a positive association with HCO3, Na and K whereas Staphylococcus and Kocuria showed a negative correlation with these factors. It was also noted that Staphylococcus and Kocuria showed a negative association with several parameters namely nitrate, calcium, bicarbonates, electrical conductivity, total dissolved solids, salinity, inorganic carbon, total carbon, total hardness, and magnesium of the samples. Interestingly, it was found that bacterial genera Pseudomonas showed the opposite trend with bacterial genera Arthrobacter in case of most of the samples. The pattern of this organism is self-explanatory with the geochemical approach. Implementing correlation analysis within the geochemical factor and microbial genera, showed that Arthrobacter has a positive association with geochemical factors TC, IC, NO3, Cl-, HCO3, Na and a negative correlation with TOC and pH. Pseudomonas showed the opposite trend with these geochemical factors i.e., positive association with TOC and pH and a negative correlation with TC, IC, NO3, Cl-, HCO3, Na (Figure 4). This indicates that the geochemical factor plays a significant role in shaping microbial patterns in this ecosystem.
The interrelation among the samples with respect to their geochemical properties and bacterial community composition were analysed by statistical analyses. PCA performed on selected water geochemistry parameters revealed that samples LBSTc1, LBSTc2 and LBWDc4 formed a cluster, whereas samples KSSMc1, KSSTc2, KSSTc3, KSSTc4, LBWDc1 and LBWDc3 were related to these samples (Figure 5a). Based on abundance of bacterial genera, a biplot PCA was performed. Axes 1 and 2 of the resulting bi-plot gave 39.7% and 23.8% of the total variability. It was also observed that sample LBSTc1, KSSTc4, LBSTc2, LBWDc4 and LBWDc1 grouped together as Staphylococcus and Paenarthrobacter whereas KSSMc1, KSSTc7, KSSTc8, KSSTc2 and LBWDc2 formed another separate cluster including Pseudomonas, Flavobacterium (Figure 5b).