Hot springs of Indian Himalayas: potential sources of microbial diversity and thermostable hydrolytic enzymes

Microbial communities in hot springs at high elevations have been extensively studied worldwide. In this sense, the Indian Himalaya regions is valuable ecosystems for providing both the extreme 'cold' and 'hot' sites for exploring microbial diversity. In the present study, a total of 140 thermophilic bacteria were isolated from 12 samples collected from Manikaran and Yumthang hot springs of Indian Himalayas. The bacterial isolates were studied for phylogenetic profiling, growth properties at varying conditions and potential sources of extracellular thermostable hydrolytic enzymes such as protease, amylase, xylanase and cellulase. Based on production of extracellular hydrolases, 51 isolates from Manikaran (28) and Yumthang thermal springs (23) were selected and identified using 16S rRNA gene sequencing which included 37 distinct species of 14 different genera namely Anoxybacillus, Bacillus, Brevibacillus, Brevundimonas, Burkholderia, Geobacillus, Paenibacillus, Planococcus, Pseudomonas, Rhodanobacter, Thermoactinomyces, Thermobacillus, Thermonema and Thiobacillus. Out of 51 hydrolase producing bacteria, 24 isolates showed stability at wide range of temperature and pH treatments. In present investigation, three thermotolerant bacteria namely, Thermobacillus sp NBM6, Paenibacillus ehimensis NBM24 and Paenibacillus popilliae NBM68 were found to produced cellulase-free xylanase. These potential extracellular thermostable hydrolytic enzymes producing thermophilic bacteria have a great commercial prospect in various industrial, medical and agriculture applications.


Introduction
Microbes were present in every conceivable ecological niche, includes from the tropics to the poles and hot springs to underwater hydrothermal vents. Particularly, prokaryotic communities under thermophilic habitats have been undergone the physiological adaptations to high temperature and chemical stress. Recently, these communities have attained the focus of applied research not only in terms of biotechnological prospects but also to understand the use of primitive analogues of biomolecules existed during early Earth environments (Raddadi et al. 2015;Saxena et al. 2016). Microbial communities in hot springs at low elevations have been extensively studied worldwide, however in few investigation has been done to explore the diversity in natural hot springs/thermal lakes in India. Particularly the Himalayas, though it has both extreme 'cold' and 'hot' ecosystems, but many of these valuable gene pools were remain unexplored due to inaccessible for human activities.
The microbial diversity of cold and snow caped lakes such as cold deserts of Himalaya India (Sahay et al. 2013;Saxena et al. 2014Saxena et al. , 2015Yadav et al. 2015a, b), High-Arctic snow (Harding et al. 2011) and cold springs on the Qinghai-Tibetan Plateau, China (Yang et al. 2015) were studied. Apart from cold habitats, Himalayas have unique hot springs at high elevations, such as Soldhar and Ringigad hot springs, Uttaranchal Himalaya (Kumar et al. 2004), Chumathang hot spring, NW Indian Himalayas , Vashisht, Khirganga and Kasol hot springs (Shirkot and Verma 2015) and tattapani hot spring in NW Indian Himalayas (Priya et al. 2016).
Thermal springs are hot spots of biodiversity of microbes which can be utilized as source of novel genes, molecules and hydrolytic enzymes for agricultural, medical and industrial processes (Saxena et al. 2016). Novel thermophilic microbes have isolated and characterized from thermal extreme environments of world such as Thermotoga elfii (Ravot et al. 1995), Thermotoga hypogeal (Fardeau et al. 1997), Thermoanaerobacter uzonensis (Wagner et al. 2008) and Herbinix luporum (Koeck et al. 2016). Manikaran and Yumthang thermal springs are the hottest in the country, with a temperature range of 89-95°C, which can be potential sources of novel genes, molecules and microorganisms. Despite intensive studies on terrestrial thermal springs, very little is known about bacterial diversity of thermal springs at high elevation. Hence, a comprehensive approach is needed to analyze the bacterial diversity of such niche, in terms of their phylogenetic profiling and its potential bioresources for extracellular hydrolytic enzymes.
Thermostable enzymes, isolated from thermophiles, have found potential commercial and industrial significance due to their inherent stability under harsh industrial processes  as well as stable performance at high temperature (Moraïs et al. 2016). Natural thermal environments or extreme niches are providing support to the primitive form of life; such habitats became special inherent resources of bioactive molecules for modern biotechnologists. Therefore, every country has putting research efforts towards exploration and conservation of the bio-resources available in their natural ecosystems. Potential microbes from hot springs offer a major advantage of preserving those strains for future studies and exploring them in due course for potential biotechnological applications in medical, industrial and agriculture processes. The present investigation deals with culturable thermophilic bacteria and their identification using 16S rRNA gene sequencing. Following culturable technique, isolation, characterization, phylogenetic profiling and the hydrolytic enzymes production of thermo-adapted culturable microbes was performed for samples collected from hot springs of Indian Himalayas.

Study sites and sample collection
Water and sediment samples were collected from Manikaran and Yumthang hot springs, Indian Himalayas (Table 1). Manikaran hot spring is located in the Beas and Parvati valley geothermal system, at an elevation of 1760 metres with coordinate 32°02 0 00 00 N:77°20 0 48 00 E. Yumthang thermal spring is located in the North Sikkim district of Sikkim, India at an elevation of 3564 metres with coordinate 27°49 0 36 00 N:88°41 0 45 00 E. The temperatures and pH of samples were recorded at sampling sites. The temperature and pH of Manikaran hot springs ranges from 89 to 95°C and 7.8 to 8.2 respectively whereas the temperature and pH of Yumthang hot springs ranges from 43 to 63°C and 6.5 to 6.8 respectively. Six different samples from each hot springs collected and transferred into sterilized thermostatic bottles. All the samples were kept in sterilized thermostatic flask and were transported to the laboratory in a minimum time.

Isolation and enumeration of thermophilic bacteria
The population of culturable thermophilic bacteria in the 12 different water and sediments were enumerated through enrichment using the standard serial dilution plating technique. For enrichment, 10 mL of each water sample was inoculated directly into 50 mL of nutrient broth, where as in case of sediment samples, 5 g of sediment was mixed with 10 mL of corresponding filter sterilized water and inoculated into 50 mL of nutrient broth. All the samples were incubated at 60°C for 2 h. Aliquots of 100 lL of each enriched samples appropriately diluted and spread on six different culture media plates namely, thermus medium (TM: Yeast extract, 4 g; polypeptone peptone, 8 g; NaCl, O; 20 mg/L) and phosphate buffer, 0.5 mL/L (KH 2 PO 4 , 5.44 g; Na 2 HPO 4 Á12H 2-O, 43 g/L)]; yeast extract tryptone (YET: Casein enzymic hydrolysate, 6 g; yeast extract powder, 3 g/L), Thermus thermophilus medium (TTM: Yeast extract, 4 g; proteose peptone, 8 g; NaCl, 2 g/L), nutrient agar (Peptone, 5 g; NaCl, 5 g; beef extract, 1.5 g; yeast extract, 1.5 g/L) and R2A medium (R2AM: Casein enzymic hydrolysate, 0.25 g; peptone, 0.25 g; casein acid hydrolysate, 0.5 g; yeast extract, 0.5; glucose, 0.5 g; starch soluble, 0.5 g; K 2 HPO 4 , 0.03 g; MgSO 4 Á7H 2 O, 0.5 g; sodium pyruvate, 0.03 g/L) and incubated at 60°C for 6-15 days. Colonies that appeared were purified by repeated streaking to obtain isolated colonies using respective medium plates. The pure cultures were maintained at 4°C as slant and glycerol stock (20%) at -80°C for further use.

Culture conditions and growth pattern of bacterial isolates
Effects of temperature and pH on growth of all the representative isolates were investigated. All the bacterial isolates were grown for 24 h at 40°C, after that 300 lL of bacterial broth were inoculated into micro well plates having thermus liquid medium and incubated in temperature controlled Automated Microbiology Growth Analysis System (Oy Growth Curve Ab Ltd, Finland). The optical density at 600 nm was measured at regular intervals. All the isolates were analyzed in triplicates and growth curves were derived using the analysis software provided with the instrument. CFU have been recorded for each selected isolated after 24 h by inoculating 100 lL of broth on thermus medium.

Screening for thermostable enzymes producing bacterial strains
All the 140 bacterial isolates were screened for the production of extracellular thermostable hydrolases such as protease, amylase, xylanase and cellulase. The primary screening of all isolates for enzymes production was carried out as described earlier (Sahay et al. 2013). The all isolates were inoculated in the basal medium plates (Yeast extract, 1 g; KH 2 PO 4 , 1 g; MgSO 4 Á7H 2 O; 0.1 g; CaCl 2-H 2 O, 0.05 g; NaCl, 5 g; Na 2 CO 3 , 10 g and agar 15 g/L) containing starch (2.5 g/L), xylan (10 g/L), carboxy methyl cellulose (CMC) (5 g/L) and skimmed milk (10% w/v) for amylase, xylanase cellulase and protease enzymes production respectively. Purified bacterial strains were grown in 150 mL flasks containing 50 mL of nutrient broth and 5 lL of culture was spot inoculated on basal medium plates and incubated at 45, 55 and 65°C for 3-5 days. Plates were observed for the formation of clear zone for protease. The clear zone around the colony was observed in flooded with 0.1% Congo red solution for 15 min at room temperature followed by further treatment with 1 M NaCl for cellulase and xylanase activity. The cultures were tested for amylolytic activity using basal medium supplemented with 0.025% starch. After incubation for 5 days, in each plate 0.6% KI solution was added for 30 s. The formation of zone around cultures indicated the amylase activity. The stability of each enzyme was estimated at different temperature and pH. Selected strains were inoculated into 250 mL flasks containing 100 mL basal medium having corresponding polymer as substrate and incubated at its respective optimum growth temperature and pH for 3-5 days. An aliquot of the crude enzyme (cell free extract) was pre-incubated at different temperatures (45, 55, 65, 75 and 85°C) for 30 min at pH 7.0 and activity was measured at its respective optimum temperature. The stability of enzyme activity (SE) at variable temperatures and pH was calculated as per the formula described in our earlier studies (Sahay et al. 2013). All the enzyme assays were conducted in triplicates and data was subjected to analysis of variance (ANOVA) using software SPSS ver. 10.

Results and discussion
The physico-chemical analysis showed specific variations among sediment and water samples collected from Manikaran and Yumthang hot springs. The samples from both springs were acidic in nature; however there was a wide variation in the temperatures of the two springs. In Manikaran, the maximum temperature was as high as 99°C; while in Yumthang hot spring maximum temperature was 63°C. Total six culture media were employed to retrieve the culturable bacteria from both hot springs. A total 140 bacteria were isolated from Manikaran and Yumthang hot springs. The variation was observed in bacterial species obtained from two thermal springs of distinct geographic locations. Similarly, in recent reports on natural extreme environments the culture-based approaches provided thermophiles, with highly significant for biotechnological applications Huang et al. 2011;Khiyami et al. 2012;Sahoo et al. 2015;Sen and Maiti 2014;Stefanova et al. 2016;Yadav et al. 2015c).
Thiobacillus denitrificans, from both springs showed wide pH ranges i.e. 3-8; 4-8, 4-9, 5-8 or 5-9 (Table 3). However, the isolates such as NBM24, NBM6, NBY38, NBY4, NBY5, NBY33, NBY36, NBY16, NBY26, NBY37, and NBY55, 11 isolates have showed a narrow pH range of 5-8. Out of the 51 isolates from both springs, 11 isolates from Manikaran showed tolerances to the pH range 3-8; while 23 (15 from Manikaran ? 8 fromYamthang) showed growth at pH range of 4-8 however only 3 isolates from Yumthang thermal springs have showed broad pH range 4-9 and 5-9. Comparative studied has been done for each isolate for pH and temperature tolerances, it was observed that thermotolerant isolates with growth temperature range 45-65°C showed the pH range between 3 and 9 (Table 3). Extreme environments of high temperatures can be a source for novel species of microbes, as they can tolerate extremes of temperature stress. In recent years, several studies have been conducted to look for the diversity of microbes in extreme high temperatures environments, such as geothermal areas of Deception island, in the South Shetland Archipelago (Llarch et al. 1997); Antarctic Geothermal Soils (Logan and Allan 2008); Fumarole Bay, Deception Island (Muñoz et al. 2011); permafrost volcanic sedimentary rocks (Mironov et al. 2013); Geothermal Habitats in Antarctica (Herbold et al. 2014); Hot springs of Bakreshwar, Balrampur, Chumathang and Vashist, India (Kumar et al. 2014a, b); different hot springs in India (Srivastava et al. 2014;Yadav et al. 2015c).
All the 140 were examined for their ability to produce extracellular hydrolytic enzymes at different temperatures and it was found that protease, amylase, cellulase and xylanase were exhibited by 30, 30, 23 and 18 strains  (Table 3). Among 140, 51 bacterial isolates produced one or more enzymes of protease, amylase, cellulase and xylanase (Fig. 1a, b). Among 51 bacterial strains only, 4, 6, 5, and 3 produced protease, amylase, cellulase and xylanase respectively. Among selected isolates, only 5 (9.8%) bacterial strains (NBM31, NBM71, NBY4, NBY38 and NBY44) were efficient producers of all four type thermostable enzymes (Fig. 1a, b). The selected isolates showed the stability in enzymes activities at 45°C and/or 65°C (Table 3). The culturable bacterial isolates of both hot springs showed thermostable enzyme activities with wide range of pH variation (Table 3). Our present results were in support with the earlier study that demonstrated thermostable cellulase production at an optimum temperature of 50°C by Brevibacillus thermoruber (Derekova et al. 2008). There are many reports on production of thermostable cellulase and xylanase from bacterial isolates from thermal springs/high temperature habitats (Gerasimova and Kuisiene 2012; Kumar et al. 2014b;Pandey et al. 2013;Suman et al. 2015). In general the cellulases and xylanases are of special interest due to abundance of cellulose and hemicellulose in nature, moreover hydrolysis of these substrates require temperature tolerant/thermostable enzymes (Bajaj and Abbass 2011). Thermophilic microorganisms with the ability to produce proteases and amylases have also been reported from geothermal sites or thermal springs (Kumar et al. 2014b). In the present investigation, strains capable of producing thermostable proteases and amylases were identified. The thermophilic bacteria produced thermo-active enzymes, which is of great interest for both fundamental research and industrial applications. Different thermo-active enzymes production by microbes, in this study may find applications in various industries viz. food, detergents, pharmaceutical, biofuels, etc.
Xylanases are the endoactive enzymes which are generally produced in the medium containing xylan and also containing xylanase hydrolysate as the carbon source and attack the xylan chain in a random manner, causing a decrease in degree of polymerization of the substrates and liberating shorter oligomers, xylobiose and xylose. Cellulase free xylanase are of paramount significance in some of industries (Adhyaru et al. 2014;Pandey et al. 2013). Treatment with xylanase at elevated temperature disrupts the cell wall structure, facilitates lignin removal in the various stages of bleaching of paper, Therefore xylanase must lack cellulolytic activity. In present investigation, three thermotolerant bacteria namely Thermobacillus sp NBM6, Paenibacillus ehimensis NBM24 and Paenibacillus popilliae NBM68 were found to produced cellulase-free xylanase (Fig. 1a). There are many report on cellulase-free xylanase production by thermophilic bacteria e.g. Fig. 2 Phylogenetic analysis of thermo-active hydrolytic enzymes producing bacteria obtained from a Manikaran hot springs; b Yumthang hot springs. The 16S rRNA gene sequence based dendrogram representing isolates were constructed by neighbor-joining method. Accession numbers of each isolate was given in parenthesis, and reference sequences having C97% similarity were used as taxonomic representatives. The bootstrap values (1000 replications) were indicated at the nodes Paenibacillus sp. N1 (Pathania et al. 2012) and Cellulosimicrobium cellulans CKMX1 (Walia et al. 2015). Recently, interest in xylanase has markedly increased due its wide variety of biotechnological applications such as pre-bleaching of pulp, improving the digestibility of animal feed stocks, modification of cereal-based stuffs, bioconversion of lignocellulosic material and agro-wastes to fermentable products, clarification of fruit juices and degumming of plant fibers.
The members of phylum Firmicutes were most dominant among the identified bacteria isolated from both thermal springs. The members of phylum Bacteroidetes and proteobacteria (a, c) were less dominant. The present investigation provides evidences that Bacillus and Bacillus derived genera were ubiquitous and predominant under harsh environments of temperatures. Similarly earlier reports also indicated the abundance of members of Firmicutes in extreme environment (Sahay et al. 2011;Verma et al. 2014Verma et al. , 2016aYadav et al. 2016b). Members of the Firmicutes (mainly members of families Bacillaceae and Paenibacillaceae) are widely used in agriculture as plant growth promoting and disease suppressing bacteria, besides their use in industry as a source of enzymes and in medicine (Yadav et al. 2016a, b). Further, in general the bacteria belong to the genera Bacillus and Thermus were mostly reported as aerobic, heterotrophic thermophiles and found in thermal systems with neutral to alkaline pH (Spanevello and Patel 2004). Though Thermus spp. may be predominant heterotrophs in many hot springs (Hjorleifsdottir et al. 2001) in present investigation none of the Thermus species were obtained.
In conclusion, thermo-adapted bacteria have attracted the attention of the scientific community due to their ability to produce thermostable hydrolytic extracellular enzymes.
Microbial extracellular enzymes with optimal activity at high temperature provide opportunities to study the adaptation of life in thermal/high temperatures habitats and the potential for biotechnological exploitation, besides those efficient in multiple plant growth promotion traits at high temperatures. Diverse thermo-active enzymes detected in this study may be used to serve various industrial, agricultural and medicinal purposes.