Bacterial diversity analysis of Yumthang hot spring, North Sikkim, India by Illumina sequencing
© The Author(s) 2017
Received: 25 October 2016
Accepted: 6 August 2017
Published: 20 August 2017
Hot springs harbor rich bacterial diversity that could be the source of commercially important enzymes, antibiotics and many more products. Most of the hot springs present in Northeast of India are unexplored and their microbial diversity analysis could be of great interest to facilitate various industrial, agricultural and medicinal applications. The present study is an attempt to analyze the comprehensive bacterial diversity of Yumthang hot spring, Sikkim located at an altitude of 11, 800 ft. with a close proximity of Tibet 27° 47′ 30″ N 88° 42′ E using culture independent approach i.e. 16S rRNA gene amplicon metagenomic sequencing.
The temperature and pH of the hot spring was recorded as 390–410 C and 8 respectively. Metagenome comprised of 1, 381,343 raw sequences with a sequence length of 151 bp and 55.62% G + C content. Metagenome sequence information is submitted at NCBI, SRA database under accession no. SRP057072. A total of 9, 95, 955 pre-processed reads were clustered into 1, 999 representative OTUs (operational taxonomical units) phylogenetically comprising of 17 bacterial phyla including unknown phylum indicating 99 families. Hot spring bacterial community is dominated by Proteobacteria (54.33%), Actinobacteria (32.19%), Firmicutes (6.03%), Bacteroidetes (2.87%) and unclassified bacteria (2.91%) respectively out of the total reads.
Several bacterial and archaeal sequences remained taxonomically unclassified, indicating potentially novel microorganisms in this hot spring ecosystem. Metagenomics of this habitat will facilitate identification of microorganisms possessing industrially relevant traits.
KeywordsBacterial diversity Illumina Sikkim Hot springs 16S rDNA
Geologists have spotted and studied many thermal springs in the various regions of Indian subcontinent [1–3]. 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.
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).
The V3 hypervariable region of the 16S rRNA gene was amplified using 341F/ 518R primer combination 5’CCTACGGGAGGCAGCAG 3′ and 5’ATTACCGCGGCTGCTGG 3′ . 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.
QIIME data analysis package was used for 16S rRNA data analysis . 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 . 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
Top ten OTU’s based on total read count number in the hot spring sample
OTU Table Id
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 . Thiobacillus sp., belonging to phylum Proteobacteria observed as abundant, has been reported from Yumthang in an earlier study . 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 . 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 [23–29]. 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 . Thermophilic actinobacteria are biotechnologically important producers of several enzymes such as DNA polymerases, pullulanases, amylases, xylanases, lipases and proteases . 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. , Rhodococcus has been also reported from alkaline hot springs of the world . 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. .
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.
The authors are grateful to the Head Department of Zoology, Kumaun University, Nainital, India for providing basic infrastructural facility and administrative assistance.
Financial support was provided by SERB, Govt. of India Vide Project SB/FT/LS-335/2012 for the design of the study, sample collection and analysis of data.
Availability of data and materials
Sequence data that support the findings of this study have been deposited in the Sequence Read Archive (SRA) service of the National Centre for Biotechnology Information (NCBI) database under the accession number SRP057072 https://www.ncbi.nlm.nih.gov/sra/?term=SRP057072.
AKA conceived organized and wrote the paper. AKA and SDM analyze the data; SSB, BRK and NSK critically analyzed the study and helped in drafting the article as well as edited the manuscript. AKA obtained funding for the original project idea. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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