Full Text: 1 Introduction Hot springs diversity have been studied worldwide, which are reported as a normal source of several thermophilic bacteria. Geothermal springs are significant spots of microbial biodiversity, having the capacity to produce various enzymes for industrial, agriculture and medical research processes and are also helpful to get novel genes as well as molecules (Saxena et al., 2016; Sahay et al., 2017). Thermophiles are heat-loving microbes, therefore they can grow at 45° C to 75° C with optimum temperature range between 50° C and 60 ° C. Some of the thermophilic microorganisms tolerate a higher temperature for their survival and growth, whereas other thermophiles tolerate a comparatively lower temperature (Hartmann et al., 1989; Pelczar et al., 1993; Panikov et al., 2003; Eze et al., 2011). The special attraction of hot water springs is the ecological variation of different types of microorganisms and their molecular characteristics (Adiguzel et al., 2011; Genc et al., 2015). Thermophilic bacteria represent both Gram-positive and Gram-negative group and they also belongs to aerobic and anaerobic groups of bacteria. Due to their various valuable applications, researchers have focused their research to find out new genus and species of microorganisms around the world (Yoneda et al., 2013; Cihan et al.,  2014; Aanniz et al., 2015). There are several hot springs available in Gujarat. Some studies have been carried out on thermophilic bacteria in TuvaTimba, Lasundra, Tulsishyam, Dholera and Unai (Kikani & Singh 2011; Chudasama 2012; Joshi et al., 2013; Gohel et al., 2013; Dudhagara et al., 2014). Present study was conducted at Dholera hot water spring and focus on seasonal variations of thermophilic organism’s isolates from this ecosystem. Information regarding the microbial community of Dholera hot spring is available in scarcity. The aim of present study was to characterize the thermophilic bacteria from Dholera hot spring in three different seasons and the interest was to find out novel species at this site. 2 Materials and Methods 2.1 Sample collection Hot spring water samples were collected from Dholera hot spring (22°15'0"N   72°11'27"E), Gujarat. Dholera is an ancient port-city in Gulf of Khambhat, 30 km from Dhandhuka city of Ahmedabad district, Gujarat. Water samples were collected in sterile glass bottles from Dholera hot water spring in three different seasons: monsoon (September), winter (January) and summer (April).  Samples were transported in a thermocol box to the laboratory and processed on the same day of the sampling and then stored at room temperature. 2.2 Physicochemical analysis Temperature and pH of collected samples were recorded at the time of sampling using portable pH meter.  Conductivity, TDS and salt of the collected samples were measured in the laboratory using Oakton Thermo, Multi-parameter PCS Testr 35, and Thermo Fisher Scientific. 2.3 Isolation of bacteria Samples were processed for isolation of bacteria on the same day of the sample collection. Thermophilic microorganisms were isolated directly from water samples by serial dilution technique using various media like Nutrient agar medium (0.5% Peptone, 0.3% Beef extract, 0.5% NaCl, 2.0% Agar), Luria-Bertani medium (1.0% Tryptone, 1.0% NaCl, 0.5% yeast extract, 2.0% Agar), ATCC Thermus medium (0.5% NaCl, 0.5% Peptone, 0.4% Beef extract, 0.2% Yeast extract, 2.0% Agar), Starkey`s sulphate reducing medium (Himedia M1981), Sulphate reducing medium (Himedia M803) and Medium77 medium (0.5% K<sub>2</sub>HPO<sub>4</sub>, 1.0% NH<sub>4</sub>Cl, 0.1% CaCl<sub>2</sub>.2H<sub>2</sub>O, 0.1% MgSO<sub>4</sub>.7H<sub>2</sub>O, 5.0% Sodium lactate, 1.0% Yeast extract, 5.0% FeSO<sub>4</sub>.7H<sub>2</sub>O, 1.0% Sodium thioglycolate, 1.0% Ascorbic acid, 2.0% Agar ( Patil et al., 2014). All the plates were incubated at 45 ± 2°C and 50 ± 2°C. After the growth, morphologically distinct colonies were selected and subcultured on respective medium to get pure isolates. Then from these well isolated purified colonies, 20% glycerol stocks of bacterial cultures were prepared for preservation and further study on the isolates. 2.4 The conventional method for identification and characterization of the isolates The selected isolates were observed for morphological and growth characteristics by the conventional methods as outline below. The bacterial isolates were characterized by Gram staining and observed under light microscope. The thermophilic isolates were identified by conventional, physiological methods and characterised based on biochemical identification tests. These tests are characterized based on Gram nature, shape, temperature, pH, catalase production test, casein utilization test, starch utilization test, citrate utilization test, Sugars profile like D-Glucose, D-Fructose, Maltose, Mannose, Sucrose, Mannitol, Lactose, Xylose, Galactose, H<sub>2</sub>S production test and Sulphate reduction test as per the Bergy’s manual (Hensyl, 1994).  2.5 Diversity indices A range of diversity indices profile, cluster analysis and similarity in diversity were generated using statistical software Paleontological Statistics (PAST) considering the results of biochemical tests (Hammer et al., 2001). 2.6 Genomic DNA isolation Bacterial broth cultures (2-5 ml) were grown in log phase at 45 ± 2 °C and 50 ± 2°C for 24 hours. Overnight cultures (2.0 ml) were centrifuged at 10,000 – 15,000 X g for 15 minutes. The supernatant was decanted and pellet was collected and re-suspended in 1 ml of TE buffer. The DNA was quantified by NanoDrop spectrophotometer by taking absorbance at 260 and 280 nm (Nucleic acid software). The size of DNA was estimated by agarose gel (0.5% W/V) electrophoresis subsequent staining with ethidium bromide and visualisation in U.V. illumination by Gel Documentation System (Gene snap software). Genomic DNA was isolated from the obtained pallets from pure cultures using the miniprep method described by Wilson with some modifications (Moore et al., 2004; Wilson, 1987). The obtained DNA pellets stored at 4 °C for further analysis. 16S rRNA gene sequence analysis was done at Chromous Biotech (Bangalore). 3 Results 3.1 Physicochemical characteristics Physicochemical characteristics of water samples of Dholera hot spring are listed in Table 1. Dholera water spring was covered with cement concrete construction, hence the soil sample was not available. The temperature of the water was 40° C to 50° C in Dholera. The TDS (Total Dissolved Salt) value varied from 3.1 to 3.7 ppt (parts per thousand) in Dholera. Observed pH value was 7.1 to 7.9 at Dholera sampling point. Conductivity of the studied water samples were reported between 4.4 to 4.9 mS (milli-Siemens). Whereas seasonal physicochemical variations were concerned, the temperature in summer was at high of 50 ° C and at low of 40 ° C in winter, which is significant. Overall seasonal differences in conductivity, TDS and salt concentration in Dholera samples were in the range of 10%. 3.2 Microbial analysis Total 18 morphologically different isolates were isolated and purified from the water samples collected from Dholera hot springs during summer, monsoon and winter season using Nutrient agar medium, Luria-Bertani medium, ATCC Thermus medium, Starky`s medium, Sulphate reducing medium and Medium77 medium. Out of these isolates; eight isolates were Gram-positive while rest ten were Gram-negative. A wide range of pigmented colonies was observed as shown in table 2. The colours of the pigmented colonies observed were distributed over cream, brown, light brown, black and yellow. 3.3 Metabolic activity based on the biochemical test Various metabolic activities were assessed to have a broad view of the characterization of bacteria, on the behalf of these isolated strains were identified. Out of 18 isolated isolates, catalase production was showed by 8 isolates. Further, 18, 18 and 9 isolates were able to utilize casein, starch and citrate respectively. H₂S production and sulphate reduction properties were reported in nine isolates while sugars metabolism activity varied in all the isolates. The results of the water samples are summarized in Figure 1. Catalase production was dominant in summer and monsoon seasons isolates, while it was indicated significantly lower in winter season`s isolates. On the other hand, all the three seasons isolates showed positive results for the casein and starch utilization. Citrate utilization was dominant in all three seasons. Sugars profile like fructose, maltose and glucose were positively utilized in all Dholera summer season`s isolates. Rest of the monsoon season`s isolates indicated positive results for utilization of fructose and glucose. Galactose and glucose were also positive in all isolates of winter season`s samples. In the summer and winter season`s isolates, xylose showed positive results in only one isolate. H₂S production and Sulphate reduction were dominant in all three seasons. 3.4 Cluster analysis and diversity indices based on metabolic activity Diversity study of various isolates was carried out in terms of 15 biochemical tests on the basis of their results dendrograms were prepared, and are shown in Figure 2. The blue colour label indicates summer season, green colour indicates the monsoon season and pink colour indicates winter season. All the isolates obtained from water samples collected from Dholera were divided into 2 major group as A and B (Figure 2). B group showed sulphate reducing isolates in all three seasons based on their biochemical results.  Morphologically distinct isolates from Dholera thermal ecosystem were studied to determine their similarity. From Dholera samples total 18 colonies were selected from water samples.  Irrespective of the seasonal collection distinct isolates range from 6 to 9 in each sample (Data not shown). Among the different isolates studied the maximum similarity between any 2 isolates was 86 - 87 per cent. Except for DW – 49 and DW – 50 which showed 100 per cent similarity and they were identified as Bacillus subtillis based on 16S rRNA partial gene sequence analysis. Diversity indices data in terms of Taxa_S, Individuals, Dominance, Simpson, Shanon, Menhinick, Margalef, Berger-Parker and Chao-1 were also studied for all the samples collected in the different seasons from the Dholera hot spring ecosystem (Table 3). 3.5 Molecular identification of selected organisms From the total 18 isolates 6 colonies were selected randomly and these isolates were identified based on 16S rRNA gene sequence analysis and results are shown in Table 4. Among the 6 identified cultures three belong to Bacillus genus, one to Brevibacillus, one to Brevibacterium and one to Tepidimonas. The details of NCBI identification numbers are given in Table 4. 4 Discussion and Conclusion  In some hot springs, the physicochemical parameters have been observed to vary with the seasonal effect. While in other studies it has been reported that, the physicochemical parameters can be stable with no significant changes in seasonal effect. Moreover seasonal variation in microbial diversity is low in hot springs due to the high temperature of the springs (Ferris & Ward, 1997; Mackenzie et al., 2013; Briggs et al., 2014; Prieto-Barajas et al., 2017). The prevalent conditions in hot springs such as high water temperature, pH, sulphur concentration, salinity, conductivity and other physicochemical parameters are known to permit the development of selected bacterial communities (Nayak, 2013). In the current study, biochemical tests determined the metabolic activity of the bacterial cells (Goh et al., 2012; Nayak, 2013). Diversity indices tools have been used for the population of bacterial diversity, species richness and also better understanding about microbial community (Kim et al., 2017). Kikani et al. (2015), calculated the various diversity indices of Tulsi Shyam hot spring, Gujarat. They have given information about the evenness in their distribution, community structure and species richness (Kikani et al., 2015). 16S rRNA gene sequencing techniques have been used for the identification of bacteria and phylogenetic analysis as the "gold standard" (Ludwig & Schleifer 1999). It has been reported that molecular-based identification of thermophilic bacteria by 16S rRNA gene sequencing is useful at the genus level identification (Sahin et al., 2009). It has earlier been reported that some thermophilic Bacillus species are a source for useful enzymes for industrial and commercial purposes (Rahman et al., 1994; Rao et al., 1998; Dudhagara et al., 2014). In previously published reports, Bacillus spp from the genus Bacillus have been claimed to be largest and the best studied genus (De Boer et al., 1994; Kolsto et al., 2009; Nayak, 2013). Species of genus Bacillus have been found in Lasundra, TuvaTimba, Unai hot springs of Gujarat and these bacillus species have been used for various applications like production of enzyme, biosurfactant, and thermostability etc (Kikani & Singh, 2011; Joshi et al., 2013; Gohel et al., 2013; Dudhagara et al., 2014). According to Hadad et al. (2005) thermophilic bacterium Brevibacillus brostelensis isolated from soil, and this strain plays an important role in biodegradation of polythene (Hadad et al.,  2005). B. brostelensis, thermophilic bacteria has been reported from Saudi Arabia hot spring and possesses enzyme having ability to degrade polythene substrates (Khalil et al., 2018). Tepidimonas taiwanensis strain was first reported in a Southern Taiwan hot water spring and it was claimed to be a novel species. Among the Tepidimonas genus, this strain was able to produce alkaline protease enzyme for industrial application  (Chen et al., 2006). The focus of this preliminary study was a comparison of the seasonal diversity of Dholera hot spring. Further present investigation also focused on the identification of seasonal diversity to get novel species. Based on bacterial identification study, almost similar numbers of isolates were found in all seasons of Dholera hot spring.16S rRNA sequence-based molecular identification shows a good amount of correlation with the biochemical identification of thermophilic bacteria. The presence of T. taiwanensis species is reported for the first time in the Dholera hot springs of Gujarat. Based on our morphological and biochemical studies the isolates divided in two major groups such as sulphate reducers and sulphate non-reducers. Acknowledgement Authors are thankful to “Xavier Research Foundation” for supporting our research and providing grant and good lab facilities. Conflict of interest Author declares that there is no conflict of interest.