Diversity and phylogenetic profiling of niche-specific Bacilli from extreme environments of India

Abstract

The diversity of culturable, aerobic and heterotrophic Bacillus and Bacillus-derived genera (BBDG) was investigated in various extreme environments (including thermal springs, cold deserts, mangroves, salt lakes, arid regions, salt pans and acidic soils) of India. Heat treatment followed by enrichment in different media led to a total of 893 bacterial isolates. Amplified ribosomal DNA restriction analysis (ARDRA) using three restriction enzymes AluI, MspI and HaeIII led to the clustering of these isolates into 12–74 groups for the different sites at 75 % similarity index, adding up to 559 groups. Phylogenetic analysis based on 16S rRNA gene sequencing led to the identification of 392 bacilli, grouped in two families, Bacillaceae (89.03 %) and Paenibacillaceae (10.97 %), and included 13 different genera with 75 distinct species. It was found that among the thirteen genera, nine (Bacillus, Halobacillus, Lysinibacillus, Oceanobacillus, Pontibacillus, Salinibacillus, Sediminibacillus, Thalassobacillus and Virgibacillus) belonged to Bacillaceae and four (Ammoniphilus, Aneurinibacillus, Brevibacillus and Paenibacillus) belonged to Paenibacillaceae. Novel isolates tolerant to low and high pH and temperature, salt and low moisture were identified. The major outcome of the present investigation was the identification of niche-specific species and also the ubiquitous presence of selected species of BBDG, which illustrate the diversity and pervasive nature of BBDG in extreme environments.

Introduction

Extreme environments represent unique ecosystems that harbor novel biodiversity. India is one among 12 mega-biodiversity countries and 25 hotspots of the richest and highly endangered eco-regions of the world (Myers et al. 2000). Microbial communities are found in most diverse conditions, including extremes of temperature, salinity, water deficiency and pH. In order to survive under such extreme conditions, these organisms, referred to as extremophiles, have developed adaptive features that permit them to grow optimally under one or more environmental extremes, while poly extremophiles grow optimally under multiple conditions (Rothschild and Mancinelli 2001). These extremophiles can grow optimally in some of the earth’s most hostile environments of temperature (−2 to 20 °C, psychrophiles; 60 to 115 °C, thermophiles), salinity (2–5 M NaCl, halophiles) and pH (< 4, acidophiles and > 9, alkaliphiles) (Van den Burg 2003). In an effort to understand the diversity and distribution of cultural Bacillus and Bacillus-derived genera (BBDG) in a diverse range of extreme environments, an investigation involving isolation, identification and characterization employing biochemical and molecular techniques was undertaken. The niches explored included hot springs (Manikaran, Chumathang, Bakreshwar, Balarampur and Vashisht) and cold deserts (Leh and Rohtang Pass), areas with high salinity (Sambhar Lake, Chilka Lake and Rann of Kutch), acidity (Andaman and Nicobar Islands and Kollam), water deficient/drought stress (Jaisalmer), as well as mangroves (Bhitarkanika and Sunderbans) in India.

Thermal springs represent unique ecological niches and harbor both mesophilic and thermophilic members, especially those belonging to the genus Bacillus and related genera (Cihan et al. 2012). Phylogenetic characterization of microflora has been undertaken for geothermal springs in different parts of the world (Derekova et al. 2008). In India, Bakreshwar, Balarampur, Chumathang, Manikaran and Vashisht hot water springs represent an unusual niche with temperatures in the range of 60 to 100 °C; these springs can be potential sources of novel genes and microorganisms (Kumar et al. 2013). Prospecting cold habitats has led to the isolation of a great diversity of microorganisms (Sahay et al. 2013). Leh (Jammu and Kashmir) and Rohtang Pass (Himachal Pradesh) represent cold deserts and a niche for cold adapted microorganisms that have immense significance in the field of biotechnology, because of their distinct metabolism. Psychrophilic microorganisms are also potential sources of novel pigments (as food additives), enzymes, antifreeze compounds—which can be valuable in agriculture as inoculants (plant growth-promoting bacteria)—or bio control agents in extreme habitats (Khan and Patel 2007; Srinivas et al. 2009).

Extremes of high (alkaline) and low (acidic) pH also influence the buildup of microbial population, and in turn, soil productivity. If the soil is acidic, the availability of essential nutrients, particularly, phosphorus, calcium, magnesium, and molybdenum, is affected. Very few reports are available on the distribution and diversity of bacteria in acidic soils (Yadav et al. 2011; Verma et al. 2013). Another useful extreme environment is the mangrove ecosystem, which is mostly nutrient-deficient, especially in terms of nitrogen and phosphorus. In spite of this, mangroves can be highly productive, which can be attributed to microbial activity leading to major nutrient transformations. Sunderbans (West Bengal) and Bhitarkanika (Odisha) are important mangroves of India that represent salt tolerant, complex and dynamic ecosystems. Microbial research in saline environments has also attracted the interest of researchers due to various biotechnological applications (Sahay et al. 2011, 2012; Pandey et al. 2013). Soda lakes and deserts represent the most stable naturally occurring alkaline environments on Earth. Sambhar Lake (Rajasthan), Chilka Lake in Odisha, and the Great Rann of Kutch (Gujarat) are typical saline environments in India, and very few reports on microbial diversity in these habitats are available (Sahay et al. 2012). Low moisture conditions coupled with high temperatures in arid deserts lead to enrichment of microbial communities that can survive extreme variations in temperature and moisture. Such environments encompass typically poor soils with low organic content and limited amounts of bioavailable inorganic nutrients. The microbiota of desert ecosystems is not only responsible for the productivity, biogeochemical cycling of elements and ecosystem balance, but also for soil neogenesis and improvement of soil structure. Diversity of 16S rRNA and nifH genes derived from the rhizosphere of endemic drought tolerant grass Lasiurus sindicus has been reported from the samples collected from Jaisalmer (Rajasthan) (Chowdhury et al. 2009). However, the cultural diversity of bacteria in the Thar Desert has not been characterized.

The genus Bacillus is a heterogeneous collection of aerobic or facultative anaerobic endospore-forming bacteria that are ubiquitous in many environments. The sequencing of 16S rDNA led to the identification of five phylogenetically distinct groups within the genus Bacillus (Ash et al. 1991). Since 1990, several new genera, such as Amphibacillus (Niimura et al. 1990), Paenibacillus (Ash et al. 1991), Alicyclobacillus (Wisotzkey et al. 1992), Aneurinibacillus, Brevibacillus (Shida et al. 1996), Virgibacillus (Heyndrickx et al. 1998), Gracilibacillus, Salibacillus (Waino et al. 1999), Geobacillus (Nazina et al. 2001), Filobacillus (Schlesner et al. 2001), Jeotgallibacillus, Marinibacillus (Yoon et al. 2010) and Ureibacillus (Fortina et al. 2001), have been sorted out. Bacillus species are phenotypically and genotypically heterogeneous (Claus and Berkeley 1986). Members of the families Bacillaceae and Paenibacillaceae are widely used in agriculture as plant growth-promoting and disease-suppressing agents, besides their use in industry as a source of enzymes and in medicine. The importance of this group is reflected in the large number of studies targeting their phenotypic and genotypic diversity in different ecological niches (Suihko and Stackebrandt 2003, Sass et al. 2008, Ettoumi et al. 2009, Yadav et al. 2011; Kumar et al. 2013) in food (Oguntoyinbo et al. 2010) and industry waste (Freitas et al. 2008). Bacillus thuringiensis is currently used in the biological control of insects (Perez et al. 2007).

The present investigation was undertaken towards exploring the diversity and pervasive nature of BBDG in extreme environments and identifying niche-specific species, using a combination of culture-based, biochemical and molecular approaches.

Material and methods

Sampling sites

Water, soil and sediment samples were collected from different extreme environments of India (Fig. 1), whose details are given in Table 1. A total of 152 samples were collected from fifteen sites comprising various habitats. The sites surveyed included those exhibiting extreme conditions of pH, temperatures, salinity and drought. Samples were collected in sterile polythene bags/bottles, labeled, transported on ice and stored at 4 °C until analysis. The pH and conductivity of the samples was recorded on site.

Fig. 1

Map of India depicting sampling locations

Table 1 Geographic details and physico-chemical characteristics of collection sites

Enrichment and isolation

The population of BBDG in the water, sediments and soil samples were enumerated through enrichment using the standard serial dilution plating technique. One gram of sediment/soil or 1 ml of water sample was added to 9 ml of sterile distilled water and heated in a hot water bath at 80 °C for 15 min to kill the vegetative cells. The samples were diluted and appropriate dilutions were spread on nutrient agar (NA) plates amended with methyl red (0.02 %). In addition to NA medium (HiMedia Laboratories, Mumbai, India), other media and conditions were used for the isolation of different categories of extremophiles (details given in Table 2). Selective isolation of Bacillus thuringiensis was done using sodium acetate (0.25 and 0.75 M) buffer (HiMedia Laboratories, Mumbai, India), followed by Luria broth enrichment and heat treatment at 80 °C for 15 min. The samples were then diluted and appropriate dilutions were spread on nutrient agar and T3 agar medium plates (Travers et al. 1987).

Table 2 Media and the conditions employed for isolation of different categories of extremophiles

The nutrient agar and soil extract agar plates having pH of 3–5 and 9–11 were used to isolate acidophilic and alkaliphilic BBDG, and were incubated at 30 °C for 2–3 days. To isolate psychrophilic and thermophilic BBDG, plates were incubated at 4 °C and 45 °C, and populations were counted after 15–20 days (Larkin and Stokes 1966) and 2–3 days, respectively. For isolation of halophilic BBDG, aerobic enrichment cultures were prepared by adding 1.0 g of each sample into separate Erlenmeyer flasks and 100 ml of chemically defined medium or halophilic medium, followed by incubation on a shaker (Kuhner LT-X, Dinkelbergstrasse 1, Switzerland) at 30 °C. An aliquot of 100 μL from each enriched sample was spread-plated on four different media listed in Table 2. Plates were incubated at 30 °C for a maximum period of 5–7 days. Colonies that appeared were purified by repeated re-streaking to obtain isolated colonies using respective medium plates. The pure cultures were maintained at 4 °C as slant and glycerol stocks (20 %) at −80 °C for further use.

PCR amplification of 16S rDNA and amplified rDNA restriction analysis (ARDRA)

Genomic DNA was extracted by the modified Pospiech and Neumann method (1995). The primers pA (5′-AGAGTTTGATCCTGGCTCAG-3′) and pH (5′-AAGGAGGTGATCCAGCCGCA-3′) were used for the amplification of 16S rDNA (Edwards et al. 1989). The amplification was carried out in a 100 μl volume, and amplification conditions as described earlier were used (Sahay et al. 2011). After amplification, the PCR products were resolved by electrophoresis in 1.2 % agarose gel in 1X TAE buffer (Bangalore Genei, Bangalore, India). Gels were stained with ethidium bromide (10 mg ml⁻¹), (Hengstmann et al. 1999), visualized on a gel documentation system (Alpha-Imager), and gel images were digitalized. PCR-amplified 16S rDNA products were purified with a Quiaquick purification kit (Qiagen). Aliquots of purified 16S rDNA PCR products were digested separately with three restriction endonucleases, AluI, HaeIII and MspI (Bangalore Genei, Bangalore, India), in 25 μl reaction volumes, using the manufacturer’s recommended buffer and temperature. Restricted DNA was analyzed by horizontal electrophoresis in 2.5 % agarose gels. The gels were visualized and gel images were digitalized. Strong and clear bands were scored for similarity, and clustering analysis was undertaken using the software, NTSYS-2.02e package (Numerical taxonomy analysis program package, Exeter software, USA). Similarity among the isolates was calculated by Jaccard’s coefficient (Jaccard 1912) and dendrogram was constructed using the UPGMA method (Nei and Li 1979).

16S rDNA sequencing and phylogenetic analysis

The nucleotide sequences of purified 16S rDNA were di-deoxy cycle sequenced with fluorescent terminators (Big Dye, Applied Bio systems) and run in a 3130xl Applied Bio systems ABI prism automated DNA sequencer. The DNA sequence was double-checked by sequencing both strands using primers pA and pH for forward and reverse reactions, respectively. The partial 16S rDNA sequences of the isolated strains were compared with those available in the databases. Identification at the species level was determined using a 16S rRNA gene sequence similarity of ≥97 % with that of a prototype strain sequence in the GenBank (Benson et al. 2010). Sequence alignment and comparison was performed, using the program CLUSTAL W (Thompson et al. 1994) with default parameters and the data converted to PHYLIP format. Minor modifications were done manually on the basis of conserved domains, and positions containing more than 50 % gaps were removed. One sequence from each group was selected as a representative operational taxonomic unit (OTU). The phylogenetic tree was constructed on the aligned data sets using the neighbor-joining method (Saitou and Nei 1987) implemented in the program MEGA 4.0.2 (Tamura et al. 2007). Bootstrap analysis on 1,000 random samples taken from the multiple alignments was performed as described by Felsenstein (1981).

The partial 16S rDNA sequences were submitted to NCBI GenBank (Benson et al. 2010), and accession numbers were assigned as given in Table 3.

Table 3 Enumeration of isolates using standard techniques, Gen Bank Accession numbers and classification based on 16S rDNA-PCR–RFLP profiles

Tolerance of isolates to extremes of pH, temperature, salinity and drought

The screening of all the isolates for pH tolerance was carried out by spot inoculating the cultures onto nutrient agar medium with pH ranging from 3 to 11, with an increment of 1. The pH in the medium was adjusted by addition of acid phthalate buffer (pH 3–4), neutralized phthalate buffer (pH 5.0), 50 mM phosphate buffer (pH 6.0–8.0)], bicarbonate buffer (pH 9.2–10.6) or sodium hydroxide/potassium chloride buffer (pH 12.0–13.0) (HiMedia Laboratories, Mumbai, India). The isolates from saline habitats (Sambhar Lake, Chilka Lake, Rann of Kutch, Bhitarkanika and Sunderbans) were screened for tolerance to salt (5 to 25 % NaCl concentration) by spot inoculating each of the isolates onto nutrient agar plate supplemented with NaCl to total concentrations of 5, 10 15, 20 and 25 % (w/v). Isolates from drought habitat were screened for tolerance to low water potential on polyethylene glycol (PEG-8000)-infused plates of −0.5, 0.7 and −1.2 MPa water potential, following the procedure described by Verslues and Bray (2006). The isolates were screened for temperature tolerance by incubating the culture spot inoculated plates at different temperatures of 5, 15, 20 and 25 °C (for low temperature tolerance) and at 40, 45, 50, 55 and 60 °C (for high temperature tolerance) for 72–96 h. The cultures tolerant to 60 °C were screened further by inoculating in broth and incubating in shaker at 150 rpm for 72 h at 75 and 90 °C. The optical density of the broth was measured at 600 nm and compared with the growth at 37 °C.

Statistical analysis

In order to compare the bacterial diversity among fifteen different habitats, the 16S rRNA gene sequences of the isolates showing ≥ 97 % sequence similarity were grouped into the same OTU (phylotype). The software Shannon–Wiener Diversity Index/Shannon Entropy Calculator (http://www.changbioscience.com/genetics/shannon.html) and Rarefaction Calculator (http://www2.biology.ualberta.ca/jbrzusto/rarefact.php) were used to calculate Shannon index (H), Evenness (J) and the Simpson’s index (D).

Results

Abundance and diversity analyses of BBDG

The population of BBDG was enumerated in different samples collected from fifteen sites of India having extreme environmental conditions (Table 1). The abundance of BBDG in the samples varied from 4.5 × 10³ to 4.0 × 10⁶ CFU g⁻¹ sediment or ml⁻¹ water, with the highest values in samples from Chilka Lake, followed by Kollam, Kerala (9.5 × 10⁵ CFU g⁻¹ sediment or ml⁻¹ water), and the lowest values in were in samples from Sunderbans, followed by Chumathang (1.1 × 10⁴ CFU g⁻¹ sediment or ml⁻¹ water) (Table 3). The diversity of morphotypes was highest at Sunderbans (99) and Rann of Kutch (99), and lowest (24) at Kollam (Table 3).

PCR amplification of 16S rDNA and amplified rDNA restriction analysis (ARDRA)

PCR amplification of 16S rDNA followed by ARDRA with three restriction endonucleases was carried out to look for the species variation among the morphotypes selected. The 16S rDNA amplicons were digested with restriction enzymes, which generated profiles having 3 to 7 fragments ranging in size from 100 to 820 base pairs. ARDRA results revealed that among the restriction endonucleases, AluI was more discriminatory as compared to MspI and HaeIII. A combined dendrogram was constructed for each sampling site to determine the percent similarity among the isolates. At a level of 75 % similarity (Table 3), the isolates were grouped into clusters; and the number of clusters ranged from 12 (for Vashisht thermal springs) to 74 (for Leh cold desert). The total number of clusters was 559, summed up for all the sites.

16S rRNA gene sequencing and phylogenetic analysis

16S rRNA gene sequencing and phylogenetic analysis of a representative isolate from each cluster revealed that all the isolates showed > 97 to 100 % similarity with the sequences within GenBank (Table 3). A total of 392 isolates were identified as BBDG. The remaining 167 isolates (which included genera such as Desemzia, Exiguobacterium, Jeotgalicoccus Planococcus, Sporosarcina, and Staphylococcus) showed no similarity with BBDG; hence, these were excluded from this study. One sequence from each group was selected as a representative operational taxonomic unit (OTU), and all the isolates were classified into 75 OTUs using a ≥ 97 % sequence similarity cut-off value. The phylogenetic tree of 75 identified species of BBDG was constructed to determine their affiliations (Fig. 2). Analysis of the 16S rRNA sequences revealed that 61 strains belonged to Bacillaceae, whereas 14 strains belonged to Paenibacillaceae. The 61 Bacillaceae members were further distributed into 16 clusters, the major clusters being the Bacillus cereus group (Bacillus cereus, Bacillus anthracis, B. thuringiensis, B. mycoides, B. bombysepticus and B. pseudomycoides) and the B. subtilis group (B. altitudinis, B. pumilus, B. safensis, B. amyloliquefaciens, B. subtilis, B. vallismortis, B. mojavensis, B. licheniformis and B. tequilensis). Halobacillus, Pontibacillus, Oceanobacillus manasiensis and Virgibacillus sp. representing halophilic bacilli isolated from salt lake were present in one cluster; while moderately salt tolerant bacilli including B. baekryungensis, B. barbaricus, B. decolorationis, B. halodurans, B. hwajinpoensis and B nanhaiensis were present as a separate cluster. B. aryabhattai, B. megaterium, B. flexus, B. horikoshii, B. simplex and B. psychrosaccharolyticus, despite being salt tolerant bacilli, were present in a separate cluster. Fourteen strains of Paenibacillaceae were distributed in three different clusters, three species of Brevibacillus constituting one cluster, 11 species of Paenibacillus formed the second, and one species each of Aneurinibacillus and Ammoniphilus formed the third cluster.

Fig. 2

Phylogenetic tree showing the relationships among 75 BBDG, 16S rRNA gene sequences with reference sequences obtained through BLAST analysis. The sequence alignment was performed using the CLUSTAL W program and trees were constructed using Neighbor joining with algorithm using MEGA4 software (Tamura et al. 2007). One thousand bootstrap replicates were performed. Bootstrap values are indicated on the branches. The tree was rooted using E. coli as the outgroup

Diversity analyses

The species-wise distribution of the BDDG isolates from the present investigation is given in Fig. 3, and in Table 4 in terms of their abundance in the different environments. Bacillus cereus was among the most commonly recorded species. Two typing methods (ARDRA profiling and 16S rRNA gene sequence analysis) adopted for examining the diversity of BBDG provided a similar level of resolution. Shannon’s Diversity index was highest (H′ = 3.04) for Sunderbans, followed by Manikaran hot springs (H′ = 2.73), whereas Kollam recorded the lowest value (H′ = 1.73). The lowest species richness was recorded in BBDG of Chumathang and Vashisht hot springs, while Sunderbans recorded the highest species richness (Table 5). The individual rarefaction curves for all the samples of fifteen sites indicated that the bacterial populations were the least diverse in Chumathang hot springs followed by Vashisht hot springs, and most diverse in Sunderbans followed by Rann of Kutch (Fig. 4).

Table 4 Distribution of predominant BBDG in the different extreme environments

Table 5 Diversity indices for the isolates from 15 locations in India exhibiting extreme environmental conditions

Fig. 3

Abundance of different Bacillus and Bacillus derived genera in the samples surveyed

Tolerance of isolates to extremes of pH, temperature, salinity and drought

All the isolates were screened for tolerance to a range of salinities, pH, temperatures and PEG-mediated water deficit (drought) (Fig. 4). All the 196 bacterial isolates from Chilka Lake, Sambhar Lake, Rann of Kutch, Bhitarkanika, and Sunderbans were tolerant to 5 % NaCl, while 87, 72 and 31 isolates could tolerate 10, 15 and 20 % NaCl, respectively (Fig. 5a). Two isolates, Virgibacillus halodenitrificans ABK-2 and Bacillus marisflavi AB-18 from Rann of Kutch could grow even at 30 % NaCl concentration. In general, the isolates obtained from Sambhar Lake and Rann of Kutch, showed tolerance to higher levels of salinities (20 % NaCl or more). Among the representative isolates from each cluster, all except CS-39 were able to tolerate 10 % NaCl concentration, 25 were able to tolerate 15 %, whereas eight isolates could tolerate 20 % NaCl concentration. Three isolates, Bacillus halodurans ABSL-8, B. methanolicus ABSL-11 and Ammoniphilus sp. ABSL-12, were obtained from Sambhar Salt Lake; one isolate from Bhitarkanika mangrove soil, Bacillus mojavensis AB-10, and two isolates from Sunderbans mangrove soil, Halobacillus litoralis S-91 and Bacillus hwajinpoensis S-93, were able to grow at 20 % NaCl and pH 9.0. Eight isolates, Bacillus sp. NSP22.2, Bacillus sp. SB47, Thalassobacillus devorans strain MSP14, Bacillus sp. NSP10, Bacillus sp. MSP18.3, Salinibacillus aidingensis strain MSP4 and Bacillus licheniformis strain NSP4 obtained from Rann of Kutch, were obligate halophiles, as they required 5 % NaCl in the medium for their growth. All isolates from Andaman and Nicobar Island and Kollam were acidotolerant and could grow at pH 5 (Fig. 5b). Seventy-one bacterial isolates obtained from the thermal springs of Manikaran, Bakreshwar, Vashisht, Balrampur and Chumathang were tolerant to temperatures of 55 °C or more (Fig. 5c). Isolates from Manikaran thermal spring could grow at temperatures ranging from 45 to 90 °C. Only two isolates, Paenibacillus pabuli M-10 and Paenibacillus tylopili M-18 from Manikaran hot springs, could tolerate temperatures as high as 90 °C. Most of isolates from the hot water springs were alkalitolerant and could grow at pH 9.

Fig. 4

Rarefaction curves of observed OTUs in the soil/sediment samples from 15 sites in India

Fig. 5

Enumeration of bacterial isolates from a Chilka Lake, Sambhar Lake, Rann of Kutch, Bhitarkanika and Sunderbans on the basis of their salt and pH tolerance; b from Andaman and Nicobar Islands and Kollam, based on their pH and salt tolerance; c from thermal springs of Manikaran, Bakreshwar, Vashisht, Balrampur and Chumathang on the basis of temperature and pH tolerance

All the isolates obtained from Leh and Rohtang Pass were psychrotolerant and could grow at 10 °C (Fig. 6a). Two isolates, Paenibacillus xylanexedens L-76 and Paenibacillus terrae L-102 from Leh, and one isolate, Bacillus psychrosaccharolyticus R-2 obtained from Rohtang Pass, grew well at 4 °C and are psychrophilic, as they could not grow beyond 20 °C. Interestingly, isolate R-2 could tolerate 5 % NaCl and a pH of 5.0. Among the 24 isolates obtained from Jaisalmer, 17 were able to grow on PEG-infused plates with water potential of −0.5 Mpa (Fig. 6b). Three representative isolates, Bacillus mycoides J-4, B. megaterium J-5 and B. endophyticus J-13 obtained from Jaisalmer, were tolerant to moisture stress (−0.5 Mpa and 10 % NaCl).

Fig. 6

Distribution of bacterial isolates based on their degree of tolerance and belonging to a Cold deserts of Leh and Rohtang Pass, with respect to temperature and pH; b Hot/arid desert of Jaisalmer, with respect to water stress and pH

Discussion

Microorganisms represent the richest gamut of molecular and chemical diversity in nature, as they comprise the simplest, yet dynamic, forms of life. Interest in the exploration of microbial diversity has been spurred by the fact that microbes are essential for life as they perform numerous functions integral to the sustenance of the biosphere, including nutrient cycling and environmental detoxification, which involve processes such as augmentation, supplementation and recycling of plant nutrients, so vital to sustainable agriculture. Microorganisms abound in all kind of habitats, viz. with extremes of pH, temperature, salinity and water stress. More recently, this largely unexplored reservoir of resources has become the focus of investigations for innovative applications useful to mankind.

With the progress of exploration of extreme environments, the richness of microbial diversity is becoming increasingly evident. Among bacteria, aerobic, endospore-forming BBDG are pervasive in the environment and play a significant role in agriculture, medicine and industry. Extreme environments can be a source for novel species of Bacillus, as these bacteria can tolerate extremes of pH, temperature, salinity and moisture stress. In recent years, several studies have been conducted to look for the diversity of BBDG in different environments, such as geothermal areas of Deception island, in the South Shetland Archipelago (Llarch et al. 1997); the Gulf of Mexico (Siefert et al. 2000); board and paper products (Suihko and Stackebrandt 2003); the biosphere reserve in Egypt (Mohamed et al. 2006), steel plant wastes (Freitas et al. 2008); deep sea hyper-saline anoxic basins within the Eastern Mediterranean Sea (Sass et al. 2008); Avord Basin, Oregon (Smith et al. 2009); marine sediments (Ettoumi et al. 2009); acidic soils (Yadav et al. 2011) Lonar Lake (Tambekar and Dhundale 2012); geothermal regions of Turkey (Cihan et al. 2012) and the hyper-arid Atacama Desert, Chile (Paulino-Lima et al. 2013). Such studies have illustrated that a large number of aerobic endospore-forming Gram positive Bacillus species are present in diverse environments, some of which possess unique capabilities for adapting to extreme environments.

Preliminary screening revealed the presence of many BBDG that can grow in a wide range of environments at pH 5.0–12.0, temperatures between 4 and 90 °C, in salinity from 0 to 30 % NaCl and at moisture stress ranging from 0 to –0.75 Mpa. Many such isolates were also found to tolerate multiple stresses. The population count of BBDG showed variations among the sites and the media employed; and the counts varied from 4.5 × 10³ to 4.0 × 10⁶ CFU g⁻¹ sediment or CFU ml⁻¹ water. In general, for most of the sites, the population was in the range of 1.1 × 10⁴ to 8.8 × 10⁴, indicating that BBDG predominate most of the extreme environments. In earlier studies, the populations of Bacillus in acidic soils of Kollam, Kerala were reported to range from 5 × 10⁴ to 300 × 10⁴ cells g⁻¹ soil (Yadav et al. 2011), while samples from Sambhar Salt Lake recorded a total bacterial count in the sediment samples ranging from 32 to 59 × 10⁵ CFU g⁻¹, whereas in water samples it ranged from 14 to 38 × 10⁵ CFU ml⁻¹ (Sahay et al. 2012). In deep sea hyper-saline anoxic sediments in the Mediterranean, a high number of endospores, ranging from 3.8 × 10⁵ to 1.2 × 10⁶ g⁻¹ dw sediment, were reported (Ventosa et al. 1998). Microbial abundance in snow cover has been reported to range from 10³ to 10⁵ per ml in melted snow and varies with altitude and latitude; though it was less abundant in Antarctic than in mountainous and Arctic snow and increased with altitude.

A total of 893 putative Bacillus isolates were obtained from 15 different extreme environments, employing different media. Morphological variations among the isolates obtained on different media further confirmed that members of the genus Bacillus exhibit a wide range of nutritional requirements. On the basis of restriction fragment analysis with restriction enzymes AluI, MspI and HaeIII, all the isolates grouped into 559 clusters. Among the restriction enzymes, Alu1 was found to be more discriminatory and yielded several fragments. In earlier studies (Yadav et al. 2011; Vardhan et al. 2011; Cihan et al. 2012), AluI was found to be ideal for restriction analysis and clustering of Bacillus isolates. Sequencing of 16S rRNA gene of the representative isolates from 559 clusters identified 392 as BBDG; these were taken up for phylogenetic analysis. Partial sequencing of the smaller subunit of 16S rRNA gene assigned all the 392 isolates to 75 different species, grouped in thirteen genera of Phylum Firmicutes and belonging to two families (Bacillaceae 89.03 %, and 10.97 %, Paenibacillaceae). Priest (1993) also reported a global distribution of species of Bacillus and Paenibacillus. Multiple species of Bacillus and Paenibacillus were reported from both bulk and rhizosphere soils, and their counts ranged from 10³ to 10⁶ cells ml⁻¹ (Vargas-Ayala et al. 2000).

It is well known that 16S rDNA sequences are reliable tools to establish phylogenetic relationships among bacteria, and in the present study they proved valuable in the identification of BDDG. However, a large number of isolates were classified as Bacillus sp. with no species assigned to them. Among the Bacillaceae, species of Bacillus cereus appeared to be the most predominant species recovered from all extreme environments, except from Sambhar Salt Lake. The other predominant species were Bacillus megaterium, B. subtilis, B. firmus and B. pumilus (Table 3). The wide occurrence of B. subtilis and B. cereus has been reported in different studies (Ettoumi et al. 2009). Among 96 bacilli isolated from marine sediments, 68 % were identified as species of B. subtilis, B. licheniformis, B. pumilus and B. cereus (Ettoumi et al. 2009). In another study, 23 marine isolates were clustered, based on 16S rRNA gene sequencing, into four species belonging to Bacillus licheniformis, B. cereus, B. subtilis and B. pumilus (Miranda et al. 2008). The diversity of Bacillus genotypes in soil samples from El-Omayed Biosphere reserve in Egypt also revealed that a majority of the isolates were closely related to the members of the B. cereus/B. thuringiensis group (Mohamed et al. 2006). Bacillus megaterium has also been reported to be among the most abundant in some soils (Liu and Sinclair 1992). Diversity indices further illustrated that these diverse environments posses a high degree of genetic diversity.

The cold deserts represent hot spots of biodiversity, and several novel cold-tolerant bacterial species have been isolated from these regions (Shivaji et al. 2005). Significant prokaryotic diversity has been detected, including heterotrophic bacteria, cyanobacteria and eukaryotes, with many related to known psychrophilic and psychrotolerant species (Amato et al. 2007). Among the 145 isolates obtained, BBDG exhibited great variations. Some niche-specific bacilli as Bacillus muralis (L-74), Lysinibacillus fusiformis (L-2) and Paenibacillus terrae (L-57) from Leh, and Bacillus psychrosaccharolyticus (R-2), Paenibacillus lautus (R-27) and Virgibacillus halodenitrificans (ABR-18) from Rohtang Pass, were isolated. It is well documented that the most frequently encountered bacterial genera in cold environments belong to Proteobacteria, Cytophaga—Flavobacterium-Bacteroidetes (CFB) group and low and high G/C Gram-positive genera (Amato et al. 2007). The presence of these genera in these cold environments suggests their adaptability to low temperature, which may be attributed to the presence of pigments or polyunsaturated fatty acids that have been implicated in cold adaptation (Shivaji et al. 2007). In our study, all the isolates were capable of growing between 4 and 30 °C, indicating that psychrotolerant bacteria are predominant in the sediments of Leh and Rohtang Pass. In the present study, many of the isolates obtained from cold deserts showed pigmentation and produced copious amount of exopolysaccharides. Bacteria living in cold aquatic environments (e.g., Antarctic and Arctic sea ice) are known to produce high amounts of exopolymeric substances (Nichols et al. 2005). It is suggested that these exopolysaccharides help in adhesion to wet surfaces and the formation of the biofilm matrix, which traps nutrients, protects the cell against unfavorable environmental conditions, and mediates biochemical interactions (Nichols et al. 2005). Virgibacillus halodenitrificans (ABR-18) from Sarkund Lake in Rohtang Pass was found to grow best under cold and saline conditions, consistent with the properties of its habitat (Doran et al. 2003, 2008), and its phylogeny is predictive of a cold active and halotolerant phenotype.

Thermal springs represent extreme niches that have been a source of biotechnologically important microorganisms best illustrated by Thermus aquaticus. In the last decade, several attempts have been made for phylogenetic characterization of microflora from thermal springs in different parts of the world (Reysenbach et al. 2000; Derekova et al. 2008; Singh et al. 2010; Kumar et al. 2013). Recently, the draft genome sequence of Thermus sp. isolated from Manikaran thermal springs has been published (Dwivedi et al. 2012). In the present study, 195 isolates from Indian hot water springs (Manikaran, Balarampur, Vashisht, Chumathang and Bakreshwar) were isolated and identified. In general, isolates obtained from Manikaran showed tolerance to higher temperatures. Two isolates, Paenibacillus pabuli (M-10) and Paenibacillus tylopili (M-18), could grow at temperatures > 80 °C. There were many species and derived genera that were niche-specific and typically isolated from Manikaran. Some BBDG from the present study include Aneurinibacillus danicus (M-35), Bacillus beijingensis (M-74), B. vireti (M30), Brevibacillus agri (M-73), Paenibacillus pabuli (M-10), P. tylopili (M-18) and Lysinibacillus xylanilyticus (M-52). Bacillus fusiformis (B-10) from Bakreshwar and Brevibacillus from Vashisht and Balrampur were identified as niche-specific bacilli. Aneurinibacillus danicus and Brevibacillus sp. have been earlier reported as thermophilic bacteria (Goto et al. 2004). Bacillus licheniformis and B. megaterium were previously reported to be the most dominant bacilli in thermal springs (Llarch et al. 1997). Most of the thermophilic bacteria isolated from Indian springs were alkalitolerant. Earlier reports also suggest the occurrence of thermo-alkali tolerant xylanase-producing bacteria from Manikaran thermal springs (Singh et al. 2010).

In the present study, a large number of halophilic or halotolerant species, such as Bacillus halodurans (ABSL-8), Bacillus methanolicus (ABSL-11), Ammoniphilus sp.(ABSL-2), Halobacillus trueperi (ABSL-21), Bacillus vallismortis (ABSL-23) and Halobacillus dabanensis (ABSL-29) from Sambhar Lake, Marinococcus halophilus (ABK-3) from Rann of Kutch, and Pontibacillus sp. (AB-2) from Chilka Lake, were identified. Sambhar Lake has been previously investigated (Sahay et al. 2012). Similarly, many other BBDG have been reported from saline alkaline habitats, such as Bacillus vallismortis from Death Valley, California (Spring et al. 1996), Ammoniphilus, halo-alkalitolerant bacilli from the rhizosphere of sorrel (Rumex acetosa) and from decaying wood, Finland (Zaitsev et al. 1998), Bacillus halodurans from a Kenyan soda lake (Hashim et al. 2004), Halobacillus dabanensis and Oceanobacillus manasiensis sp. nov. from salt lakes in Xinjiang, China (Wang et al. 2010), and Pontibacillus from a sea urchin (Chen et al. 2009). Three species of the genus Halobacillus (H. halophilus, H. litoralis and H. trueperi) were reported to require NaCl ions for growth (Spring et al. 1996).

Sunderbans, the world’s largest coastal wetland comprising mangrove forests, and Bhitarkanika, the second largest mangrove ecosystem of India, are hot spots of biodiversity. A total of 184 bacteria were isolated from these Indian mangroves and characterized for their salt tolerance. Three isolates, Bacillus vietnamensis (S-28), B. barbaricus (S-31), B. marisflavi (S-14) from Sunderbans, and B. atrophaeus (B-71) from Bhitarkanika mangroves were able to tolerate 15 % NaCl concentration, while two isolates Halobacillus litoralis (S-91) and Bacillus hwajinpoensis (S-93) from Sunderbans and one isolate Bacillus mojavensis (AB-10) from Bhitarkanika could tolerate 20 % NaCl concentration. Some niche-specific bacilli from Sunderbans were identified as Bacillus alcalophilus (S-77), B. aquimaris (S-51), B. cibi (S-22), B. horikoshii (S-45), B. hwajinpoensis (S-93), Halobacillus litoralis (S-91), Paenibacillus amylolyticus (S-78), and Bacillus vietnamensis (S-28). Bhitarkanika mangrove was also represented by specific BBDG, such as Bacillus atrophaeus (B-71), Bacillus methylotrophicus (B-11), and Bacillus bombysepticus (B-80). Bacillus horikoshii, B. atrophaeus, and B. methylotrophicus have earlier been reported to tolerate 8–12 % NaCl concentration. Bacillus horikoshii was isolated from coastal saline soils, Gujarat, India (Yousuf et al. 2012). Bacillus hwajinpoensis represent novel members of Bacillus rRNA group, which were earlier reported from sea water of the East Sea and the Yellow Sea in Korea (Yoon et al. 2004). Bacillus vietnamensis sp. nov., a moderately halotolerant, aerobic, endospore-forming bacterium, was initially isolated from Vietnamese fish sauce (Noguchi et al. 2004), whereas Halobacillus litoralis and Halobacillus trueperi, moderately halophilic strains, were reported from hyper-saline sediments of the Great Salt Lake in Utah (Spring et al. 1996). However, the culture-independent molecular analysis of bacterial communities in the mangrove sediment of Sunderbans, India revealed only the presence of one sequence type that showed similarity with Bacillus subtilis (Ghosh et al. 2010). An earlier diversity study carried out at another location—Bhitarkanika, in India—revealed Bacillus as one of the most predominant genera (Mishra et al. 2012).

Andaman and Nicobar Islands and Kollam, Kerala region were selected for isolation of BBDG tolerant to acid pH. A total of 74 BBDG were isolated from the two regions. Isolates, three each from Kollam, Bacillus amyloliquefaciens (A-2), B. pumilus (A-5) and B. megaterium (A-6), and Andaman and Nicobar Islands, Lysinibacillus sp. (AN-4), Bacillus pseudomycoides (AN-13) and Bacillus aryabhattai (AN-21), could grow at <pH 5.0. In an earlier study, many different species of BBDG, Bacillus humi, B. megaterium, B. drentensis, B. pocheonensis, B. aestuarii, B. arbutinivorans, B. niacini, were isolated from the rhizosphere of rice grown in Kollam, Kerala (Yadav et al. 2011). Niche-specific bacilli identified from Andaman and Nicobar Islands and Kollam were Bacillus pseudomycoides and B. amyloliquefaciens, respectively.

There is very little knowledge on rhizosphere microbiology of the desert plants. A total of 56 bacteria were isolated from rhizosphere samples of Jaisalmer desert. Species of Bacillus found to tolerate low water potential were identified as Bacillus tequilensis (J-12) and Bacillus mycoides (J-4). Some BBDG specific to Jaisalmer desert identified were Bacillus mycoides (J-4) and Bacillus endophyticus (J-13).

The comprehensive analyses of diversity of Bacillus and Bacillus derived genera by prospecting extreme environments helped in the development of a huge database including baseline information on the distribution of BBDG in different niches and identifying niche-specific microbes. This database also helped in identifying novel bacteria capable of solubilizing K and Zn under different abiotic stresses (unpublished data), besides those efficient in the production of hydrolytic enzymes of industrial significance such as cellulases and xylanases. The cultures tolerant to low and high temperature, salinity, acidic pH and water stress represent a rich bioresource for useful genes and alleles, which can aid in the generation of abiotic stress-tolerant transgenics.