Ann Microbiol (2015) 65:431–441 DOI 10.1007/s13213-014-0876-1 ORIGINAL ARTICLE Predominance of Bacillus sp. in soil samples of the southern regions of Western Ghats, India Gowdaman Vasudevan & Venkatachalam Siddarthan & Prabagaran Solai Ramatchandirane Received: 5 August 2013 /Accepted: 12 March 2014 /Published online: 17 April 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract The aim of this study was to determine the bacterial samples assessed by both cultivation-dependent and diversity in soils of the southern region of the Western Ghats, a cultivation-independent methods. ‘biodiversity hotspot’, and thereby futher our understanding of the microbial communities in this ecological niche. The Keywords Western Ghats . Diversity index . 16S rRNA . diversity and phylogeny of bacterial populations in soil sam- ARDRA . Metagenomic DNA . Bacillus species ples collected from various locations of the Tamil Nadu and Kerala regions of Western Ghats were compared using both cultivation-dependent and cultivation-independent methods. Introduction A total of 171 bacterial strains were isolated based on their morphological characteristics and their diversity indices cal- India is considered by UNESCO (2012) to have two of the culated. The distinctive amplified ribosomal DNA restriction ‘mega diversity hotspots’ in the world, namely, the Himalayas analysis (ARDRA) pattern of each isolate was determined, and the Western Ghats. The Western Ghats is a forested tract and representative isolates were then subjected to 16S rRNA of relatively smooth but very old mountain range that extends gene sequencing. On the basis of their sequence similarity, the from Central Maharashtra to the southern tip of Kerala. The isolates were distributed among three different genera belong- region supports a wide range of endemic taxa with nearly ing to Firmicutes (83.3 %), Proteobacteria (8.3 %) and high 1,500 species of flowering plants (38 %), 126 species of G+C Gram-positive bacteria (8.3 %). The highest and the amphibians (78 %), 157 species of reptiles (62 %), 508 species lowest values for the diversity indices were obtained for of birds (4 %) and 137 species of mammals (12 %) identified metagenomic DNA extracted from isolates BWGA and to date (Bawa et al. 2007). The diversity of flora and fauna of BVP, respectively; these were used for 16S rRNA gene library the Western Ghats has been reasonably well documented, but construction and analysis. Based on their phylogenetic analy- rather fewer studies have examined fungal diversity in the area sis, the predominant members of the habitat were found to (Raviraja 2005) and fewer yet have looked at the diversity of belong to the phylum Firmicutes (84.62 %). Firmicutes was bacteria. Soil bacteria are an essential component of the biotic the dominant bacterial phylum detected by both approaches, community in natural forests and are largely responsible for but the culture-independent approach detected a considerably ecosystem functioning due to their participation in most nu- higher number of uncultivable bacteria. In conclusion, in our trient transformations (Hackl et al. 2004). Although the main study of the bacterial diversity of this Western Ghats region, diversity of life has been proven to be microbial, the vast we fund that the genus Bacillus was predominant among the majority of soil bacteria still remain unidentified because only a minor percentage of naturally occurring microorganisms can be cultured (Pace 1997). Electronic supplementary material The online version of this article The diversity of microorganisms and the role they play ‘in (doi:10.1007/s13213-014-0876-1) contains supplementary material, ’ which is available to authorized users. situ remains largely unknown. For almost a century, culture- dependent methods used to study bacterial diversity have been : : * G. Vasudevan V. Siddarthan P. Solai Ramatchandirane ( ) continuously optimized to detect diverse microorganisms. Molecular Microbiology Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore 641046, Tamil Nadu, India However, only a small proportion of the bacteria present in e-mail: [email protected] extreme environments are readily cultivable, limiting further, 432 Ann Microbiol (2015) 65:431–441 more detailed study. In addition, culture-dependent methods been not many reports on bacterial diversity. Therefore, the are selective and biased based on the type of media used, the aim of our study was to estimate bacterial diversity and nutrients provided and the culture conditions. This has led in identify the predominant soil bacteria in the southern regions more recent decades to the development of culture- of the Western Ghats using both culture-dependent and ad- independent methods to study the enormous diversity of un- vanced culture-independent methods in order to obtain an cultivable organisms and emphasized the need for comple- overview of the bacterial diversity. Although the culture- mentary approaches for the analysis of soil bacterial diversity independent approach is considered to be the superior tech- (Vaz-Moreira et al. 2011). nology for studying bacterial diversity, culture-dependent The relatively recent application of molecular methods to methods are considered to potentially be of more practical investigate unculturable microbes from diverse environments importance (Burmølle et al. 2009). has altered commonly accepted views of microbial diversity. Research based on the extraction of total community DNA from environmental samples followed by PCR, cloning and sequencing of 16S rRNA genes has become conventional, Materials and methods often comprising one of the first steps in the study of the bacterial diversity of an environment of importance (Oline Site description and sampling et al. 2006). The recovery and analysis of 16S rRNA genes directly from environmental DNA provides a means for in- The present-day Western Ghats constitute a continuous vestigating microbial populations in any habitat, eliminating chain of small to medium-sized mountain ranges running the dependence on the isolation of pure cultures (Amann et al. between 8 and 21°N latitude and 73 and 77 °E longitude. 1995b; Ahmad et al. 2009). The southern part of the Western Ghats region encom- Only limited investigations have been carried out on bac- passes the states of Tamil Nadu and Kerala, where many terial diversity in various forest ecosystems of the Western reserve forests can be found. Soil samples from 12 differ- Ghats. The southern regions of the Western Ghats are mostly ent sampling sites throughout the southern regions of located in the states of Tamil Nadu and Kerala. These two Western Ghats (Fig. 1) were obtained by first removing states are characterized by their abundance of forested areas, the top surface soil and then collecting samples at a soil which are largely unexplored to the fullest. To date, there have depth of about 10 cm. A small amount of each sample was Fig. 1 Experimental area depicting Western Ghats and the locations of the sampling sites Ann Microbiol (2015) 65:431–441 433 used for serial dilution plating, and the remaining was Sigma-Aldrich, St. Louis, MO). The ARDRA profiles of stored at 4 °C until further use. different isolates were compared manually and the observed DNA polymorphisms scored as dominant markers and con- Isolation and morphological characterization of bacteria verted to a binary matrix. The data were used to derive similarity measures in terms of Jaccord’s coefficient in all Enumeration of soil bacteria was achieved by serial dilu- possible pairwise combinations. The similarity matrix was tion and plating on different types of solid media at various used for cluster analysis using the UPGMA method in the strengths, such as nutrient agar (NA), yeast starch agar and NTSys-PC 2.1 software package (Exeter Publishing, soil extract agar (SEA) [see Electronic Supplementary Setauket, NY). Material (ESM) Table 1] (Bianchi and Armand 1982). Following incubation at 37 °C for 24–72 h, the isolates Extraction and purification of metagenomic DNA were selected based on their cell morphology, colony mor- phology, Gram reaction, among others, and the results Soil DNA was extracted from 5 g of each soil sample with documented. 13 ml of extraction buffer (100 mM Tris–HCl pH 8; 100 mM EDTA pH 8, 100 mM sodium phosphate, 1.5 M NaCl, 1 % Diversity analysis CTAB) and incubated for 30 min at 37 °C before being centrifuged at 6,000 rpm for 5 min. The supernatant was A range of diversity indices (ESM Table 2) were used to collected and used for DNA isolation as described by Zhou characterize the bacterial community studies, including the et al. (1996). Metagenomic DNA consists of humic acids and ubiquitous Shannon index, the evenness index derived from other phenolic compounds that inhibit its downstream appli- the former and Simpson’s dominance index and its associated cations. Hence, purification is important for further process- equitability index (McCaig et al. 1999;ChoandKim2000; ing. Purification was preceded by treatment with a modified Dunbar et al. 2000). method of Ausubel et al. (1987). Genotypic characterization of isolates Construction of library and sequence analysis Genomic DNA was isolated from bacterial cultures grown in Purified PCR products were ligated with pTZ57R/T vector Luria Bertani (LB) broth (Hi Media, Mumbai, India) using (Fermentas) following the manufacturer’s instructions. The standard protocols (Sambrook et al. 1989). The 16S rRNA ligation reaction