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Microbial diversity of soil from two hot springs inUttaranchal Himalaya

Bhavesh Kumar, Pankaj Trivedi, Anil Kumar Mishra, Anita Pandey*,Lok Man S. Palninn

GB Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora, 263 643 Uttaranchal, India

Accepted 13 January 2004

AbstractSoil samples collected from two hot springs, Soldhar and Ringigad, both located in theGarhwal region of Uttaranchal Himalaya were analysed for their physical, chemicaland microbial components. The alkaline pH, total absence of carbon and nitrogen, andhigh temperature were features common to soil samples from both sites. The Soldharsamples contained higher amounts of Cu, Fe and Mn. Ringigad soil was devoid of Cu,but had much higher phosphate. While the optimum incubation temperature forisolating the maximum microbial counts from soil samples from the two sites was501C, microbial growth in broth was also observed when incubated at 801C.Microscopic examination revealed three types of microbial populations, i.e., bacteria,yeast and filamentous organisms. The soil samples were found to be dominated byspore forming rods. Out of 58 aerobic isolates, 53 were gram positive bacilli. Grampositive anaerobic oval rods were also observed up to 601C. Soil dilution platesrevealed the presence of antagonistic and phosphate solubilizing populations.& 2004 Elsevier GmbH. All rights reserved.

Introduction

Microorganisms are important components in allecosystems; their ubiquity is mainly due to smallsize and easy dispersal, the ability to grow andmultiply also under anaerobic conditions, theirmetabolic versatility and flexibility to utilize a broadrange of nutrients, and the ability to tolerate andcolonize otherwise unfavourable locations, e.g.,those with extremes of temperature, pH, redox

potentials, salinity, and humidity. The microorgan-isms that thrive under extreme environments, frompolar deserts to geothermal springs, are referred toas extremophiles (Stolp, 1988; Delong, 1998; Seack-bach, 2000; Staley and Reysenbach, 2002).

Hot springs are manifestations of geologicalactivity and represent extreme environments, thatemerge in the Himalayan region. Outlet tempera-ture range about 451C and above. Soil samples fromtwo hot springs, Soldhar and Ringigad, located in

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KEYWORDSMicrobial diversity;Hot springs;Thermophile;Bacilli;Filamentous commu-nities;Uttaranchal Himalaya

*Corresponding author. Anita Pandey, G.B. Pant. Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora, 263643 Uttaranchal, India.E-mail address: anita@gbpihed.nic.in (A. Pandey).nnPresent address – Senior Scientific Advisor - Biotechnology & Project Director, State Biotechnology Programme, Govt. of

Uttaranchal, Biotech Bhawan, PO Haldi, Pantnagar, 263 146 Uttaranchal, India.

0944-5013/$ - see front matter & 2004 Elsevier GmbH. All rights reserved.doi:10.1016/j.micres.2004.01.004

Microbiological Research 159 (2004) 141–146

the Chamoli district of the Garhwal region inUttaranchal Himalaya, were collected and analysedfor their physical, chemical and microbial compo-nents.

Materials and methods

Sampling sites

Soil samples were collected from two hot springs,Soldhar (latitude 391 290 2500, longitude 791 390 2900;altitude 1900m amsl) and Ringigad (latitude 301 330

1400, longitude 791 400 0.0600; altitude 1850m amsl),both located in the Chamoli district of the Garhwalregion of Uttaranchal Himalaya. The approximatearea of both these sites was about 45m2. While thehot water outlet (901C) was present in the middleof the mound at Soldhar, the mouth of the hotspring was located along slope towards a cliff atRingigad creating a temperature gradient along theslope. At this site the soil temperature around thewater outlet was 781C. While Soldhar was devoid ofany vegetation, growth of algal mats and a speciesof fern was recorded at Ringigad. The details of thetwo sites can be seen in the location map (Fig. 1).

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Figure 1. Location map. Bar¼ 1 cm.

142 B. Kumar et al.

Physical and chemical analyses of soilsamples

The soil samples were analysed for physical(particle size, colour, pH and water holdingcapacity) and chemical (carbon, nitrogen, phos-phorus, sulphur, potassium, zinc, copper, iron andmanganese) properties using standard methods(Allen, 1974; Carter, 1993). Carbon content andtotal nitrogen were determined by the wet-oxida-tion method and Kjeldahl technique (TecatorKjeltec Auto 1030 Analyser), respectively. Phos-phorous (P) and sulphur (S) were determined usinga spectrophotometer (Uvikon 931, Italy). The totalpotassium (K) was determined by flame photometry(Systronics Mediflame 127). Zn, Cu, Fe and Mn wereestimated by atomic absorption spectrophotometry(AAS vario6), following the method by Carter(1993). The colour of the soil was determined usingthe Munsell soil colour chart (Munsell, 1992). Alldeterminations were conducted using triplicatesamples.

Enumeration of soil microbes

For the enumeration of soil microbial population,isolations were carried out on non-selective as wellas selective media using the serial dilution techni-que (Johnson and Curl, 1972). Tryptone yeastextract agar, potato dextrose agar, and actinomy-cetes isolation agar were used for the isolation ofbacteria, fungi and actinomycetes, respectively.Thiosulphate agar, sulfate reducing medium andsulphur medium were used for selective isolation ofS-metabolizing microorganisms. Pikovskaya med-ium was used for detecting the phosphate solubiliz-ing microbes. The Petri dishes were incubated at211, 281, 501, 601, 751 or 801C in the dark. Forincubation at 501C and above, the medium wasgelled with 2.0% agar (instead of 1.6%, w/v).Isolations were also carried out on diluted tryptoneyeast extract medium (1/2, 1/4 and 1/8 strength).Observations were recorded after one week ofincubation. For isolating obligate thermophilicorganisms, a mixture of 1 g soil and 9ml tryptone

yeast extract broth in culture tubes were incubatedat 751 or 801C. The tubes were observed forturbidity 36 h after incubation. Enumeration ofanaerobic microorganisms was carried out onappropriate medium using anaerobic chambersand incubated at 211, 281, 421, 501 or 601C, andthe observations were recorded after one week. Allexperiments were conducted in triplicate. Allmedia used were from Hi Media, Bombay.

Microscopic observations

Representative colonies from agar plates werepurified through repeated subculture and thensubjected to microscopic examination. Smearswere also prepared from the broth cultures formicroscopic observations.

Physiological properties

The temperature, pH and salinity tolerance ofisolates was determined by incubating cultures atdifferent temperatures and by modifying pH andsalt concentration of the media, respectively. Fordetection of antimicrobial activity of isolates,inhibition zones formed on the dilution plates wererecorded.

Results and discussion

The physical and chemical properties of soil arepresented in Table 1; the soil from both sites wasslightly alkaline, light brown in colour with thesame particle size of 460 mm). The water holdingcapacity of the soil from Soldhar was almost threetimes that of Ringigad; carbon, nitrogen andpotassium could not be detected in the soilsamples. The soil from Soldhar had higher amountsof Cu, Fe and Mn; Cu was absent from the Ringigadsample. Soil samples from the Ringigad site werefound to possess a higher phosphorus content.

While the microbes could be isolated from thesoil samples on various media (Table 2) across abroad temperature range (21–801C), the optimum

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Table 1. Physical and chemical properties of soil samples from two hot springs in Garhwal Himalaya

Study site Physical properties Chemical constituents

WHC (%) pH Particle size % (dry wt. basis) ppm

C N P S K Zn Cu Fe Mn

Soldhar 193.0 8.2 460 m F F 0.037 0.001 F 2.443 1.784 159.8 10.3Ringigad 61.7 8.1 460 m F F 0.120 0.002 F 2.716 F 11.7 0.12

F¼ not detected.

Microbial diversity of soil from two hot springs in Uttaranchal Himalaya 143

temperature for the isolation of microbial popula-tions was 501C. Moderate numbers were isolated ondiluted tryptone yeast extract agar plates, whichalso helped to enumerate a few colonies that weremorphologically different from those that devel-oped on full strength medium. Actinomycetes orfungal colonies could not be observed in any of themedia used. On potato dextrose and actinomycetesisolation agar, usually used for preferential isola-tion of fungi and actinomycetes, only bacterialpopulations were observed. No growth was ob-served on various selective media used, namelythiosulphate agar, sulphate reducing medium andsulphur medium, indicating the absence of Smetabolizing populations. Some filamentous organ-isms were isolated from soil samples from bothsites, using broth culture at 751C or 801C. Anaero-bic populations were isolated at 21–501C from soilsamples of the two sites (Table 3).

A total of 59 (58 aerobic and 1 anaerobic)morphologically distinct representative colonieswere obtained as pure cultures. On the basis ofmicroscopic examination, three types of microbialpopulations were observed (bacteria, yeasts andfilamentous organisms from Soldhar, and bacteriaand filamentous organisms from the Ringigad). Outof 58 aerobic isolates, 53 isolates were found to bebacilli (4–6 mm in length and 0.8–1.6 mm in width;Fig. 2A). The anaerobic colonies also showed

similar rod shaped bacteria (3.5–6.0 mm in lengthand 0.9–1.4 mm in width; Fig. 2B). Most bacilli wereobserved with an endospore. Yeast colonies fromSoldhar samples revealed the presence of branchedmycelium with spores (spore size 3–7 mm; Fig. 2C).Filamentous organisms revealed distinct structures,and were of three types in Soldhar samples: (i) longfragmented filaments (fragment number varyingfrom 9 to 49; fragment size from 1.6 to 8.0 mm inlength and 0.6 mm in width; terminal fragmentshowing bulbous ends; Fig. 2D); (ii) spiral branchedfilaments (46 mm in length and 0.8 mm in width; Fig.2E); and (iii) long thin filaments (42 mm in lengthand 0.5 mm in width; Fig. 2F). Only one type offilamentous growth showing branched spiral fila-ments with cytoplasmic inclusions was observed insamples collected from Ringigad (37.5 mm in lengthand 1.6 mm in width; Fig. 2G). Out of the four typesof filamentous growth, only two (shown in Figs. 2Dand F) could be cultured and are being maintainedin broth at 601C. The filaments shown in Figs. 2Eand G were obtained only during the initialisolation in broth but could not cultured aftertransfer. A number of soil dilution plates exhibitedinhibition zones on TY agar plates indicating thepresence of antagonistic bacteria. Formation ofclear zones around some bacterial colonies onPikovskaya medium indicated the presence ofphosphate solubilizing populations in the samples.

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Table 2. Microbial populations in soil samples from Soldhar and Ringigad, incubated at different temperatures

Microbial populations (cfu� 104 g�1)

Media Soldhar Ringigad

Bacteria Yeast Bacteria Yeast

241C 301C 501C 601C 751C 801C 241C 301C 241C 301C 501C 601C 751C 801C 241C 301C

TYA 27 38 44 15 3 2 13 10 24 37 49 20 3 1 F FTYA (1/2 strength) 20 23 31 17 3 1 F F 28 27 39 29 2 F F FTYA (1/4 strength) 18 25 38 12 2 2 F F 32 33 38 33 1 F F FTYA (1/8 strength) 9 13 28 31 4 2 F 3 21 39 13 39 F 1 F FPDA 23 29 38 7 1 F 31 33 27 31 35 36 2 F F FAIA 49 40 50 8 F F 17 5 43 44 48 43 F F F F

cfu¼ colony forming units; TYA¼Tryptone yeast extract agar; PDA¼Potato dextrose agar; AIA¼Actinomycetes isolation agar.

Table 3. Anaerobic populations in the soil samples from Soldhar and Ringigad, incubated at different temperatures

Microbial populations on anaerobic agar

Study site Cfu� 103 g�1 Cfu� 102 g�1 Cfu� 101 g�1

211C 281C 421C 501C 601C

Soldhar 20 25 8 14 8Ringigad 10 18 7 11 6

cfu¼ colony forming units.

144 B. Kumar et al.

The isolates have been grouped on the basis oftheir temperature, pH, and salt requirements(Table 4). The isolations conducted at mesophilicand thermophilic temperatures indicated suitabil-ity of such environments for growth and multi-plication of thermophiles. Colonization ofendospore forming bacilli under harsh environ-ments is well known (Sneath, 1986). Occurrenceof pink filamentous community in Octopus springand black filamentous community associated withthermal springs at Calcite spring in Yellow NationalPark has been recorded. Thermal areas protected

in the Yellow National Park, USA have receivedattention concerning microbial ecological research(Reysenbach et al., 1994, 2000; Reysenbach andCady, 2001).

Isolates belonging to the mesoplilic range aremaintained by routine subculture or stored in 10%glycerol in a deep freezer (�201C). Isolates ofthermophilic range could be maintained by pro-longed incubation at the optimum temperature onagar plates (agar concentration 2%), till the agargets dried (usually after 3 months). The culturescould be easily revived by inoculating small piece of

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Figure 2. Microscopic observations. A Aerobic gram positive bacteria. B Anaerobic gram positive bacteria. C Yeast cellswith mycelia. D Long chains of filaments with terminal bulbous ends. E Spiral branched filaments. F Long thin filaments.G Branched filaments with cytoplasmic inclusions. Bar¼ 2.5 mm.

Microbial diversity of soil from two hot springs in Uttaranchal Himalaya 145

dried agar on fresh medium and incubation at theoptimum temperature.

The extremophiles, in particular thermophilesand alkalophiles, have been a subject of exten-sive investigation. These are particularly soughtfor the production of industrially useful enzymeslike protease, cellulase, cellulase free xylanase,and amylase, and for their industrial applica-tions in the detergent, leather, pulp andpaper, and other industries (Pennisi, 1997;Srinivasan and Ingale, 1999; Budhiraja et al.,2002). For example, Thermus aquaticus was thefirst such organism to be used for the production ofTaq polymerase, followed by Pyrococcus furiosis,used for the production of Pfu (Mani and Sakker,2001).

High temperature, alkaline nature and poornutrient status of the soil at both sites seem toprovide a unique environment for the developmentof specialized microbial communities. Identifica-tion of areas with extreme environments including,the isolation of extremophiles, and investigationson the associated microbial wealth for biotechno-logical applications are of great importance, bothfor basic and applied research. The Indian Hima-layan region is particularly valuable in terms ofproviding both the extreme ‘cold’ and ‘hot’ sitesfor exploring microbial diversity. To the best of ourknowledge this is the first report of microbialdiversity associated with hot spring sites in theUttaranchal Himalaya. The isolates obtained fromthe two sites are maintained in the laboratory forfurther investigations.

Acknowledgements

The Department of Biotechnology and the UnionMinistry of Environment and Forest, Govt. of India,New Delhi are thanked for providing financialsupport. Ms. Kiran Lata Tripathi is acknowledgedfor drawing the location map. VPKAS, Almora isthanked for technical assistance.

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Table 4. Grouping of the total aerobic isolates based ondifferent physiological parameters

Temperature Rangea Number of strains

Thermotolerant 30–551C 46

Thermophile 45–651C 9

Hyperthermophile 55–851C 3

Halophile 0.5–2.0% 32.0–5.0% 1610% 38

Alkaliphiles 8–9 3110 1211 15

Acdiophiles 6 295 174 12

aBased on Seackbach (2000).

146 B. Kumar et al.