Indian Journal of Microbiology

, Volume 58, Issue 2, pp 234–238 | Cite as

Psychrotolerant Sphingobacterium kitahiroshimense LT-2 Isolated from Dhundi Glacier, Himachal Pradesh: Origin Prediction and Future Application

  • Shivika Sharma
  • Subhankar Chatterjee
Short Communication


A psychrotolerant bacterium, isolated from Dhundi Glacier, Himachal Pradesh (India) was identified as Sphingobacterium kitahiroshimense LT-2 on the basis of biochemical, molecular and phylogenetic analysis. Sphingobacterium kitahiroshimense was first reported from Japan and was isolated from the city of Kitahiroshima, Hokkaido, Japan. In this report we have discussed about the origin of our strain and predicted that air masses and dust associated microbial cells transportation phenomena may be applicable for the origin of this species in this region. Enzymes and secondary metabolites secreted by the genus Sphingobacterium have enormous potentiality regarding their biotechnological application. Preliminary study of our strain based on metabolic profiling through HPLC showed many new metabolites were secreted by the bacterium when grown in presence of different sugar medium at 28 °C. As far as our knowledge this is the first report about Sphingobacterium species isolated from this region. This preliminary finding will help to draw an idea about the bacterial population in this Himalayan Glaciers (in HP) as well as biotechnological application of this strain can be explored further.


Psychrophiles Psychrotolerant bacteria Sphingobacterium Secondary metabolites Cold active enzymes Dust associated microbial cells transportation 

The organisms thriving in extreme ecosystems have prosperous resource of various chemical and novel natural products with interesting bioactivities. Among them, psychrophiles which can be found in Arctic, Antarctic and Himalaya are “cold-loving” microorganisms living at sub-zero temperatures undergo certain adaptations by using variety of mechanisms to sustain their cell cycle [1]. These include the production of cold-shock and anti freeze proteins, synthesis of cold tolerant enzymes for the regulation of metabolic pathways, alterations in membrane composition to increase membrane flexibility, ample translation and proper protein folding under cold conditions. These adaptations are often accompanied by certain unique modifications to both gene regulation and metabolic pathways, increasing the possibility of finding unique functional metabolites of pharmaceutical importance [2]. The micro organisms dwelling in the cold habitat are also found to be the rich source of secondary metabolites which mainly showed antibacterial and free radical properties. The psychrophilic microorganisms are of two types viz. Stenopyschrophiles (true psychrophiles) that grow at definite low range of temperature (0–15 °C) and cannot grow at higher temperature range and Eupsychrophiles (psychrotolerant or psychrotrophs) which can grow at low temperature conditions and can tolerate up to mesophilic temperature range (20–25 °C) [3]. Very little study has been done till date on the bacterial diversity of the Himalayan glaciers whereas this region is considered as a place of one of the most underutilized resources. The psychrophilic and psychrotolerant cultivable bacterial strains are dominant in the Himalayan ecosystem. Approximately 45% of cultivable bacterial strains from this region belonged to the Proteobacteria group in which γ-Proteobacteria and β-Proteobacteria constitutes up to 39 and 31% respectively. The second most predominant class is Firmicutes (32%) followed by Actinobacteria (16%) and Bacteroidetes (6%). The most abundant strains of microorganisms in Himalayan range belonged to the genus Bacillus (30%) followed by Pseudomonas (24%) and Arthrobacter (12%) [4].

In the present study, a psychrotolerant bacterium identified as Sphingobacterium kitahiroshimense LT-2 (accession number MG027863), was isolated from Dhundi Glacier (nearby soil) which is geographically located between 32°21′53.47″N and 77°03′52.77″E in Beas basin in Manali on North of Kullu in Himachal Pradesh (India) situated at an elevation of 4038 m on the eastern slope of Rohtang pass in the western Himalayas. The strain LT-2 was isolated by using the dilution-plating technique on Nutrient Agar Plates incubated at 15 °C and characterized on the basis of biochemical analysis and molecular characterization by Sequencing of 16S rRNA gene. Phylogenetic analysis and multiple sequence alignment of data were done using the software packages MEGA 5 [5] using CLUSTALW [6] respectively. Evolutionary distances and clustering were based on the neighbour joining [7] and maximum-likelihood methods [8]. On the basis of neighbour-joining algorithm the strain LT-2 showed 99.64% similarity with Sphingobacterium kitahiroshimense (Fig. 1). The biochemical analysis showed that it is gram negative, rod shaped, non spore forming, catalase and oxidase positive aerobic bacterium. The smooth, circular and creamy white colonies developed on nutrient agar plates after 3–4 days. This bacterium is psychrotolerant and can withstand temperature range in between 4 and 37 °C.
Fig. 1

Phylogenetic tree showing the relationship between Sphingobacterium kitahiroshimense LT-2 (Query_LT2) and other related strains, 16S rRNA gene sequences with reference sequences obtained through BLAST analysis. Phylogenetic analysis and multiple sequence alignment of data were done using the software packages MEGA 5 using CLUSTALW respectively. Evolutionary distances and clustering were based on the neighbour joining and maximum-likelihood methods. One hundred bootstrap replicates were performed. Bootstrap values are indicated on the branches

The family Sphingobacteriaceae comprises of certain psychrotolerant microorganisms such as members of genus Sphingobacterium in which cells comprises of high concentration of sphingophospholipids which helps the microorganisms to adapt in cold conditions [9]. The first discovery of this genus Sphingobacterium was proposed by Yabuuchi et al. [10]. The family members of this genus have been isolated from different sources and up to 32 species of Sphingobacterium have been identified [11]. The main fatty acid composition of Sphingobacterium comprises of iso-C15:0, iso-C15:0 2-OH, C16:1ω7c and C17:0 3-OH and biochemical characteristics includes catalase and oxidase positive and negative for gelatinase and indole production [9].

Only one published data on Sphingobacterium kitahiroshimense was reported which was isolated from the city of Kitahiroshima, Hokkaido, Japan [12]. It will be an interesting piece of work with novelty to know the phenomenon of transfer (if any) of this bacterium strain from Japan to Dhundi glacier and vice versa. The microbial communities in troposphere of Himalayan range in Himachal Pradesh are feebly characterized due to the harsh conditions and various difficulties in collecting biomass. A very little study has been done on spatial and temporal variability of these tropospheric microbial groups, mechanism of adaptation of these psychrophiles to low temperature conditions and viability phenomenon of these microbes. The effects of atmospheric processes such as tropical cyclones, hurricanes on microbial groups that can precipitate or aerosolize microbial cells and can cause alteration in composition and functions of microorganisms [13]. In Himalayan range of Himachal Pradesh, there is prevalence of strong winds with higher rate of precipitation during the start of winters. The range of dust concentrations at 5000 m of glacier is in between 8 and 100 µg m−3 up to 800 µg m−3 with an annual dust fluctuation spanning from 770 to 1030 mg m−2 [14]. The microbial groups detected at the middle troposphere altitude (6000 m) get entrapped with the deposition of dust particles constituting dust traps. The strong wind currents, cyclones and hurricanes in the Himalayan range may help in the transfer of dust associated microbes from long distance. Previous studies also supported the fact that air masses and dust associated microbial cells transported with them and generally occurred from lower altitudes and were brought up during the hurricanes/cyclones [13]. Based on these theories, it can be said that transportation of viable microbes can be possible across the continents and same phenomena may also be applicable for the present case. Figure 2 summarises the mechanism of transportation of the microbes across the different continent [15].
Fig. 2

Mechanism of transportation of the microbes across the different continent

The secondary metabolites secreted by psychrophiles/psychrotolerant bacteria have diverse biological activities ranging from cytotoxic, antioxidant, antiviral and antibacterial [16]. It was reported that, Sphinobacterium sp. have a set of enzymes to regulate the metabolic pathways in extreme conditions such as lipases, isocitrate dehydrogenase, cellulases, superoxide dismutase, oxidases, pectinase and esterase etc. [17]. Cold active enzymes can withstand extreme range of temperature, pressure and these cold tolerant enzymes have a great importance at commercial levels as these acts as catalyst in various biotechnological industries. The cold tolerant enzymes are used in the field of food industry (pectinase, α-galactosidase), lignin degradation (superoxide dismutase), detergent formulation industries (lipases, cellulases), bioremediation (oxidases), biotransformations (methylases and aminotransferases) and in pharmaceutical industries (amylases, Chitinase A) [17] (Fig. 3). The bacterium identified in the present study may contribute to the production of novel antibiotics or other biologically active secondary metabolites which can play a potential role in future healthcare system or in biotech industry. A preliminary study has been done based on the metabolic profiling of the secondary metabolites secreted by this bacterium in various sugars medium at 28 °C. HPLC profiling of these metabolites indicated that few metabolites were exclusively secreted when bacterial culture was grown in these mediums at 28 °C (Fig S1). The characterization of these metabolites and biological activity study are in progress.
Fig. 3

Various biotechnological applications of the enzymes of Sphingobacterium sp.

As far as our knowledge this is the first report about Sphingobacterium species isolated from this region. The findings from this preliminary study are important in a sense that it can give valuable idea about the bacterial population in Himalayan Glaciers (HP) as well as the bacterium isolated from this study can be utilised further for biotechnological application.



Financial support for this work was provided by SERB-DST, Govt. of India (PDF/2016/000818). We are thankful to Ms. Riju Parmar and Dr. Anurag Linda for their help in strain collection. We gratefully acknowledged IMTECH, Chandigarh for their support regarding the identification of the strain.

Compliance with Ethical Standards

Conflict of interest

Authors declare no conflict of interest.

Supplementary material

12088_2018_712_MOESM1_ESM.tif (166 kb)
Supplementary Fig. 1 (S1): HPLC profile of secreted secondary metabolites of Sphingobacterium kitahiroshimense LT-2 grown in presence of Glucose at 28 °C. Peaks exclusively appeared in the Glucose medium have also been shown. The secondary metabolite profiling was carried out on UltiMate 3000 UHPLC+ system, Thermo Scientific; The HPLC system was equipped with HYPERSIL GOLD C18 reverse phase column (250 mm × 4.6 mm; particle size: 5µ, Thermo Scientific). The analyte was eluted in multistep gradient modes using mobile phase of methanol (A) and water (B): 0–2 min 0–60%A; 2–30 min from 60% A to 90%A; 30–40 min 90%A; 40–45 min 90% A to 60% A; 45–50 min 60% A to 0% A at a flow rate of 0.8 ml/min. This was followed by washing and re-equilibration steps. A quality control sample was injected at regular interval to monitor the stability of the method. The analyte was detected using Diode Array Detector at 270 nm wavelengths. The data so generated was analyzed by using Chromeleon software. (TIFF 165 kb)


  1. 1.
    Tian Y, Li YL, Zhao FC (2017) Secondary metabolites from polar organisms. Mar Drugs 15:28. CrossRefPubMedCentralGoogle Scholar
  2. 2.
    Amico SD, Collins T, Marx JC, Feller G, Gerday C (2006) Psychrophilic microorganisms: challenges for life. EMBO Rep 4:385–389. CrossRefGoogle Scholar
  3. 3.
    Maayer PD, Anderson D, Cary C, Cowan DA (2014) Some like it cold: understanding the survival strategies of psychrophiles. EMBO Rep 15:508–517. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Gangwar P, Alam SI, Bansod S, Singh L (2005) Bacterial diversity of soil samples from the western Himalayas, India. Can J Microbiol 55:564–577. CrossRefGoogle Scholar
  5. 5.
    Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4888. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. PubMedGoogle Scholar
  8. 8.
    Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376. CrossRefPubMedGoogle Scholar
  9. 9.
    Liu R, Liu H, Zhang CX, Yang SY, Liu XH, Zhang KY, Lai R (2008) Sphingobacterium siyangense sp. nov., isolated from farm soil. Int J Syst Evol Microbiol 58:1458–1462. CrossRefPubMedGoogle Scholar
  10. 10.
    Yabuuchi E, Kaneko T, Yano I, Moss CW, Miyoshi N (1983) Sphingobacterium gen. nov., Sphingobacterium spiritivorum comb. nov., Sphingobacterium multivorum comb. nov., Sphingobacterium mizutae sp. nov., and Flavobacterium indologenes sp. nov.: glucose non-fermenting Gram-negative rods in CDC Groups IIK-2 and IIb. Int J Syst Bacteriol 33:580–598. CrossRefGoogle Scholar
  11. 11.
    Long X, Liu B, Zhang S, Zhang Y, Zeng Z, Tian Y (2016) Sphingobacterium griseoflavum sp. nov., isolated from the insect Teleogryllus occipitalis living in deserted cropland. Int J Syst Evol Microbiol 66:1956–1961. CrossRefPubMedGoogle Scholar
  12. 12.
    Hidetoshi M, Hiromichi K, Takahumi O, Atsushi S, Kazuyoshi K, Isao Y (2008) Sphingobacterium kitahiroshimense sp. nov., isolated from soil. Int J Syst Evol Microbiol 58:1576–1579. CrossRefGoogle Scholar
  13. 13.
    DeLeon-Rodriguez N, Lathem TL, Rodriguez-R LM, Barazesh JM, Anderson BE, Beyersdorf AJ, Ziemba LD, Bergin M, Nenes A, Konstantinidis KT (2013) Microbiome of the upper troposphere: species composition and prevalence, effects of tropical storms, and atmospheric implications. Proc Natl Acad Sci USA 110:2575–2580. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Stres B, Sul WJ, Murovec B, Tiedje JM (2013) Recently deglaciated high-altitude soils of the Himalaya: diverse environments, heterogenous bacterial communities and long-range dust inputs from the upper troposphere. PLoS ONE 8:e76440. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Boetius A, Anesio AM, Deming JW, Mikucki JA, Rapp JZ (2015) Microbial ecology of the cryosphere: sea ice and glacial habitats. Nat Rev Microbiol 13:677–690. CrossRefPubMedGoogle Scholar
  16. 16.
    Berdy J (2005) Bioactive microbial metabolites. J Antibiot 58:1–26. CrossRefPubMedGoogle Scholar
  17. 17.
    Choi HA, Lee SS (2012) Sphingobacterium kyonggiense sp. nov., isolated from chloroethene-contaminated soil, and emended descriptions of Sphingobacterium daejeonense and Sphingobacterium mizutaii. Int J Syst Evol Microbiol 62:2559–2564. CrossRefPubMedGoogle Scholar

Copyright information

© Association of Microbiologists of India 2018

Authors and Affiliations

  1. 1.Bioremediation and Metabolomics Research Group, Department of Chemistry and Chemical Sciences, School of Physical and Material SciencesCentral University of Himachal PradeshKangraIndia
  2. 2.Department of Environmental Sciences, School of Earth and Environmental SciencesCentral University of Himachal PradeshKangraIndia

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