In India, the geothermal exploration began in early 1973 by Geological Survey of India and they reported more than 350 hot springs having temperature range varying above 40°C-100°C throughout the entire sub-continent region. Based on the tectonic movements, the hot springs of India were categorized into orogenic and non-orogenic [15]. Sikkim naturally hosts many hot springs. It is a major tourist attractive state of India where nature is in its juvenile form and a refreshing season greets its visitors. Previous studies through culture independent studies was done on the water samples from some hot springs of Sikkim located at Polok, Borong, Reshi and Yumthang, showed bacterial diversity at both the phylum and genus levels. Those hot spring water samples, were abundant in Proteobacteria (Polok-47%; Borong-63%; Reshi-76%) and Yumthang hot spring was predominant with Actinomycetes (98%). The most abundant genera in the hot spring water of Sikkim were Acidovorax, Acinetobacter, Exiguobacterium, Flavobacterium, Ignavibacterium, Paenisporosarcina, Paracoccus, Pseudomonas, Rhodococcus, Serratia, Sulfuritalea, Thermodesulfovibrio, Thermus, and Thiobacillus [16–19].
The high throughput sequencing assembly (Table 2), obtained gave 234612 reads for NYS (October), 209837 reads for NYS (December), 219384 reads for OYS (October), 238388 reads for OYS (December), 231568 reads for TAR (October), and 254002 reads for TAR (December). A total of 3803 OTUs were obtained from the six soil samples, with the minimum length of an OTU being 291bp and maximum being 492bp. The mean OTU length obtained was 450.3bp. Statistical analysis based on PERMANOVA revealed significant differences in OTUs composition between the six locations (F-value = 3.534, r2 = 0.469, p<0.1).
Table 2
Metagenomics assembly data and statistics
Sample-ID | Input reads | Filtered | Percentage of input passed filter | Denoised | Merged | Percentage of input merged | Non-chimeric | Percentage of input non-chimeric |
NYS-MUD_OCT | 234612 | 128171 | 54.63 | 103589 | 77953 | 33.23 | 17301 | 7.37 |
NYS-MUD_DEC | 209837 | 64907 | 30.93 | 48754 | 34036 | 16.22 | 9833 | 4.69 |
OYS-MUD_OCT | 219384 | 114717 | 52.29 | 95282 | 70813 | 32.28 | 15801 | 7.2 |
OYS-MUD_DEC | 238388 | 76214 | 31.97 | 55434 | 36351 | 15.25 | 12592 | 5.28 |
TAR-MUD_OCT | 231568 | 124428 | 53.73 | 99576 | 75310 | 32.52 | 19871 | 8.58 |
TAR-MUD_DEC | 254002 | 84522 | 33.28 | 65339 | 44825 | 17.65 | 18408 | 7.25 |
Venn diagram analysis was done to understand the distribution of shared OTUs at phylum and genus level of the hot spring soil bacterial diversity (Figure 5). Less proportions of OTU were shared between the six hot springs (17 at phylum level and 8 at genus level). The distribution of shared OTUs across the sediments revealed less overlap among each other. Thus, it showed higher variation and diversity among the samples. Box-plot analysis for comparing the alpha diversity indices and Chao 1 values, for the month of October vs. December, among all the hot spring soil sediments, higher species variedness and species richness was obtained in the month of December both at phylum and genus level (Supplementary Figure SF1). Alpha diversity denotes the species variedness and Chao value depicts the species richness among the environmental samples. As, the monsoon season is from the month of June to September, at the Sikkim Himalayas, hence during the first month of the post-monsoon season i.e. in October, there is immense diversity of the species in the hot spring soil ecosystem. This might be due to the fact that during rain in these high altitudes, frequent geomorphological changes occurs that enhances the bacterial diversity. Also, with heavy rainfall, the aquifers discharge is substantially high and hence more enrichment of the bacterial ecology occurs. With the recede in rainfall, during the later stages of post-monsoon, i.e., from November onwards these high altitudes starts experiencing snowfall and winter onsets. Thus, with less groundwater aquifer discharge, and also drastic change in the atmospheric conditions, the bacterial diversity at the month of December changes, which is evident from the box-plot.
Hot spring soil ecology is usually governed by complex uncultured bacteriomes. The hot spring soil ecology of the Sikkim Himalayas, had higher percentages of Gram negative bacterial phylum such as Proteobacteria and Bacteroidetes as compared to that of Gram positive bacterial phylum of Firmicutes and Actinobacteria. Interestingly, in the hot spring water samples also similar observations were reported [16, 17, 19]. The resident signature soil flora such as Planctomycetes and Chlorobi, which are commonly found in sulfur rich hot springs were also found here. Photosynthetic flora of Chlorobi was also prevalent in these hot spring soil samples. Temperature plays a very crucial role in the diversity [20]. During the month of December, there were drastic changes in the abundance percentage for majority of the phylum and genera as compared to that from the month of October. At phylum level, the abundance of majority of the phyla such as Proteobacteria, Thermi, Bacteroidetes, Acidobacteria, Planctomycetes, Actinobacteria, Candidate Phylum OP8, Firmicutes and Chlorobi, decreased during the month of December except Chloroflexi (Supplementary Figure SF2) and at genus level, except some unclassified Bacteroidales all the other major genera had higher abundance percentage increase in December (Supplementary Figure SF3). In the various other hot spring soil sediments also, similar observation was found. The hot spring soil of Atri, Bor Khleung, NYS, and Eritrea all had similar range of temperature varying from 50°C to 70°C. The Atri hot spring soil of Orissa and Solfatara Crater, Italy as studied by Sahoo et al. (2015) and Crognale et al. (2018) [21, 22] had the highest abundance of Chloroflexi (52%; 26%) and they lied within the same temperature ranging from 45°C to 65°C. Globally, the hot spring soils are rich in Proteobacteria which seems a common characteristic feature in soil ecology. Bacteriodetes was also relevantly abundant in Taptapani, Bor Khleung, NYS, OYS and TAR. Our findings on Planctomycetes and Verrucomicrobia abundance (5%-8%) was similar to that in Eritrea and Bor Khleung samples. Jakrem and Tapovan was the only hot spring soil which was highly robust with Thermi and Firmicutes [3, 18]. Global comparisons with the various resident soil phylum, the studied hot spring soils of Sikkim had lower abundance percentage of Chloroflexi, Nitrospirae, Proteobacteria and Firmicutes and it was rich in Thermi, Actinobacteria Planctomycetes and Verrucomicrobia.
The hot springs of North-east India i.e. from Sikkim (NYS, OYS and TAR) and Meghalaya (Jakrem) was highly abundant in Thiobacillus, Chloroflexus and Sulfuritalea whereas Eritrea had completely different genera in higher percentages such as Proteiniphilium and Proteiniclasticum [23] which were not reported from Indian Himalaya geothermal regions. Tapovan of Uttarakhand had lower abundance of Flavobacterium, Meiothermus and Acinetobacter [3] which were in higher percentages at Sikkim hot spring soil. At Solfatara Crater, it was predominant with Acidithiobacillus, Sulfobacillus and Leptospirillum [22].
The variation of the genera and phylum depends on various abiotic parameters such as pH, temperature, dissolved oxygen and other physic-chemical components present in the hot springs. Taking them as unity factor, EdgeR and DESEQ2 was performed for calculating the differential abundance analysis. Many low abundance classes or ranks, get omitted during computation. Thus, these statistical tools were used to eliminate this discrimination. Thiobacillus, Planctomyces and Arthronema and phylum Cyanobacteria, Acidobacteria, and Armatimonadetes had the highest variation/fluctuations in their relative abundance percentage in NYS, OYS and TAR, throughout both the months (Figure 6). This may be due to the change in temperature [10, 20, 24] and as well as the variability of ground aquifer discharge with the onset of winter during the post-monsoon season. Meiothermus, Sulfuritalea, Desulfobulbus and Azosprilum, and phylum Thermi, Firmicutes and Bacteroidetes could easily withstand these climatic variations and did not had much effect on their relative abundance percentage. This might be due to their physiological and cellular adaptation to the geothermal environment.
Correlation matrix represented through heat map analysis (Figure 7) was done to compare the unculturable bacterial diversity present in soil among the nine different hot springs of the world – Atri (55°C-58°C) and Taptani (45°C-50°C) (Orissa, India), Eritrea (50°C-80°C), Solfatara Crater (40°C-70°C, Pozzuoli, Italy), Bor Khlueng (50°C-58°C, Thailand), Jakrem (45°C-48°C, Meghalaya, India), New Yume Samdung (NYS) (57°C-61°C), Old Yume Samdung (OYS) (45°C-57°C) and Tarum (TAR) (44°C-49°C) (Sikkim, India). The phylum Proteobacteria, Planctomycetes, Bacteroidetes, Chloroflexi, Actinobacteria and Verrucomicrobia were positively correlated and had similar abundance percentages in these hot spring soil sediments as reported by various researchers [18, 19, 21–23, 25]. These phyla are commonly associated with the soil flora and are an important contributors of bacterial diversity in the hot springs worldwide. The hot spring soil sediments of Manikarnan (Himachal Pradesh, India), as reported by Mahato et al. (2019), [26] was abundant in Acidothermus, Alishewanella, Arthrobacter, Bacillus, Bifidobacterium, Brevundimonas, Burkholderia, Chloroflexus, Frankia, Meiothermus, Nocardia, Rhodothermus, Thermobaculum, and Thermosynechococcus. And in the studied hot springs of Sikkim, we found majority of the genera present were Thiobacillus, Roseiflexus, Meiothermus, Planctomyces etc. In other hot springs also, there were various genera but mostly belonged to Proteobacteria. Another important aspect was the discovery of many unclassified genera, which suggests that novel flora might be habituating these hot springs.