Chapter-3

Materials & Methods

35

3.1 Study area

Cyanobacterial samples were collected from 9 hot water springs and 22 locations in

cold desert (Lahaul-Spiti) of North-Western Himalayas.

3.1.1 Hot water springs

Six hot water springs at Tattapani, Manikaran, Kheer Ganga, Vashisht, Kasol, and

Kalath in Himachal Pradesh and 3 hot water springs; Surya Kund at Yamunotri, Janki

Chatti and Gauri Kund at Kedarnath in Uttarakhand were visited for the collection of

cyanobacterial samples (Fig. 1, Table 4).

3.1.1.1 Tattapani hot water springs

Tattapani (31º 14ʹ 57.10ʹʹ N, 77º 05ʹ 21.83ʹʹ E) is situated at Shimla-Mandi Highway 50

km from Shimla at an altitude of 675 m on the right bank of Satluj river in Mandi

district of Himachal Pradesh. These hot water springs emerge from a stretch of about 1

km along the river bank. Tattapani has developed into a major Hindu pilgrimage centre.

3.1.1.2 Manikaran hot water springs

Manikaran (32º 01ʹ 37.18ʹʹ N, 77º 20ʹ 42.43ʹʹ E) is situated at an altitude of 1710 m on

the bank of river Parvati about 35 km from Bhuntar in Kullu district of Himachal

Pradesh. Several hot water springs emerge at various locations spreading to a distance

of about 1.5 km from the old bridge on Parvati river to Brahmaganga along the river.

The springs at Manikaran come out with pressure and are very hot. An experimental

geothermal energy plant has also been set up here by government of India.

3.1.1.3 Kheer Ganga hot water spring

Kheer Ganga (31º 59ʹ 30.16ʹʹ N, 77º 30ʹ 35.17ʹʹ E) is situated at an altitude of 2865 m

about 25 km upwards from Manikaran, Kullu district and about 15 km from village

Varshoni. At this location hot water spring emerges out from the rocks on the mountain

top. This place is approachable only by tracking through a thick forest in five to eight

hours.

3.1.1.4 Kasol hot water spring

Kasol (32º 00ʹ 45.45ʹʹ N, 77º 18ʹ 56.93ʹʹ E) is situated at an altitude of 1595 m, about 32

km from Bhuntar on way to Manikaran, Kullu district of Himachal Pradesh. The hot

water spring is about 1 km upstream on the right bank of Parvati river towards

Manikaran and is connected to Kasol by footpath. Hot water emerges only at one

location at Kasol.

Chapter III: Materials and Methods

36

Fig. 1. Location of hot water springs of North-Western Himalayas.

3.1.1.5 Vashisht hot water spring

Vashisht (32º 15ʹ 13.94ʹʹ N, 77º 11ʹ 24.43ʹʹ E) is situated at an altitude of 2020 m near

Manali, Kullu district of Himachal Pradesh. The village is 3 km from Manali at the

foot hills of laterite and conglomerate rocks. The hot water kunds (ponds) are in the

centre of the village.

3.1.1.6 Kalath hot water spring

Kalath (32º 13ʹ 18.01ʹʹ N, 77º 11ʹ 29.90ʹʹ E) is situated at an altitude of 1806 m, 6 km

away from Manali on Kullu-Manali highway on the right bank of Beas river, Kullu

district of Himachal Pradesh.

37

Table 4. Name and coordinates of selected hot water springs of North-Western

Himalayas.

Location Latitude Longitude Altitude (m)

Tattapani 31o14’57.10’’ 77

o05’21.83’’ 675

Manikaran 32o01’37.18’’ 77

o20’42.43’’ 1710

Kheer Ganga 31o59’30.16’’ 77

o30’35.17’’ 2865

Kasol 32o00’45.45’’ 77

o18’56.93’’ 1595

Vashisht 32o15’13.94’’ 77

o11’24.43’’ 2020

Kalath 32o13’18.01’’ 77

o11’29.90’’ 1806

Surya Kund 30o59’50.99’’ 78

o27’44.38’’ 3188

Janki Chatti 30o58’33.37’’ 78

o26’15.18’’ 2565

Gauri Kund 30o43’50.67’’ 79

o03’59.13’’ 3505

3.1.1.7 Surya Kund hot water spring

Surya Kund (30º 59ʹ 50.99ʹʹ N, 78º 27ʹ 44.38ʹʹ E) is situated at an altitude of 3188 m at

famous Hindu piligrim Yamunotri, Uttarkashi district of Uttarakhand. This hot water

spring is unique being close to freezing glacier of Yamunotri but the water is boiling

hot. This place is accessible by 13 km trek from the town of Hanuman Chatti followed

by 6 km trekking from Janki Chatti. There are two trekking routes from Hanuman

Chatti to Yamunotri, the one along the right bank of Yamuna river and other route

which lies on the left bank of Yamuna river goes via Kharsali.

3.1.1.8 Janki Chatti hot water spring

Janki Chatti (30º 58ʹ 33.37ʹʹ N, 78º 26ʹ 15.18ʹʹ E) is situated at an altitude of 2565 m

enroute to Yamunotri 7 km from the town Hanuman Chatti, Uttarkashi district of

Uttarakhand. This hot water spring is inside a temple dedicated to Lord Shiva.

3.1.1.9 Gauri Kund hot water spring

Gauri Kund (30º 43ʹ 50.67ʹʹ N, 79º 03ʹ 59.13ʹʹ E) is situated at an altitude of 3505 m,

on the way to Kedarnath, 28 km from Ukhimath, Uttarkashi district of Uttarakhand.

This hot water spring is inside a temple

3.1.2 Cold desert area

Cold desert area of the North Western region of India comprises Lahaul-Spiti, parts of

Kinnaur and Chamba districts of Himachal Pradesh along with Ladakh region of

Jammu and Kashmir and Uttarkashi area of Uttarakhand.

38

Lahaul-Spiti cold desert investigated during the present study is part of the

trans-Himalayan region of India. Lahaul-Spiti covers an area of ca. 13,883 km2 and is

situated between 31°44’57” and 32°59’57” N and 76°29’46” and 78°41’34” E.

Geographically it is the largest district in the state of Himachal Pradesh. It has

Chamba on its west and Kangra, Kullu and Kinnaur districts on its south. Climatic

conditions are typical of dry temperate and alpine zones. Altitudinal range is between

2,400 m-6,400 m, rainfall is scanty, and the area remains covered with snow for more

than 6 months in a year. Temperature during winter falls nearly to -30 ºC and even the

temperature of summer nights is sub zero. Except for the periods of rain or snowfall,

the air is dry and strong winds blow most of the year.

Lahaul-Spiti district is divided into Lahaul Valley and Spiti Valley. The two

valleys remain cutoff from each other for more than six months due to heavy snowfall

at Kunzum Pass (4550 m). Rohtang Pass (3980 m) is the gateway to Lahaul Valley

and it connects the Valley with the Kullu district. The Lahaul region has three valleys,

the Chandra Valley, the Bhaga Valley and the Chandra-Bhaga Valley. Spiti is at

higher altitude with more rugged terrain than Lahaul. The valley is a remote high area

situated on Tibetan plateau, with almost no rain and remains snow-covered for more

than six months. Spiti river passing through Spiti Valley originates from the Kunzum

Pass and descends into Sutlej River. Samples were collected from 22 sites, including

the four high altitude lakes Chandra Tal, Deepak Tal, Surya Tal and Sissu Lake and

two glaciers, Glacier I (near Batal) and Glacier II (near Trilokinath), from cold desert

region of Lahaul-Spiti (Fig. 2, Table 5).

3.2 Sample collection

Water, soil and cyanobacterial mats were collected between July 2007 and November

2011from the selected sites. Necessary information such as name of sampling site,

form of sample, date, time, weather conditions, co-ordinates and altitude etc. were

noted at each site.

3.2.1 Collection of algal samples

Samples were collected in the form of water sample, soil/stones and algal mats with

the help of sterilized spatula, forceps, blade or scalpel. Algal mats were handpicked or

lifted gently with a flat knife/blade. Planktonic algae were collected with the help of

dropper/forceps/mesh. Sterilized polythene bags and plastic bottles were used for the

collection of algal samples.

39

Fig. 2. Sampling locations in the cold desert area of Lahaul-Spiti in North-Western

Himalayas

3.2.2 Collection of water/soil samples

For the analysis of physico-chemical parameters, water samples were collected in 1 L

pre-sterilized plastic bottles, while soil samples were taken in sterilized polythene

bags. Some characteristics of water were noted on the site at the time of sampling

while rest of the physico-chemical characteristics were studied in the laboratory.

40

Table 5. Name and coordinates of selected localities of cold desert area of North-

Western Himalayas.

3.3 Physico-chemical analysis of water samples

Water temperature, pH and conductivity were noted/measured in the field during

sampling. Water temperature was noted by a thermometer at the site of collection.

The pH of the samples was measured by a digital pH meter (Orion; Thermo Fisher

Scientific, USA) and electrical conductivity determined by a conductivity bridge

(Systronics, USA). Water samples in the laboratory were analyzed for hardness,

alkalinity, chloride, calcium, sodium, potassium, magnesium, total phosphate,

ammonium nitrogen and nitrate nitrogen by standard methods available in literature

(Mackereth et al., 1978; APHA, 2005).

Location Latitude Longitude Altitude (m)

Soil/mats in flowing water

Gondla 32o30’29.59’’ 77

o01’27.82’’ 3163

Dorni 32o21’30.27’’ 77

o18’42.14’’ 3310

Gramphoo 32o23’34.27’’ 77

o15’37.14’’ 3315

Batal 32o21’31.24’’ 77

o37’10.14’’ 3995

Tandi 32o33’21.05’’ 76

o58’31.52’’ 2955

Lossar 32o26’22.15’’ 77

o45’24.22’’ 4060

Bar 32o02’45.45’’ 78

o05’23.95’’ 3315

Kaja 32o13’40.29’’ 78

o04’15.21’’ 3668

Tabo 32o05’37.54’’ 78

o23’00.64’’ 3285

Lari 32o04’49.32’’ 78

o25’13.72’’ 3323

Koksar 32o24’50.91’’ 77

o14’05.81’’ 3130

Udaipur 32o43’27.37’’ 76

o39’54.34’’ 2640

Trilokinath 32o41’02.35’’ 76

o41’38.92’’ 2922

Darcha 32o40’27.23’’ 77

o11’51.44’’ 3340

Keylong 32o34’19.44’’ 77

o02’04.47’’ 3145

Kunzam 32o23’46.23’’ 77

o38’11.77’’ 3145

Glaciers

Glacier I 32o37’24.31’’ 76

o37’43.60’’ 4255

Glacier II 32o23’42.23’’ 77

o33’59.38’’ 4584

Lakes

Chandra Tal 32º28’30.65” 77º37’1.42” 4300

Suraj Tal 32º45’02.09” 77º24’52.79” 4853

Deepak Tal 32º45’11.02” 77º15’20.03” 3752

Sissu Lake 32º28’36.48” 77º07’37.94” 3010

41

3.3.1 Estimation of total hardness

Total hardness of water was determined by the EDTA method in alkaline condition.

EDTA and its sodium salts form a soluble chelated complex with certain metal ions.

Calcium and magnesium ions develop wine red colour with Erichrome Black T in

aqueous solution at pH 10.0 ± 0.1. When EDTA is added as a titrant, calcium and

magnesium divalent ions get complexed resulting in sharp change in colour from wine

red to blue which indicates end-point of the titration.

Reagents:

(i) Buffer solution: 16.9 g of NH4Cl was dissolved in 143 mL of NH4OH and 1.25 g

of magnesium salt of EDTA was added to it, and diluted to 250 mL.

(ii) Inhibitor: 4.5 g of hydroxylamine hydrochloride was dissolved in 100 mL of 95%

ethyl alcohol.

(iii) Erichrome Black T indicator: 0.5 g of dye was mixed with 100 g NaCl.

(iv) 0.01M EDTA: 3.723 g of EDTA sodium salt was dissolved in 100 mL distilled

water

(v) Standard calcium carbonate solution (1mL = 1 mg CaCO3)

Procedure:

Fifty milliliter filtered water sample was taken in a 250 mL conical flask and 2 mL of

buffer solution was added to it followed by 1 mL of inhibitor. 0.2 g Erichrome Black

T indicator was added to it and mixed by shaking, then it was titrated with 0.01M

EDTA till wine red colour of solution was changed to blue, volume of EDTA solution

used was noted as ‘A’. Blank was titrated against 0.01 M EDTA solution and volume

of EDTA used was noted as ‘B’. Calcium carbonate solution was used as standard and

volume of 0.01 m EDTA titrant used was noted as ‘D’.

Calculations:

Total hardness as CaCO3 mg L−1 =C × D

mL of sample × 1000

Where, C = (A-B) volume of EDTA used for sample

D = mg CaCO3 equivalent to 1 mL EDTA titrant

3.3.2 Estimation of total alkalinity

Alkalinity of water sample was estimated by titrating the sample with standard

solution of H2SO4, at pH 8.3 using Phenolphthalein as an indicator (carbonate

42

alkalinity) and then titrated further to second end point to pH 4.5 using methyl orange

as an indicator (total alkalinity).

Reagents:

(i) 0.02 N Sulphuric acid

(ii) Phenolphthalein indicator: 0.5 g of phenolphthalein in 50 mL of 95% ethanol and

50 mL of distilled water, added to it 0.02 N NaOH drop wise until faint pink

colour appeared.

(iii) Methyl orange indicator: 0.1 g of methyl orange in 200 mL of distilled water.

Procedure:

Fifty milliliter filtered water sample was taken in a 250 mL conical flask. To it, 0.2

mL of phenolphthalein indicator was added, after shaking colour changed to pink, it

was titrated with 0.02N H2SO4 until the pink colour disappeared. The disappearance

of pink colour was noted as end point ‘A’. This indicated phenolphthalein carbonate

alkalinity. To determine the total alkalinity, 2-3 drops of methyl orange were added to

the same sample and continued titration till the colour was changed from yellow to

orange. The amount of titrant used was noted as ‘B’.

Calculations:

Phenolphthalein carbonate alkalinity mg L−1 =mL of titrant ′A′

mL of sample × 1000

Total alkalinity mg L−1 =mL of titrant ′B′

mL of sample × 1000

3.3.3 Estimation of chloride content

Chloride content in water sample was determined by titration with standard silver

nitrate solution, using potassium chromate as indicator.

Reagents:

(i) Potassium chromate indicator solution: 5 g of K2Cr2O7 in distilled water, AgNO3

was added to it till definite red precipitate was formed and then allowed to stand

for 12 h, filtered and was diluted to 100 mL.

(ii) 0.0141M Silver nitrate titrant: 2.395 g AgNO3 were dissolved in 1000 mL of

distilled water.

Procedure:

Fifty milliliter of filtered water sample was taken in a conical flask. To it, 1 mL of

K2Cr2O7 indicator solution was added and mixed thoroughly. It was titrated against

43

0.0141M AgNO3 solution. Appearance of pinkish yellow colour was noted as end

point ‘A’. Volume of titrant used for blank was noted as ‘B’.

Calculations:

Chloride mg L−1 =(A − B) × M × 35.45

mL of sample × 1000

A = mL of titrant used for sample

B = mL of titrant used for blank

M = Molarty of AgNO3

3.3.4 Estimation of calcium content

Calcium content in water samples was determined by titration with standard EDTA

titrant. Murexide (ammonium purpurate) indicator forms pink solution with Ca2+

.

When titrated with EDTA, pink solution is changed to purple.

Reagents:

(i) Murexide indicator: 10 mg ammonium purpurate was mixed with 5 g NaCl by

grinding to fine powder.

(ii) 0.01 M EDTA titrant: 3.723 g of sodium salt of EDTA was dissolved in 100 mL

distilled water

(iii) 2 N Sodium hydroxide: 8 g NaOH were dissolved in 100 mL distilled water.

Procedure:

Fifty milliliter of filtered water sample was taken in a conical flask. To it, 2 mL of 2 N

NaOH solution and 0.2 gm of murexide indicator were added and mixed by shaking,

pink colour appeared. It was titrated with 0.01 M EDTA and appearance of purple

colour was noted as end point.

Calculations:

Calcium mg L−1 =mL of titrant used × 400.5 × 1.05

mL of sample

3.3.5 Estimation of magnesium content

The concentration of magnesium in water was determined as the difference between

total hardness and amount of calcium in water, multiplied with factor 0.244.

Magnesium (mg L-1

) = (A-B) × 0.244

Where, A = total hardness (mg L-1

) and B = amount of calcium (mg L-1

)

44

3.3.6 Estimation of total dissolved solids (TDS)

Residue remained after the evaporation and subsequent drying of a known volume of

water sample in an oven at specific temperature 100-150 °C represents total solids.

Requirements:

(i) Electrically heated temperature controlled oven

(ii) Petri-plate

Procedure:

Fifty milliliter of filtered water sample was taken in pre weighed Petri-plate (W1) and

placed in oven at 150 ºC for 24 h. After 24 h, Petri-plate was removed from oven to

room temperature for cooling, and again weighed (W2). The weight of petri-plate was

taken in milligram (mg).

Calculations:

Total dissolved solids mg L−1 =(W2 − W1)

mL of sample × 1000

3.3.7 Estimation of total phosphate

Organically combined phosphorus and all other forms of phosphates including

polyphosphate were first converted to orthophosphate by digestion with strong acid

(Perchloric acid). In acidic condition, orthophosphate reacts with ammonium

molybdate to form molybdophosphoric acid. The blue colour developed after addition

of ammonium molybdate was measured at 660 nm in Spectrophotometer D20 (Thermo

Scientific, USA).

Reagents:

(i) 5% ammonium molybdate: 5 g of ammonium molybdate was dissolved in 100 mL

distilled water.

(ii) 0.2% aminonaphthol sulphonic acid: 0.5 g of 1,2,4-aminonaphthol sulphonic acid,

30 g sodium bisulphite and 6 g crystalline sodium sulphite were dissolved in

distilled water by shaking and final volume made to 250 mL, Kept it overnight

and filtered it before use.

(iii) Phenolphthalein indicator: Dissolved 0.5 g of Phenolphthalein in 500 mL of 95%

ethyl alcohol. Added to it 500 mL distilled water. Then Added drop-wise 0.02 N

NaOH till faint pink colour appeared.

(iv) Perchloric acid

(v) 2 N NaOH

45

Procedure:

Fifteen milliliter filtered water sample was evaporated in 50 mL test tube at 100 oC.

Cooled and added 1 mL of perchloric acid, and mixed well. Again kept at 100 oC to

evaporate the perchloric acid. Residue was allowed to cool, and then 15 mL of

distilled water was added to it, followed by one drop of phenolphthalein indicator. It

was titrated against 2 N NaOH until faint pink colour was just restored. Then 2 mL of

above solution was taken and 1 mL of molybdate and 0.5 mL of sulphonic acid were

added to it. The reagents were thoroughly mixed and allowed to stand for 5 minutes,

blue colour developed. Absorbance was recorded at 660 nm. Concentration of

phosphate was calculated from standard curve made with KH2PO4.

3.3.8 Estimation of nitrate content

Nitrate reacts with phenol disulphonic acid and produces a nitro-derivative which in

alkaline solution develops yellow colour.

Reagents:

(i) Phenol disulphonic acid: 25 g phenol was dissolved in 150 mL concentrated

H2SO4.

(ii) 12 N KOH: 67.3 g of KOH was dissolved in 100 mL distilled water.

Procedure:

Twenty milliliter filtered water sample was taken in 50 mL test tube and evaporated at

100 oC in an oven. The test tube was cooled at room temperature, 0.5 mL of phenol

disulphonic acid was added to it and residue was dissolved with the help of glass rod.

Then 5 mL distilled water was added to it followed by 2 mL of 12 N KOH, mixed

well by shaking and kept for 5-10 min, yellow colour developed was recorded at 410

nm. Concentration of nitrate was calculated from the standard curve prepared with

KNO3.

3.3.9 Estimation of ammonium content

Ammonium produces a yellow coloured compound when it reacts with alkaline

Nessler’s reagent.

Reagents:

(i) Nessler’s reagent: 10 g HgCl2 and 7 g KI were mixed well and dissolved in small

quantity of water, then 50 mL chilled solution of 4 N NaOH was added, and

diluted with distilled water to 100 mL final volume. Kept overnight, filtered

supernatant was stored in amber coloured bottle.

46

Procedure:

Fifty milliliter filtered water sample was taken in a conical flask. To it, 2 mL

Nessler’s reagent was added. The reagent was thoroughly mixed and was allowed to

stand for 5 minutes. A pale yellow colour developed. Absorbance was recorded at 640

nm. Concentration of ammonium was calculated from the standard curve prepared

with NH4Cl.

3.3.10 Estimation of sulphate content

Sulphate ions are precipitated in an acetic acid medium with Barium chloride so as to

form Barium sulphate crystals of uniform size. Absorbance of the BaSO4 suspension

was measured in a spectrophotometer.

Requirements:

(i) Buffer solution: Dissolved 30 g MgCl2.6H2O, 5 g CH3COONa.3H2O, 1 g KNO3

and 20 mL acetic acid (99%) in 500 mL distilled water and made 1000 mL final

volume.

(ii) Barium chloride

Procedure:

Twenty five milliliter filtered water sample was taken in 100 mL conical flask. 10 mL

buffer solution was added to it and mixed well. 0.2 g of BaCl2 crystals was added to it

and flask was constantly stirred with the help of magnetic stirrer for one min.

Turbidity appeared was recorded against the blank at 510 nm. Concentration of

sulphate was calculated from the standard curve prepared with NaSO4.

3.3.11 Estimation of potassium content

Potassium content in water samples was determined using flame atomic absorption

spectrophotometer (Shimadzu Corp., Japan).

Requirements:

(i) Atomic absorption spectrometer.

(ii) Standard solution of potassium: 0.019 g potassium chloride was dissolved in 100

mL distilled water; 1 mL = 100 μg K

Procedure:

A known volume of water sample was diluted with distilled water and used for

analysis. Standard curve was prepared by graded concentration of potassium chloride

and by noting the absorbance at 766 nm. Concentration of potassium in the sample

was determined from the standard curve.

47

Calculations:

Potassium (mg L-1

) = K (mg L-1

) from standard curve × Dilution

Dilution =mL sample + mL disilled water

mL sample

3.3.12 Estimation of sodium content

Sodium concentration in water samples was determined using flame atomic

absorption spectrophotometer. The method employed was same as for the

determination of potassium except for the absorbance was noted at 589 nm and

sodium chloride was used as standard.

(Standard solution of sodium: 0.025 g Sodium chloride was dissolved in distilled

water, 1 mL conc. HNO3 was added to it, and final volume 100 mL was made with

distilled water; 1 mL = 100 μg Na).

3.4 Standard microbiological techniques

3.4.1 Glassware and chemicals

Glassware used during the present study was of Borosil make. Glassware were dipped

in chromic acid for 24 h before washing. After washing with liquid soap and tap water

these were oven dried. Chemicals used in media preparation were obtained from

Sigma-aldrich, Merck India, and SD fine Chemicals, India.

3.4.2 Composition of nutrient medium

For propagation and maintenance of cultures, Chu-10 medium (Safferman and Morris,

1964) was slightly modified and used. The composition of medium is given in Table

6.

3.4.3 Sterilization

The nutrient medium, solutions and glassware were steam sterilized in an autoclave at

15 lbs pressure inch-2

for 15 min.

3.4.4 Inoculations

All inoculations were done over a flame in a laminar air flow chamber (Klenz Aids,

India). The chamber was initially surface sterilized with rectified spirit.

3.4.5 Isolation and purification of cyanobacterial strains

Collected algal samples were initially washed with sterilized double-distilled water

and examined under the light microscope to evaluate the content of cyanobacterial

species. Cyanobacteria from the samples of hot water springs were isolated at 42 ±2

oC, while cyanobacteria from samples of cold desert area were isolated at 15 ±2

oC.

48

Table 6. Composition of modified Chu-10 growth medium (Safferman and Morris, 1964).

Chu-10 medium (Safferman and Morris, 1964) Modified Chu-10 medium

Macronutrients g L-1

Macronutrients g L-1

Micronutrients (A6) mg L-1

Ca (NO3)2.4H2O 0.232 CaCl2.2H2O 0.232 H3BO3 0.5

MgSO4.7H2O 0.025 MgSO4.7H2O 0.025 ZnSO4.7H2O 0.05

Na2CO3 0.02 Na2CO3 0.02 MnCl2.4H2O 0.5

Na2SiO3.5H2O 0.044 Na2SiO3.5H2O 0.044 CuSO4.5H2O 0.02

K2HPO4 0.01 K2HPO4 0.01 MoO3 0.01

Ferric citrate 0.0035 Ferric citrate 0.0035 CoCl2 0.04

Citric acid 0.0035 Citric acid 0.0035

KNO3 1.10

The medium used during the present study contained 1.5 mM CaCl2.2H2O in place of Ca(NO3)2.4H2O 1.0 mM used by Safferman and

Morris (1964). Other modification includes change in concentration of ferric citrate, citric acid and additional of KNO3 10 mM and A6

micronutrients of Allen and Arnon (1955).

49

A small portion of each sample was inoculated in 20 mL sterilized liquid nutrient

Chu-10 medium for culture enrichment. The medium was supplemented with

cyclohexamide (100 µg mL-1

) to eliminate eukaryotic contaminants. Clonal and

axenic cultures of cyanobacteria were established by repeated sub-culturing on Petri

plates containing solidified Chu-10 medium (Stanier et al., 1971). Small portions of

samples were taken and serially (10, 100, 1000 time) diluted from enrichment culture.

Then 0.1 mL diluted sample was spread thoroughly over the surface of nutrient agar

plates with the help of a ‘L’ shaped glass rod. The plates were incubated under ideal

growth conditions in the BOD incubator (NSW, India) at 42/15 ±2 oC until colonies

appeared. Individual colonies were picked up with the help of a capillary tube under

binocular microscope and suspended in fresh liquid nutrient medium. After 7 days of

growth, 0.1 mL samples were again plated on nutrient agar and incubated. Distinct

healthy colonies were again picked up and inoculated in fresh liquid medium. After

establishment of unicyanobacterial isolates, cultures are being maintained in liquid

medium and agar slants.

3.4.6 Propagation of cultures

The cultures from hot water springs were propagated in the basal medium (pH 7.6) in

a fluorescent tube light fitted BOD incubator (NSW, India) at 42 ±2 °C, whereas the

cultures from cold desert area were propagated in basal medium (pH 8.0) in a BOD

incubator at 15 +2 °C. The cultures were illuminated for 14/10 h light/dark cycle, with

fluorescent tubes providing a light intensity of 45 µmol photon flux cm-2

s-1

on the

surface of culture vessels. The cultures were hand shaken at least twice a day to keep

them in a homogenous state.

3.5 Polyphasic characterization

3.5.1 Morphological identification of cyanobacteria

All cyanobacterial isolates were observed under light microscope and identified

following the taxonomic descriptions given in monographs, floras and books

(Desikachary, 1959; Anagnostidis and Komárek, 1985; Komárek and Anagnostidis,

1998, 2005; Komárek and Hauer, 2013). Measurement of filaments/trichomes/cells

was taken with the help of an ocular in a microscope whose index was predetermined.

Morphological taxonomic features included trichomes/filaments shape; cell

dimensions; presence/absence of sheath, thickness; shape, size and position of

akinetes/heterocyst, if present etc. Images were captured using a Nikon Eclipse 80i

Microscope fitted with a digital imaging system.

50

3.5.2 Molecular characterization of cyanobacterial isolates

3.5.2.1 Genomic DNA isolation

Genomic DNA of cyanobacterial isolates was isolated using HiPurATM

Genomic DNA

Miniprep Purification Spin Kit (Himedia, India) following the procedure given by

manufactures. Exponentially growing cyanobacterial cultures were pelleted at 10,000

× g for 3 min and suspended in 400 L of lysis buffer with 20 L of RNase solution.

The mixture was incubated for 10 minutes at 65 C with gentle mixing at regular

intervals and 130 L of precipitation buffer was added to the lysate. After mixing, it

was incubated on ice for 5 min. The lysate was centrifuged for 5 min at 14,000 × g

and the supernatant was loaded to Hishredder column placed in 2.0 mL collection tube

and centrifuged for 2 min at 14,000 × g. The flow-through was transferred to a new

2.0 mL collection tube, 1.5 volumes of binding buffer was added to the cleared lysate

and mixed by pippetting. Placed HI-elute Miniprep Spin column in 2.0 mL collection

tube, added 650 L of above mixture and centrifuged for 1 min at 8,000 × g. The

flow-through was discarded. Same step was repeated for the remaining mixture. The

HI-elute Miniprep Spin column was placed in a new 2.0 mL collection tube and 500

L of wash buffer was added to it and centrifuged for 1 min at 8,000 × g. The flow-

through was discarded and another 500 L of wash buffer was added, centrifuged for

3 min to dry the column. Then 25 L of elution buffer (ET) was added to the column

and incubated for 5-6 min at room temperature and centrifuged at 12,000 × g for 1

min. The step was again repeated with another 25 L of ET buffer. The quality of

DNA was checked on 0.7% agarose gel prepared in 1X TAE buffer. The eluted DNA

was stored at 20 C.

3.5.2.2 Agarose gel electrophoresis

Reagents:

Agrose, 1X TAE buffer, Ethidium bromide (50 mg mL-1

), Loading dye etc.

Procedure:

Three fifty milligram agarose was taken in 250 mL conical flask and 50 mL 1X TAE

buffer was added to it and gel solution was prepared by boiling the content. Gel

solution was allowed to cool about to 55 o

C and 4 μL ethidium bromide was added,

mixed well and poured in gel casting plate and proper comb was inserted. After

polymerization of gel, comb was removed carefully and the gel placed in the gel

running tank of electrophoresis system having 1X TAE buffer. A drop of 2 μL

51

glycerol loading dye was placed on a piece of parafilm, 4 μL of a sample was taken

and added on one of the drops of loading dye. After proper mixing with pipette,

sample was loaded in the well on the gel. After placing a cover on gel tank the system

was run on 60 mA for 30-40 min. Gel was visualized using BioRad Geldoc Photo

documentation system.

3.5.2.3 Amplified Ribosomal DNA Restriction Analysis (ARDRA)

3.5.2.3.1 Restriction digestion

Three tetra-cutter Hpa II, Hae III, Alu I and one penta cutter Hinf I restriction

endonucleases were employed for the restriction analysis of PCR product of 16S

rRNA gene of cyanobacterial isolates. Restriction reaction consisted of 1.5 µL of 10X

restriction enzyme buffer, 1U restriction enzyme, 10 µL PCR product and 18.2 mΩ

water to make final volume 15 µL. The reaction mixture was incubated at 37 ºC for 3

h for all the four enzymes. Reaction products were resolved on 2% (w/v) agarose gel

prepared in 1X TBE buffer stained with ethidium bromide (10 µg mL-1

). The

restriction patterns were visualized and photographed with Digidoc (Biorad, CA)

under UV light.

3.5.2.3.2 Analysis of PCR-RFLP

The size of restriction digest products of 16S rRNA gene resolved as bands on

agarose gel was estimated by comparing with the 50 bp DNA ladder (MBI Fermentas,

Vilinius). Fingerprints of restriction digest generated on gel were analyzed by

recording them in the binary form, i.e. 1 = presence of a band and 0 = absence of a

band. Dendrograms of these fingerprints were generated using the hierarchic

Unweighted Pair Group Means Average (UPGMA) method (Adhikari et al., 1999;

employing TREECON software v1.3b (Yves Van de Peer, University of Anterwerp).

3.5.2.4 Amplification of marker genes

3.5.2.4.1 16S rRNA gene

16S rRNA gene was amplified using cyanobacteria specific primers CYA106F and

CYA781R (Nubel et al., 1997) (Table 7). The total PCR reaction mixture was 50 µL,

comprising 200 µmol L-1

dNTPs, 50 µmol L-1

each primer, 1X PCR buffer, 3 U Taq

polymerase, and 100 ng genomic DNA. The thermo-cycling conditions involved an

initial denaturation at 94 °C for 4 min, followed by 35 cycles of 1 min at 94 °C, 1 min

at 52 °C, 2 min at 72 °C and final extension at 72 °C for 8 min. The PCR product was

resolved on 1.2% agarose gel and analyzed against 100 bp plus molecular weight

marker.

52

Table 7. Sequence of primers used in present study.

Gene Primer Sequence (5’-3’)

16S rRNA CYA 106 F

CYA 781R (a)

CYA 781R (b)

CGG ACG GGT GAG TAA CGC GTG A

GAC TAC TGG GGT ATC TAA TCC CAT T

GAC TAC AGG GGT ATC TAA TCC CTT T

cpcB-IGS-cpcA PC β F

PC α R

GGC TGC TTG TTT ACG CGA CA

CCA GTA CCA CCA GCA ACT AA

rbcL GF-AB

GR-D

GF-C

GR-E

GAR TCT TCI ACY GGT ACY TGG AC

TGC CAI ACG TGG ATA CCA CC

CTT YAC YCA AGA CTG GGC CTT C

AAC TCR AAC TTG ATT TCY TTC C

3.5.2.4.2 Rubisco large subunit (rbcL) gene

The rbcL gene was amplified using the primers GF-AB and GR-E (Tomitani et al.,

2006) (Table 7). PCR reaction mixture of 50 µL contained 100 ng DNA template, 1X

Go Taq® Green Master Mix (Promega Corporation, USA) and 50 µmol L

-1 each

primer. Amplification was done by initial denaturation at 94° C for 5 min, followed

by first 10 cycles of 10 s at 94 °C, 20 s at 50-60 °C and 40 s at 72 °C, and next 25

cycles of 30 s at 94 °C, 1 min at 52 °C and 1 min at 72 °C and final extension at 72 °C

for 7 min.

3.5.2.4.3 cpcB-cpcA intergenic spacer region (PC-IGS)

The phycocyanin IGS and flanking coding regions were amplified using the primers

PCβF and PCαR (Neilan et al., 1995) (Table 7). PCR reaction mixture of 25 µL was

comprised of 50 ng DNA template, 1X Go Taq® Green Master Mix (Promega

Corporation, Madison, USA) and 25 µmol L-1

each primer. Amplification was done

by initial denaturation at 94° C for 5 min, followed by 40 cycles of 10 s at 94 °C, 20 s

at 55 °C and 40 s at 72 °C, and final extension at 72 °C for 10 min.

3.5.2.5 DNA extraction from the gel

Amplified products of respective genes were extracted from the gel using gel

extraction kit (GeneJETTM

Gel Extraction Kit, Fermentas) following the procedure

given by manufactures. Gel containing the DNA fragments was excised using clean,

sharp scalpel. The gel slice was placed into a pre-weighed 2.0 mL tube and weight of

the gel was recorded. Sufficient binding buffer was added in 1:1 (volume: weight)

53

ratio. Gel mixture was incubated at 55 C for 10 min to completely dissolve the gel.

The solubilized gel solution was transferred to the GeneJETTM

Purification column

and centrifuged for 1 min. The flow-through was discarded and the column was

placed in the same collection tube, and 100 L of binding buffer was added to the

GeneJETTM

purification column and centrifuged for 1 min. The flow-through was

discarded and the column was placed into the same collection tube. Then 700 L of

Wash buffer was added to the GeneJETTM

Purification column and centrifuged for

1 min. The flow-through was discarded and column was placed in the same collection

tube and dried by centrifugation for 1 min to remove any residual ethanol. The

GeneJETTM

Purification column was transferred to a new 2.0 mL collection tube, 30

L of distilled water was added, incubated for 5-6 min at room temperature, and

centrifuged at 12,000 × g for 1 min. The step was again repeated with another 30 L

of distilled water. Agarose gel electrophoresis was done with 2 L of extracted DNA

to check the quality.

3.5.2.6 Sequencing of gene(s)

Purified PCR product of the marker genes was subjected to direct sequencing using a

Big-Dye Terminator Cycle Sequencer and ABI Prism 310 Genetic Analyzer (Applied

Biosystems, USA). The sequencing PCR reaction was carried out in total volume of 5

µL, which included 1 µL of 5X sequencing buffer, 1 µL of Big-Dye Terminator

Premix, 1 µL of primer (5 pmol) and 2 µL of purified DNA. Thermal cycling

conditions were initial denaturation at 96 oC for 3 min, followed by 30 cycles of 94

oC

for 10 s, 50 oC (16S rRNA) 55

oC (PC-IGS) & 52

oC (rbcL) for 40 s and 60

oC for 4

min.

The unincorporated dye terminators were removed from the sequencing

reaction using a Montage SEQ96 Sequencing Reaction Cleanup Kit (Millipore Corp,

USA). The purified sequencing products were transferred into 96-well injection plate.

The sequence was elucidated on 3130X Genetic analyzer (Applied Biosystems, USA).

The sequences obtained were analyzed with SEQUENCHERTM

and the ambiguities

were removed by visual editing. Sequences obtained were compared with the NCBI

GenBank database using the BLASTn/BLASTx (http://www.ncbi.nlm.nch.gov)

facility of the National Center for Biotechnology Information. The sequences of

uncultured organisms were excluded.

54

3.6 Phylogenetic analysis

An initial BLAST search of the NCBI GenBank database for sequences of

cyanobacterial isolates was done, which provided candidate sequences to compare the

relationships of the isolated cyanobacteria to previously characterized cyanobacterial

species. Multiple alignments were created with reference to the selected GenBank

sequences using MEGA 5.05 which implements the ClustalW multiple alignment

algorithms. Alignment positions at which one or more sequences had gaps or

ambiguities were omitted from the analysis. Using the remaining informative sites, a

phylogenetic tree was constructed by neighbor-joining (NJ) max-mini branch-and-

bound analysis using MEGA 5.05 and was used to illustrate the relationships among

generated sequences for the representative cyanobacteria, where the NJ tree branch

lengths were estimated using the maximum composite likelihood method for rooted

trees. To evaluate the robustness of branches in the inferred tree, one thousand

replicates were used for bootstrap re-sampling from which the overall NJ consensus

trees were generated. Subgroups with greater than 50% consistency in the consensus

tree are labeled at the respective nodes.

3.7 Diversity analysis

3.7.1 Relative abundance The relative abundance of a particular cyanobacterial species was calculated by

employing the following formula:

Relative abundance = X

Y× 100

Where, X = total number of samples collected

Y = number of samples from which a particular cyanobacterium type was isolated.

3.7.2 Diversity Index

Shannon-Wiener (1948), Simpson (1949), Margalef (1958), Pielou (1966) and Jaccard

(1912) indices were used to study cyanobacterial diversity of the study area.

Shannon-Wiener Index (Hʹ) = − 𝑝𝑖 ln(𝑝𝑖𝑠

𝑖=1)

Where, p is the proportion (n/N) of individuals of one particular species found (n)

divided by the total number of individuals found (N), ln is the natural log, Σ is the

sum of the calculations of each species, and s is the number of species.

55

Simpson Index (D) = 1

𝑝𝑖2𝑠

𝑖=1

In the Simpson index, p is the proportion (n/N) of individuals of one particular species

found (n) divided by the total number of individuals found (N), Σ is sum of the

calculations of each species, and s is the number of species

Margalef index (M) = 𝑆−1

ln N

Where, S = the number of species representing a particular site

N = the total number of individuals of all species within the site.

Pielou index (j) =H ʹ

ln S

Where, Hʹ = Shannon-Wiener diversity index

S = number of species.

Jaccard similarity index (J) = (100 × c)/(a + b - c)

Where, ‘a’ is the number of species in community A, ‘b’ is number of species in

community B, while ‘c’ is the number of species common to both communities.

3.8 Statistical analysis

3.8.1 Piper diagram

Similarities and differences among water samples collected from hot water springs

were analysed by preparing piper diagrams. Piper diagrams were prepared and

analysed using Software GW_Chart (Version 1.26.0.0).

3.8.2 Principle Component Analysis (PCA)

PCA is a multivariate analysis tool used to condense data. The output from the PCA is

presented in ordination diagram or two dimensional scatterplot. To reduce the

dimensionality of the data set and minimize the loss of information ordination of data

set has been done. Raw data were converted to unit less forms by uniformly adding

value 2 into whole data and then log of obtained values was taken. PCA was carried

out using the Microsoft Excel Add-Ins software XLSTAT 2012.

3.8.3 Cluster analysis

All physico-chemical parameters of water samples were used to generate

dendrograms of relationship between the sampling sites, based on their similarities.

The hierarchic Unweighted Pair Group Means Average (UPGMA) method was used

to make clustering algorithms. Cluster analysis was carried out using the Microsoft

Excel Add-Ins software XLSTAT 2012.

56

3.8.4 Canonical Correspondence Analysis (CCA)

The relationship between cyanobacterial species distribution and physico-chemical

variables was assessed by canonical correspondence analysis, followed by a Monte

Carlo Test (1000 permutations, α= 0.05) to estimate the statistical significance of

these relationships. Water temperature, pH, total phosphate, total nitrogen, and

conductivity were the environmental variables of cold desert lakes which were

included in the analysis while sodium and sulphate were also included among above

variables for hot water springs. To reduce the dimensionality of the data set and

minimize the loss of information, raw data were converted to unit less forms by

uniformly adding value 2 into whole data and log was taken. The analysis was carried

out using the Microsoft Excel Add-Ins software XLSTAT 2012.

3.9 Preservation of cyanobacteria

In vitro preservation was done by keeping cyanobacterial isolates in culture medium

contained in flasks or tubes in BOD incubators at their respective growth conditions.

Cyanobacterial agar-slants were prepared and incubated at 4 oC. After every 6 months,

cyanobacterial culture were revived from these stocks by inoculating in fresh Chu-10

medium and incubated at their respective growth conditions. After proper growth,

cyanobacterial agar-slants were again prepared and after proper growth of

cyanobacteria on slants these were again incubated at 4 oC. After every 6 month the

above procedure was repeated.