Sikkim lies in The Great Himalayan Range, which is considered as a relatively young range of mountains in the world. Sikkim is also regarded as a hot spot having huge diversity in flora, fauna and microbial community. Sikkim has several hot springs; however, till date no systematic studies have been conducted on these hot springs which provide an opportunity to study the microbiological studies. In the present study one Hot Spring and one lake were selected for microbiological studies.The hot spring is present in Lingdap, Dzongu region of North Sikkim and the lake namely Lake Tsomgo(Changu)studied is present in Sherethang, region of East Sikkim.Tsongmo Lake is located at an altitude of 12,400 ft. It lies about 37 kms away from Gangtok; this lake is one of the most important spectacular landscapes of Sikkim. The lake is highly revered by the Buddhists and Hindus as a sacred lake. The depth of the lake is 48 feet around and spreading over 1 km, the lake is oval in shape.In fact, Changu Lake is also the place of origin ofLungtse Chu River. Changu Lake is shrouded in myths and legends. It is said that in ancient times the Lamas (Buddhist Saints) used to predict the future by observing the lake’s colour. If the water of the lake had a dark colour, they predicted the future to be dark and gloomy, full of unrest. The faith-healers of Sikkim popularly known asJhakhrisalso visit this lake during Guru Purnima to offer prayers. But humans are not the only visitors to this lake it is also home to Brahmini Ducks and a favourite stop over to other species of migratory birds. Both the Hot Spring and Tsomgo Lake were seamless to study thermophilic and psychrophilic bacteria from them respectively. The main aim of the study was to isolate the thermophilic and psychrophilic bacteria from Hot Spring and Lake respectively and then screen for the amylase producing thermophilic and psychrophilic bacteria among the isolated ones.
The water samples were aseptically collected from the Hot Spring (Tatopani) and also from Tsomgo Lake in sterile containers so as to minimize the contaminations. The water sample will contain many types of microorganisms, but as only the aim of the work was to isolate thermophilic bacteria and psychrophilic bacteria hence the water sample was added to the microbiological media Thermus Broth for thermophiles and Luria Bertani broth for psychrophiles in order to enrichment the microbes. Once the enrichment was over, the samples were streaked into the Thermus Agar plates and Luria Bertani Agar plates and only the bacterial colonies were considered. Now the incubation period was set at 60°C for thermophiles and 15°C for psychrophiles and thus only the thermophilic and psychrophilic bacteria were able to survive these kinds of stressful conditions. This way thermophilic and psychrophilic bacterium was isolated. Bacterial colonies with different morphologies and culture characteristics were considered and obtained as pure culture isolates. On isolation of pure colonies they were preserved at 30 % Glycerol stock. A total of 10 bacterial isolates each in case of thermophiles and psychrophiles were taken for further screening of amylase enzyme producing bacteria.
Screening of Amylase producing Thermophilic bacteria and Psychrophilic bacteria
Amylases which dominate about 25% of enzyme trade have important role in the hydrolysis of starch in various industries. The characteristics of thermophilic organism are their capability in producing thermostable enzyme with high thermostability in production processes and they can maintain their activity longer. Liquid sugar industry needs thermostable α-amylase that can maintain its activity at high temperature, such as in the gelatinization (100-110°C) and liquefaction process (80-90°C). Therefore, continuous researches to obtain many more thermostable α-amylases are needed (68). Cold-active a-amylases isolated from living and metabolically active psychro-tolerant microorganisms confers low activation energies and high activities at low temperature which are favorable properties for the production of relatively insubstantial compounds and therefore enzymatically driven reactions can be carried out at low temperature. The low temperature stability of cold-active amylases has regarded as the most important characteristics for use in industry because of considerable progress towards energy savings but unfortunately these enzymes have lately attracted attention and their diversity, physiology and potential have largely been overlooked. Cold-active amylases cover a broad spectrum of biotechnological applications like additives in processed food industries, additives in detergents, waste-water treatment, biopulping, bioremediations in cold climates and molecular biology applications. Thus, Cold-active amylases are becoming promising enzymes to replace successfully the conventional enzyme for biotechnological industries running at low temperature and serve as a world-wide choice for biotechnologists, microbiologists, biochemists, pharmacists, biochemical and process engineers. ( 69,70,23).
In the present study10 isolates from thermophiles and 10 from psychrophiles were streaked on 1% Starch agar plates and were incubated at 60°C and 15°C respectively to screen the amylase producers, out of which, 6 thermophilic isolates labelled as; Z26, Z27, Z29, Z30, Z34 and Z35 and 4 psychrophilic isolates labelled as;CH01,CH02,CH05 and CH07 showed positive result. The positive isolates showed clear zone or halo zone surrounding their colonies inferring that they were able to hydrolyse starch with the help of enzyme amylase (Table 1).
Microbiological Observations (Morphology, Culture Characteristics and Staining)
The drop of water sample was directly mounted on cavity slides and was observed under the Compound microscope to check the presence of motility of the microorganisms present in the sample and also to observe the viable ones. There was motility in the water sample from both Hot Spring and Lake. Also some viable microorganisms were observed. Then once the isolates were isolated, even for them their motility was examined. It was observed that in case of thermophiles all the isolates were motile but interestingly in case of psychrophiles only two isolates (CH01 and CH02) were motile rest two were non-motile (Table 2).
The thermophilic isolates, when observed under compound microscope it was shown that all isolates were short rods. Similarly in case of psychrophiles all the isolates were rods except the isolate CH07 which was a coccus (Table 2). All the thermophilic isolates formed Pale Yellow coloured colonies however, all the psychrophilic isolates formed White Creamish coloured colonies. The colonies were circular having entire margins, flat elevation and opaque density (Table 3). All the thermophilic isolates formed slight clouding without ring and formed abundant sediments whereas in psychrophilic isolates formed heavy clouding without ring and also formed scanty sediments when grown in broth conditions (Table 4).
On simple staining with Methylene Blue, the general shape and size of the isolates was observed. All the thermophilic isolates were rod shaped and all the psychrophiles were rod shaped except CH07 which was a coccus (Table 5). Similarly, to understand the cellular structure of the isolates, Gram Staining was performed. It was observed that all the thermophilic as well as psychrophilic isolates were Gram positive bacteria (Table 5). On Endospore staining with Malachite Green, three of the thermophilic isolates showed the presence of endospores rest were unable to form spores. However none of the psychrophilic isolates showed presence of spores when observed under compound microscope (Table 5).
The further key to identify a bacterium and distinguish it from the rest is the detection of various enzyme activities of the isolates. Depending on the enzyme activity, they can be classified into their respective groups as per the Bergey’s Systemic Classification.
None of the bacterial isolates produced bubbles when treated with 3% H2O2. They showed negative catalase activity (Table 10).The positive catalase test infers that the microorganisms produce enzyme catalase, which degrades the by-products of aerobic respiration, like H2O2 and Superoxide Dismutase that are lethal to the cell. Similarly among ten thermophilic isolates only six isolates Z26, Z27, Z29, Z30, Z34 and Z35 were amylase positive. However in case of psychrophiles only four isolates CH01, CH02, CH05, and CH07 were amylase positive (Table 10).
Fermentative degradation of various carbohydrates by microbes under anaerobic conditions is carried out in a fermentation tube. A Fermentation tube is a culture tube that contains a Durham tube (a small tube placed in an inverted position in the culture tube) for the detection of gas production as an end product of metabolism. The Fermentation broth contains the ingredients of nutrient broth, a specific carbohydrate and a pH indicator (Phenol red), which is red at neutral pH (7.0) and turns yellow at or below a pH of 6.8 due to the production of an organic acid According to Bergey’s classification of bacteria, carbohydrate utilization is an important characteristic feature of bacteria through which differentiation and group classification can be done. Thus, carbohydrate fermentation test was carried out with four different carbohydrates Lactose, Mannitol, Dextrose, and Sucrose and each of them were substituted and checked as a carbohydrate substrate for the different isolates.All the individual thermophilic and psychrophilic isolates were inoculated in Carbohydrate Fermentation Broth and were kept at 60°C and 15°C for 48 hours. After incubation, the broths were observed for any growth and color change.
In case of thermophiles the results showed that all the isolates were unable to utilize Lactose and Sucrose and all the isolates were able to utilize Dextrose, also none of the isolates except one isolate Z29 were able to utilize Mannitol. However, in case of psychrophiles none of the isolates were able to utilize Lactose, Mannitol and Sucrose. Also Dextrose was not utilized by any psychrophiles except one isolate CH05 (Table 9).
Growth Profile of the isolates
Although the bacterial isolates were able to grow at wide range of temperatures for example in case of thermophilic bacteria they were able to grow with a range of temperatures from 40°C, 50°C, 60°C, 70°C, and 80°C when grown on Thermus broth. However, when Optical Density (O.D.) of the broth cultures were measured at 600 nm and 660 nm, most of the thermophilic isolates showed highest growth at 60°C and thus this temperature might be regarded as their optimum temperature and thus these might be true thermophilic bacteria. Similarly psychrophiles also were able to grow at wide range of temperatures from 0°C, 10°C, 15°C, 20°C and 30°C. however, two isolates CH01 and CH02 shows highest growth at 20°C and rest two isolates CH05 and CH07 isolates shows highest growth at 10°C. Thus these temperatures might be considered as the optimum temperatures for these isolates. Also it may be concluded that former two isolates are psychrotrophic bacteria as per Morita 1975.
Growth profiles of both thermophilic and psychrophilic bacteria at various pH ranges were investigated.The results have shown that four of the thermophilic isolates Z26, Z29, Z34 and Z35 showed highest growth at pH 7 i.e. neutral pH whereas, the rest two isolates Z29 and Z30 showed highest growth at pH 9 i.e. alkaline pH. Thus these pH conditions might be the optimum pH conditions for these isolates respectively. Thus it may be concluded that the two of the thermophilic isolates are Alkalophiles. Similarly in case of psychrophiles all the isolates showed highest growth at pH 7 and thus might be their optimum pH conditions. (Table 7). Also the growth profiles of all the isolates were checked against various NaCl concentrations. 0.5% NaCl and 2% NaCl concentrations were taken as control for psychrophilic and thermophilic bacteria respectively. The results have interestingly shown that the both thermophilic and psychrophilic isolates were able to grow highly at 0% NaCl concentration thus might be their optimum saline conditions. Thus this may also conclude that these isolates might be halophiles.(Table 8).
Partial purification and activity of amylase
A partially purified crude enzyme extract was prepared. The preparation was further characterized and used to check amylase activity. The α-amylase activity was checked according to Miller, 1959 using 3,5- dinitrosalicylic acid (DNS) as color reagent. The color formation occurs by the release of reducing sugars from degradation of starch. The optical density was checked at 580nm. The results showed that in case of thermophiles, four of the isolatesZ27, Z30, Z34 and Z35 were high producers of amylase and rest two Z26 and Z29 were moderate producers. The results were analysed as per colour changes an optical density readings. Similarly in case of psychrophiles only two isolates CH01 and CH07 of the four isolates were high producers of amylase whereas other two isolates CH02 and CH05 were moderate producers. The thermophilic and psychrophilic isolates were examined comparatively and it was interestingly shown that the thermophiles were high producers of amylase as compared to psychrophiles as shown in Table 11.