Background

Geologists have spotted and studied many thermal springs in the various regions of Indian subcontinent [1,2,3]. However, their microbial diversity has not been fully explored by employing modern molecular phylogenetic techniques. The present study reveals information on the bacterial community structure of Yumthang hot water spring, Sikkim, India. Illumina platform was used to sequence V3 hyper-variable region of 16S rDNA from microbial mat metagenome to profile the microbial community of this Northeastern hot spring of India.

The thermal springs of Sikkim are scattered in the Himalayan geothermal province. There are numerous natural hot springs at Sikkim; located at various locations like Polok, Reshi, Borong, Takrum, Yume Samdong, Yumthang, Zee, Shagyong Phedok and Tholug Kang of Sikkim [4]. Hot water springs are sign of geological activity and represent extreme environment. The Yumthang hot spring (27° 47′ 30″ N 88° 42′ E) is one of the less explored hot spring in Sikkim, India (Fig. 1). This hot spring is located at the Lachung river bed resulting in mixing of river water as soon as the hot water comes to the surface. This causes difficulty in measuring the hot water temperature at the site of emergence. Microbial mat along with water and sediment samples were collected in March 2014 using a hand trowel and pooled into sterile tubes, frozen in dry ice and transported to the laboratory for further analysis. Prior to sampling, the temperature and pH was measured at the precise sampling locations and recorded as 39°–41 °C and 8–8.5 respectively. The lithology of Yumthang, Sikkim is mostly composed of high grade gneisses Darjeeling gneiss and Kanchendzonga gneiss. The gneisses dominantly comprises of quartz, feldspar and biotite with minor amounts of other minerals.

Fig. 1
figure 1

Location of Yumthang hot spring, Sikkim, India (Indo-Tibetan plateu)

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Methods

Metagenomic DNA extraction

In the present investigation, the total community DNA was isolated from the microbial mat samples of the spring using FastDNA spin kit (MP Biomedicals, LLC, USA) according to the manufacturer’s protocol with some modifications to increase the yield and purity of the extracted DNA sample. The modifications included the addition of 300 μl of PPS (protein precipitation solution) to remove the protein contamination and the binding matrix pellet was re-suspended in 25 μl of DES (DNase/ Pyrogen-Free Water) to avoid over-dilution of the purified DNA. Final DNA concentration was quantified by microplate reader (BMG Labtech, Jena, Germany).

Illumina sequencing

The V3 hypervariable region of the 16S rRNA gene was amplified using 341F/ 518R primer combination 5’CCTACGGGAGGCAGCAG 3′ and 5’ATTACCGCGGCTGCTGG 3′ [5]. PCR Master Mix will contain 2 μL each primers, 0.5 μL of 40 mM dNTP (NEB, USA), 5 μL of 5X Phusion HF reaction buffer (NEB, USA), 0.2 μL of 2 U/μL F-540Special Phusion HS DNA Polymerase (NEB, USA), 5 ng input DNA and water to make up the total volume to 25 μL. Cycling conditions for the PCR reaction were 98 °C for 30 s, followed by 30 cycles of 98 °C for 10 s and 72 °C for 30 s, with a 5 s elongation step at 72 °C followed by 4 °C hold. Amplicon was excised and purified by QIA quick Gel Extraction kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s manual. Purified amplicon was paired-end sequenced (2 X 151 base pairs) on an Illumina Mi-Seq platform at Scigenome India Pvt. Ltd., Cochin, India.

Phylogenetic analysis

QIIME data analysis package was used for 16S rRNA data analysis [6]. Quality check on raw sequences was performed as per base quality score distributions, average base content per read and GC distribution in the reads. Singletons, the unique OTU that did not cluster with other sequences, were removed as it might be a consequence of sequencing errors and can result in spurious OTUs. Chimeras were also removed using UCHIME, pre- processed consensus V3 sequences were grouped into operational taxonomic units (OTUs) using the clustering program UCLUST at a similarity threshold of 0.97 [7, 8]. All the pre-processed reads were used to identify the OTUs using QIIME program for constructing a representative sequence for each OTU. The representative sequence was finally aligned against Greengenes core set of sequences using PyNAST program [9]. Representative sequence for each OTU was classified using RDP classifier and Greengenes OTUs database and the sequences those not classified were categorized as unknown.

Results and discussion

Phylum abundance results showed Proteobacteria to be abundant in the amplicon library. In the sample studied, 1, 999 OTUs represented 17 distinct phyla dominated by Proteobacteria (54.33%), Actinobacteria (32.19%), Firmicutes (6.03%), Bacteroidetes (2.87%) and unclassified bacteria (2.91%) respectively based on the total no. of reads (Table 1, Fig. 2a). Out of the total of 9, 95,955 pre-processed reads 49.80% are from the class Betaproteobacteria (Fig. 2b). The order Rhodocyclales of Betaproteobacteria constitutes 40.81% of the sequence reads followed by 40.81% of the family Rhodocyclaceae. Members under this family were mainly aerobic or denitrifying rod-shaped bacteria with diverse metabolic activities and survive in oligotrophic conditions such as aquatic habitats using photoautotrophic carbon fixation process [10, 11].

Table 1 Top ten OTU’s based on total read count number in the hot spring sample
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Fig. 2
figure 2

Relative abundance of (a) Phyla. b Class

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The phylum to genus level bacterial diversity identified in the high throughput 16S rRNA libraries from YM1 (Yumthang) hot spring is presented in Fig. 3. Proteobacteria constituted 671 OTUs i.e. 33.56% of total OTUs and 5, 41,190 reads which are 54.33% of the total reads. There are studies supporting and substantiating findings of the present study that bacterial communities in the hot springs located on the high altitude are dominated by Proteobacteria [12]. Similar observations have been made by [13, 14] that geographic locations play a significant role in bacterial community structure. Proteobacteria has also been reported from many studies based on the 16SrRNA analysis of hot springs with moderately high and very high temperatures (44–110 °C) at various geographical locations, including India [15,16,17]. Since, Proteobacteria have been found in other hot spring studies including Indian hot springs, and was also one of the abundant taxa in this study, it appears to be indigenous to this region.

Fig. 3
figure 3

Microbial community composition of Yumthang hot spring, Sikkim, India by 16S amplicon library sequencing

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The other dominant OTUs within Betaproteobacteria were OTU 395, 2102 and 501 classified under the order Hydrogenophilales, Thiobacterales and Burkholderiales respectively (data not shown). Two represented genus under the order Hydrogenophilales were Hydrogenophilus and Thiobacillus. Hydrogenophilus sp. are thermophilic, growing around 50 °C and obtaining their energy from oxidizing hydrogen. However they were previously isolated from an ice layer covering Lake Vostok in Antarctica which indicates the possibility of a geothermal system exists beneath the cold water body. The other genus Thiobacillus can oxidise the sulfur to sulfuric acid and widely used as a pesticide against pest potato scabs [18]. Thiobacillus sp., belonging to phylum Proteobacteria observed as abundant, has been reported from Yumthang in an earlier study [19]. Three thousand seventy two V3 16S rDNA reads from the amplicon library share more than 97% identity with members of sulfate reducing Desulfomicrobium sp. isolated from low temperature anaerobic enrichment culture from oil reservoir production water, China. This study also identified few Roseococcus and Alteromonas reads those are poorly described from other thermal environments. Alteromonas is reported to be a candidate genus for exopolysaccharide production [20, 21]. Thiovirga sulfuroxydans gen. Nov., sp. nov., a chemolithoautotrophic sulfur-oxidizing bacterium isolated from a microaerobic waste-water biofilm [22]. This study also identified few Thiovirga OTUs in amplicon library. It is the first description worldwide in association with hot springs. The genus Psychrobacter, a member of the class Gammaproteobacteria, is predominantly isolated from cold and/or saline environments, such as Arctic permafrost, Antarctic ice pack, estuaries, and marine fish, including Korean fermented seafood [23,24,25,26,27,28,29]. The occurrence of Psychrobacter OTUs in the amplicon library is of interest as there are no reports from hot springs all over the World.

Actinobacteria constitutes 351 OTUs i.e. 17.55% of total OTUs and 3, 20, 616 reads (32.19%) of total reads whereas 536 OTUs i.e. 26.81% of total OTUs belong to the unknown phylum. Actinobacteria edged over other microbes as a prolific producer of antibiotics and other biopharmaceuticals [30]. Thermophilic actinobacteria are biotechnologically important producers of several enzymes such as DNA polymerases, pullulanases, amylases, xylanases, lipases and proteases [31]. However, little is known about the distribution and biogeography of Actinobacteria in hot springs. The present findings revealed the presence of large number of sequence reads of Rhodococcus from bacterial phylum Actinobacteria which may embody many novel species within this industrially important genus [32, 33]. The representatives of this genus have been reported from varied environments viz. soil, sewage treatment plants, polluted and unpolluted water bodies etc. [32], Rhodococcus has been also reported from alkaline hot springs of the world [34]. Strains of Rhodococcus are well known microbes carrying out biologically relevant reactions such as desulfurization of fossil fuels, degradation of pollutants, biosurfactants and bioflocculants etc. [33].

Conclusions

The Yumthang hot spring of Indo-Tibetan plateau is home to many possibly unknown and novel microbes as indicated by the presence of 26.81% unknown OTUs out of 1, 999 OTUs.