Most advances achieved in microbial ecology over the last decade are aimed at enabling a fine description of complex microbial community composition and function in their particular environment [1]. It is well known that many important biogeochemical processes, such as carbon and nitrogen cycling, are carried out differentially throughout soil profiles by distinct microbial assemblages [2]. Phylogenetic and taxonomic characterization of these trends has been presented by Gittel et al [3]. Despite this extensive diversity and biogeochemical relevance, our understanding of the distribution of soil microbial communities and their associated processes has largely been restricted to surface soils with very few studies highlighting overall changes in microbial community composition with chronosequence [4].
Recently, microbial diversity propagating in soils of extreme cold regions has become an important area of multidisciplinary research which involves identification of phylogenetcially diverse species thriving under these ecological niches and the study of the biogeochemical events responsible for their survival [5]. Interestingly, these components are linked by a complex set of feedback pathways such that alteration in one component prompts changes in the other components. This implies that the distribution of microorganisms in the soil is relative to the differences in its physicochemical properties [6, 7].
Even with the nutrient-limited condition and extreme cold climate, soils at different depths are by no means barren or lifeless. A wide spectrum of microorganisms can thrive in these environments. In fact, many regions of Indian Himalayan states of Himachal Pradesh, Uttarakhand, Sikkim and Arunachal Pradesh have come up as hotspots of microbial diversity [5]. Environmental samples collected from glaciers, soil and hot-springs have been used to isolate microbes to screen their potential in vitro [8–10]. Such habitats host a diverse family of psychrophilic microorganisms that are evolved to survive by overcoming the negative impacts of their immediate external envrironment [11, 12]. For instance, Glaciimonas sp. PCH181 isolated from the glacial stream of Indian trans-Himalaya produces 25% polyhydroxyalkonoate of its dry cell mass. It is adapted to extreme low temperatures, limited nutrient conditions and utilizing alternate carbon sources [13].
Although a bulk of information is available about the microbial communities associated with the colder environments, several unique sites of the Himalayas are still untouched. Also, as opposed to microbial profiling data of surface soils, there is little information on the Himalayan microbial diversity across a vertical soil samples and how they link with physicochemical properties [14]. Analysis of microbial diversity and physicochemical properties might provide opportunities to expand our understanding about survival strategies of microorganisms present in this region. In this context, an understanding of microbial distribution in soil and measurement of physicochemical properties in the extreme cold conditions of the Himalayas would expediate the establishment of boundary conditions for microbial life on Earth. This would be instrumental in predicting changes in such ecosystems as a result of the increasing global temperatures [15]. Further, on account of geographic seperation and extreme climate, these regions could serve as model systems to compare and contrast the conditions present on other planetary bodies and understand if these conditions are favourable for persistence of life despite such unfavorable conditions [16]. This premise draws the corrollary of exploratory research in Himalayan region.
For studying microbial diversity in soil, several biochemical-based techniques (plate counts, carbon source utilization, fatty acid methyl ester analysis) and molecular-based techniques (denaturing gradient gel electrophoresis, guanine plus cytosine content, PCR and sequencing approaches) have been reported [17]. Sanger sequencing is a widely used method for identification of bacteria and fungi by targeted gene sequencing [18]. 16S rDNA sequences have facilitated the identification of bacteria, determination of phylogenetic relationships and discovery of novel bacterial species [19]. Similarly, for identification of fungi, internal transcribed spacer (ITS) gene sequencing has been used as the most clearly defined universal fungal barcode [20].
For study of soil physicochemical properties, analytical methods such as Atomic absorption spectrometry, Inductively coupled plasma mass spectrometry and Field emission scanning electron microscope-energy dispersive X-ray spectroscopy (FESEM-EDS) have been used previously [21–23]. These techniques provide information about the soil morphology and facilitate the measurement of its elemental concentration. FESEM-EDS is a non-destructive technique used to capture the microstructure image (FESEM) and to directly measure elemental composition using small amount (~ 500 mg) of soil (EDS) [24]. This technique provides highly sensitive qualitative and semi-quantitative results at a submicron scale [25].
In this study the focus lies on 1) the use of sanger sequencing of target gene for identification of culturable microorganims and 2) the use of FESEM-EDS for assessment of morphology and chemical components in samples across a 100cm vertical soil profile of Lam Pokhari Lake, Sikkim in Eastern Himalayas. The results from the analyses of the eleven soil samples have been presented in this paper.