38 isolation, identification and maintenance...
TRANSCRIPT
38
ISOLATION, IDENTIFICATION AND MAINTENANCE OF
CYANOBACTERIA FROM LOKTAK LAKE, MANIPUR
4.1. INTRODUCTION
The habitat of cyanobacteria varies from species to species. Often initial isolations of
cyanobacteria from the natural environments may give rise to mixed cultures. Therefore, it is
essential to purify the individual types of cyanobacteria from mixture. Several methods viz.
pipette method, centrifugation or washing method, the method by exploiting the phototactic
movement, agar plating method, serial dilution techniques and antibiotic treatment were
employed for purification of cyanobacteria depending on the degree of contamination.
Filamentous cyanobacteria were difficult to maintain in pure cultures. However, the cultures
were made pure by repeated and frequent subculturing in liquid broth. Cyanobacteria grow
slowly and it may take a long time (even months) to produce a visible growth (Castenholz,
1988). Cyanobacterial strains in culture collections are usually stored as living cultures by
transferring cells to fresh media whenever needed. The living cultures and frequent transfers
increase the risk of mixing cultures as well as possibility for the phenotypic changes of
cyanobacterial isolates, which seem to be common during prolonged cultivation (Castenholz
and Waterbury, 1989).
The physico-chemical properties were also greatly affected due to discharge of
domestic, municipal, industrial and other several factors like religious offerings, recreational
and constructional activities in the catchments areas (Panda et al., 1991). The diversity of
cyanobacteria in nature has traditionally been studied by microscopy, which usually allows
identification at the species level in contrast to many other bacteria. The untapped potential of
cyanobacteria for their scientific exploitation has been realized in recent years. It needs an
extensive screening which is expected to result in the discovery of better cyanobacterial strains
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of immense industrial interest. The acquisition of fundamental knowledge of these versatile
organisms is necessary for further progress. Evaluation of their physiological as well as
biochemical characteristics leads to the selection of more prospective strains. It is, therefore,
very essential to prepare an excellent database by determining the biochemical composition of
cyanobacteria which can be used as a potential food source. Cyanobacterial taxonomy, in
recent years, has been a subject that aroused considerable disagreement among phycologists.
The fundamental difference in the cellular organization of cyanophyceans and other algae,
however, led to the taxonomical treatment of cyanobacteria as a separate class or division
(Fritsch, 1952). Intensive investigation on cultures of cyanobacteria for the morphological
features as well as for their physiological characteristics was suggested for evolving a better
system of classification by designating reference cultures.
Isolation and maintenance of pure strains of cyanobacteria involve immense difficulty
in their characterization from environmental samples. Selective enrichment cultures often fail
to mimic the growth conditions of cyanobacteria required for proliferation in their natural
environments (Muyzer et al., 1993). It is estimated that far less than 5% of all cyanobacterial
species have been established in cultures, partly because certain species or strains are
impossible to grow on specified media (Castenholz, 1992). In addition, successful isolation and
maintenance of strains under laboratory conditions for prolonged periods can result in an
alteration of cell characteristics. Morphological characters initially observed in the fields and
used to classify each strain have been reported to change over time in culture, sometimes
resulting in strain re-assignment (Palinska et al., 1996).
There are few established techniques for culture maintenance in the field of
cyanobacterial research (Smith, 2004; Acreman, 1994; Syiem, 2005). Most common being
batch cultures and agar slants. Batch culturing requires regular transfer to fresh liquid media
that enhances the chances of contamination. The most common procedure for maintaining
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cyanobacteria is by serial transfer of actively growing cultures to a fresh media on a regular
schedule under suboptimal conditions (Lorenz et al., 2005).
In the present study, an attempt has been made to determine the distribution and
abundance of cyanobacteria from the Loktak lake of Manipur, India. Subsequently, they were
isolated, purified and maintained as unialgal cultures in the laboratory condition with a view to
understand their physiological and biochemical characterization.
4.2. MATERIALS AND METHODS
4.2.1. Description of study site
Loktak lake, a Ramsar site, located between longitudes 93º46' to 95º55' E and latitudes
24º25' to 24º42' N at the elevation of 768.5 m is the largest freshwater wetland in the north east
India and is situated in the southern part of the central plain of Manipur in Bishnupur district
(fig.1). The lake harbours many rare fishes and migratory birds, besides the maintenance of
high biodiversity. A number of streams originate from the hill ranges immediately to the west
of the lake and these streams flow directly into Loktak lake. The physico-geographical
characteristics of the lake has described separately in this thesis. Different view and activities
in the lake was shown (photoplate-1).
4.2.2. Collection of samples
Soil and cyanobacterial samples were randomly carried out from different ecological
sites of Loktak lake. In order to get maximum number of cyanobacterial species, samples from
different sites of the lake were collected in summer, rainy and winter seasons during 2010-
2012. Samples of visually conspicuous cyanobacterial growth on submerged plus exposed
soils, water plants and stones (photoplate-2) were collected in polythene bags containing native
water. A total of 60 samples were collected from different sites of the lake. This included
cyanobacteria growing as thin film or mat on surface of water or from a depth of about 5-10 cm
of water. Samples from soil surface covered with few centimetres of water, from benthic and
41
epiphytic substrata were collected. The exposed surface of roots and stems of some
angiospermic plants were collected for cyanobacterial isolation (photoplate-3). Cyanobacteria
attached firmly on moist soils were also collected by spatula from the adjoining areas of
Loktak lake. In some cases, small stones covered with cyanobacterial growth were considered
as samples. All these polythene bags were labelled giving information regarding habitat and
date of collection. Geographical details were also recorded using Global Positioning System,
GPS (Garmin eTrex Vista). The samples were processed within 48 h of their collection.
Fig.1. Map of Loktak lake
Loktak lake, Bishnupur, Manipur
Bishnupur
district
India and location of Manipur
Satellite map of Loktak lake
Manipur: Location
of Loktak lake
Loktak lake
42
5.2.3. Physico-chemical parameters of water samples
Water samples collected in plastic bottles from different sites during different season
viz. summer, rainy and winter were analyzed for different physico-chemical parameters such as
temperature, pH, nutrients (nitrate and phosphate) and total dissolved solids (APHA 1989;
Trivedi and Goel 1986). Temperature and pH were measured in the field conditions at the time
of sample collection using a mercury thermometer and pen type pH meter (Hanna Instruments)
(photoplate-3). Analysis for the remaining physico-chemical parameters was carried out in the
laboratory. Water samples were transported to the laboratory and stored in a cold room (4ºC)
before further analysis. Total dissolved solid (TDS) was determined as the residue left after
evaporation of filtered sample (Gravimetric method). Nitrate and phosphate were determined
spectrophotometrically.
4.2.4. Preparation of the medium
All the ingredients of BG-11 broth medium (Stanier et al., 1971) were prepared
(appendix). Each ingredient was added accordingly for one litre of the medium. The contents
were mixed properly with magnetic stirrer and pH of the medium was adjusted (Thermo
Scientific ORION 2 STAR) to 7.0 using 1N sodium hydroxide (NaOH) and 1N hydrochloric
acid (HCl) and sterilized in autoclave (Caltan, NSW-227) at 121ºC, 15 psi pressure for 15 min.
BG-11 solid medium was prepared by adding 15 g of agar in a litre of BG-11 broth,
autoclaved, allowed to cool to approximately 40ºC and poured into petridishes under a sterile
laminar flow (Klenzaids) before cooling to room temperature.
4.2.5. Isolation of cyanobacteria
BG-11 medium (Stanier et al., 1971) was used for isolation of cyanobacteria. 1 g of the
soil/ cyanobacterial samples was transferred to sterile 100 ml of BG-11 medium (with nitrate
source for non-nitrogen fixing and nitrate-free for nitrogen fixing cyanobacteria) in 250 ml
conical flasks. All samples were incubated in growth chamber at 28±2ºC with illumination of
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54-67 µmol photons/m2/s by cool white 40W fluorescent tubes (Philips). The flasks were
regularly monitored for the algal growth. After 10-15 days of incubation, when visible algal
growth appeared 2-3 wet mounts from each flask were prepared and observed microscopically
using trinocular research microscope (NIKON Eclipse 80i). After the microscopic observation,
it was streaked on BG-11 agar plate medium with N2 source for non heterocystous forms and
without N2 source agar plate for heterocystous forms. Inoculated plates were incubated in the
culture room which was maintained at 28±2°C illuminated with cool white 40W fluorescent
tubes at an irradiance of 54-67 µmol photons/m2/s. The culture rack was fitted with
photoperiodic automatic model timer (Saveer Biotech Limited) coupled with Biotech room
temperature controller to provide alternative light and dark phases. The plates were observed
regularly for algal growth and isolated filaments of cyanobacteria. Isolation of the
cyanobacterial strains was done by randomly picking different types of colonies developed on
the petridishes and then examined under trinocular research microscope (NIKON Eclipse 80i).
If growth of heterotrophic bacteria or any other mixed culture was observed under any of the
condition after incubation, cyanobacterial colonies were continuously cultured onto fresh plate
medium until unialgal cultures were obtained. Cyanobacterial colony grown on the surface of
the agar medium was picked up with inoculation loop and streaked through agar.
4.2.6. Identification of cyanobacterial strains
Slides of the unialga was prepared on a clean glass slide along with cover slip and
examined. Photomicrography was carried out using trinocular research microscope (NIKON
Eclipse 80i) and Carl Zeiss fluorescence microscope, Axio Scope A1 coupled with Carl Zeiss
Imaging Systems 32 software AxioVision 4.7.2 followed by taxonomical characterization of
pure cultures referring to keys given by Desikachary (1959) and Komarek and Anagnostidis
(2005).
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4.2.7. Maintenance of cyanobacterial strains
Most widely used method for laboratory maintenance of cultures is by storing them in
agar slants. This is done by inoculating pure cultures into a nutrient agar medium which were
solidified in sterile tubes. All the unialgal strains were maintained at a temperature of 19±1ºC
under 25-30 µmol photon/m2/s
of cool white 40W fluorescent tubes. All the strains were
maintained in BG-11 agar slants and broth medium. The isolated cyanobacterial strains were
sequentially assigned reference numbers having its own uniqueness and deposited to fresh
water cyanobacterial and microalgal repository of IBSD, Imphal, Manipur, India. The unialgal
cyanobacterial strains maintained in repository were subcultured for every 3-4 months
depending on the culture conditions.
4.2.8. Diversity indices
Diversity index (Shannon index, H and Simpson’s index, 1-D) was analyzed by using
PAST software with portable IBM SPSS statistics version 19.
4.2.9. Relative abundance
The relative abundance of a particular cyanobacterium was calculated by using the
following formula:
Relative abundance = X x 100
Y
Where,
X = total number of samples collected
Y = number of samples from which a particular cyanobacteria type was isolated.
4.3. RESULTS
4.3.1. Cyanobacterial strains isolated from Loktak lake
Cyanobacterial strains, either planktonic or benthic; epilithic or epiphytic were isolated
from different ecological habitats like open water, shore of the lake and phumdis. A total of 90
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strains belong to 11 genera of cyanobacteria were isolated from different sites of lake during
the period of 2010-2012. These strains represented; Anabaena (24), Nostoc (26), Calothrix
(08), Microchaete (05), Cylindrospermum (01), Westiellopsis (04), Hapalosiphon (01),
Plectonema (02), Phormidium (14), Lyngbya (03) and Limnothrix (02) were identified based on
morphological characteristics (table-2).
The number of cyanobacterial strains was more in winter than in summer and rainy
seasons in all the sites. Heterocystous forms showed more frequency of occurrence than non
heterocystous forms. It was found that Nostoc was predominant and isolated from all the sites
of Loktak lake, followed by Anabaena. Few genera such as Cylindrospermum, Hapalosiphon
and Limnothrix were isolated in less number.
Table-2: Generic representation of cyanobacterial strains in Loktak lake
Name of the cyanobacterial genera No. of strains
Anabaena Bory 24
Nostoc Vaucher 26
Calothrix Ag. 08
Microchaete Thuret 05
Cylindrospermum Kutz. 01
Westiellopsis Janet 04
Hapalosiphon Nag. 01
Plectonema Thuret 02
Phormidium Kutz. 14
Lyngbya Ag. 03
Limnothrix Meffert 02
Total no. of genera=11; Total no. of strains= 90
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All above cited cyanobacterial strains were purified as unialgal and classically
identified upto genus/species level along with their taxonomical and morphological
characteristics (table-3). Photomicrographs were shown in the photoplates-4 to 17 which were
captured in 40x and 100x objectives. Some unicellular microalgae which were observed in
natural samples and cannot be isolated in laboratory were also shown in photoplate-18.
Filament/trichome structure, constrictions, sheath, shape, presence or absence of heterocyst and
akinete were the major points considered for taxonomical characterization. Branching pattern,
row of cells in filament were main features considered for taxonomical identification of
Plectonema, Hapalosiphon and Westiellopsis. Sheath pattern was recorded as thin, hyaline,
diffluent or firm in different sheath bearing cyanobacteria. Different shape of cell was recorded
as quadrate, acute/ conical end cell, cells longer than broad (Phormidium), barrel to cylindrical,
quadratic (Nostoc, Anabaena), long and broad (Calothrix, Microchaete). Heterocyst shape
varies from spherical to sub-spherical, oval to oblong (Nostoc, Anabaena, Cylindrospermum,
Calothrix and Microchaete), oblong-cylindrical (Westiellopsis).
Different position of heterocyst was observed as intercalary (Anabaena), both
intercalary and terminal (Nostoc), basal (Calothrix, Microchaete). Akinete shape varies from
oval (Cylindrospermum), oval to oblong (Nostoc, Anabaena), sub-spherical to ellipsoidal
(Nostoc), cylindrical and next to heterocyst (Calothrix, Cylindrospermum), spherical
(Westiellopsis) and oblong (Hapalosiphon). False branching was observed in Plectonema,
however, Hapalosiphon and Westiellopsis showed true branching. Hapalosiphon always bear
single row of cells in filament while Westiellopsis having two or more rows of cell in filament
during later stage of growth. Similar morphological features were also reported by Oinam et al.
(2010) and Singh et al. (2012).
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Table-3: Occurrence and diversity of cyanobacteria in Loktak Lake, Manipur Comparative analysis of the genus-Phormidium
Name of the strains Habitat of the
strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell shape
Phormidium sp. BTA-52 Hydrophytes
Latitude: N24°30'37.8"
Longitude: E093°47'36.0"
Altitude: 772 m
bent; not
constricted
indistinct longer than broad
Phormidium tenue (Menegh.)
Gomont BTA-63
Hydrophytes
Latitude: N24°30'53.4"
Longitude: E093°47'40.4"
Altitude: 772 m
straight and
slightly
constricted at
cross-walls
diffluent longer than broad
Phormidium corium (Ag.)
Gomont BTA-64
Hydrophytes
Latitude: N24°32'31.2"
Longitude: E093°42'41.8"
Altitude: 782 m
flexuous thin quadrate
Phormidium sp. BTA-71 Waterlogged soil Latitude: N24°28'31.5"
Longitude: E093°43'32.7"
Altitude: 767 m
straight thick and
colourless
longer than broad
Phormidium sp. BTA-75 Hydrophytes Latitude: N24°36'41.8"
Longitude: E093°45'36.0"
Altitude: 792 m
straight;
granulated at
the cross-walls
thin long; apical cells
with a calyptra
Phormidium fragile
(Meneghini) Gomont
BTA-1020
Hydrophytes Latitude: N24°30'37.8"
Longitude: E093°47’42.0"
Altitude: 772 m
flexuous;
constricted at
cross-walls
diffluent quadrate; acute end
cell
Phormidium fragile
(Meneghini) Gomont
BTA-1042
Attached on surface
of boat
Latitude: N24°30'48.8"
Longitude: E093°47'31.0"
Altitude: 816 m
flexuous;
constricted at
cross-walls
diffluent quadrate; conical
end cell
48
Name of the strains Habitat of the
strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell shape
Phormidium tenue (Menegh.)
Gomont BTA-1045
Hydrophytes
Latitude: N24°30'49.9"
Longitude: E093°47'25.5"
Altitude: 779 m
straight thin and diffluent longer than
broad
Phormidium sp. (Ag.)
Gomont BTA-1048
Waterlogged soil Latitude: N24°30'58.9"
Longitude: E093°48'01.5"
Altitude: 820 m
slightly bent thin and diffluent quadrate
Phormidium fragile
(Meneghini) Gomont
BTA-1052
Hydrophytes
Latitude: N24°30'23.6"
Longitude: E093°46'57.2"
Altitude: 760 m
flexuous;
constricted at
cross-walls
diffluent quadrate
Phormidium corium (Ag.)
Gomont
BTA-1065
Moist soil Latitude: N24°31'08.2"
Longitude: E093°48’46.7"
Altitude: 825 m
long; straight
ends
thin quadrate
Phormidium tenue (Menegh.)
Gomont
BTA-1073
Waterlogged soil Latitude: N24°31'13.0"
Longitude: E093°49'01.6"
Altitude: 769 m
slightly bent diffluent longer than
broad
Phormidium tenue (Menegh.)
Gomont
BTA-1076
Moist soil Latitude: N24°30'28.1"
Longitude: E093°47'03.0"
Altitude: 766 m
slightly bent;
blunt end
thin longer than
broad
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Comparative analysis of the genus-Lyngbya
Name of the strains Habitat of the
strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell shape
Lyngbya aestuarii Liebm. ex Gomont
BTA-66
Moist soil Latitude: N24°30'37.8"
Longitude: E093°47'36.0"
Altitude: 772 m
straight; flat
end cells
firm and
lamellated
short and
granulated
Lyngbya putealis Mont. ex Gomont
BTA-1013
Moist soil Latitude: N24°30'58.9"
Longitude: E093°48'01.5"
Altitude: 820 m
curved thin constricted at
cross-walls
and granulated
Lyngbya birgei Smith G. M.
BTA-1080
Hydrophytes
Latitude: N24°31'08.2"
Longitude: E093°48'46.7"
Altitude: 825 m
straight firm and
colourless
short; rounded
ends
Description of the genus-Cylindrospermum
Name of the strains Habitat of
the strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell
shape
Heterocyst Akinete
Cylindrospermum
muscicola Kutzing ex
Born. et Flah. BTA-963
Waterlogged
soil
Latitude: N24°31'09.6"
Longitude: E093°47'56.0"
Altitude: 760 m
broad;
constricted
at cross-
walls
hyaline quadrate
to
cylindrical
oblong oval
50
Comparative analysis of the genus-Nostoc
Name of the strains Habitat of the
strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell
shape
Heterocyst Akinete
Nostoc sp. BTA-53 Hydrophytes Latitude: N24°30'37.8"
Longitude: E093°47'36.0"
Altitude: 772 m
flexuous;
curved
diffluent barrel sub-spherical oblong
Nostoc paludosum Kutzing
ex Born. et Flah. BTA-56
Moist soil Latitude: N24°36'39.1"
Longitude: E093°39'32.5"
Altitude: 770 m
entangled distinct
and
colourless
barrel to
cylindrical
broader than
vegetative
cells
oval
Nostoc sp.
BTA-60
Waterlogged
soil
Latitude: N24°34'31.5"
Longitude: E093°39'34.2"
Altitude: 769 m
flexuous hyaline barrel or
spherical
both
intercalary
and terminal;
sub-spherical
spherical
Nostoc sp.
BTA-61
Hydrophytes Latitude: N24°36'17.9"
Longitude: E093°37'16.5"
Altitude: 832 m
flexuous thin and
hyaline
quadratic spherical oblong
Nostoc commune Vaucher
ex Born. et Flah.
BTA-67
Hydrophytes Latitude: N24°29'31.6"
Longitude: E093°49'31.2"
Altitude: 778 m
flexuous
and
entangled
distinct barrel spherical oblong
Nostoc sp.
BTA-80
Hydrophytes Latitude: N24°30'29.2"
Longitude: E093°47'36.0"
Altitude: 768 m
flexuous diffluent quadratic spherical spherical
Nostoc muscorum
BTA-950
Waterlogged
soil
Latitude: N24°30'33.3"
Longitude: E093°47'10.1"
Altitude: 765 m
flexuous hyaline barrel spherical oblong
51
Name of the strains Habitat of the
strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell
shape
Heterocyst Akinete
Nostoc sp. BTA-978 Hydrophytes Latitude: N24°28'47.2"
Longitude: E093°48'28.8"
Altitude: 770 m
flexuous distinct barrel spherical ellipsoidal
Nostoc sp. BTA-979 Hydrophytes
Latitude: N24°30'23.6"
Longitude: E093°46'57.2"
Altitude: 760 m
flexuous
and loosely
entangled
indistinct barrel spherical sub-
spherical
Nostoc calcicola
Brebisson ex Born. et
Flah. BTA-984
Moist soil Latitude: N24°30'28.1"
Longitude: E093°47'03.0"
Altitude: 766 m
loosely
entangled
hyaline barrel sub-
spherical
sub-
spherical
Nostoc sp. BTA-995 Wet surface of
wall
Latitude: N24°30'24.8"
Longitude: E093°47'11.0"
Altitude: 830 m
flexuous
and loosely
entangled
indistinct cylindrical sub-
spherical
oval
Nostoc ellipsosporum
(Desm.) Rabenh. ex
Born. et Flah.
BTA-999
Waterlogged
soil
Latitude: N24°30'39.6"
Longitude: E093°47'17.5"
Altitude: 822 m
flexuous
and loosely
entangled
indistinct cylindrical sub-
spherical
ellipsoidal
Nostoc muscorum Ag.
ex Born.et Flah.
BTA-1001
Waterlogged
soil
Latitude: N24°30'41.4"
Longitude: E093°47'19.6"
Altitude: 761 m
flexuous
and loosely
entangled
distinct barrel spherical oblong
Nostoc sp. BTA-1003 Leaf of
hydrophytes
Latitude: N24°30'49.1"
Longitude: E093°47'29.0"
Altitude: 766 m
flexuous indistinct barrel oval ellipsoidal
52
Name of the strains Habitat of the
strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell
shape
Heterocyst Akinete
Nostoc spongiaeforme
Agardh ex Born. et
Flah. BTA-1005
Root of
hydrophytes
Latitude: N24°31'14.7"
Longitude: E093°49'05.4"
Altitude: 807 m
flexuous and
loosely
entangled
distinct short and
barrel
sub-
spherical
oblong
Nostoc sp. BTA-1008 Waterlogged
soil
Latitude: N24°31'08.2"
Longitude: E093°48'46.7"
Altitude: 825 m
loosely
contorted and
flexuous
indistinct barrel spherical oval to
ellipsoidal
Nostoc ellipsosporum
(Desm.) Rabenh. ex
Born. et Flah.
BTA-1011
Root of
hydrophytes
Latitude: N24°31'09.6"
Longitude: E093°47'56.0"
Altitude: 760 m
flexuous and
loosely
entangled
indistinct cylindrical sub-
spherical or
oblong
ellipsoidal
to oblong
Nostoc spongiaeforme
Agardh ex Born. et
Flah. BTA-1018
Hydrophytes
Latitude: N24°30'23.6"
Longitude: E093°46'57.2"
Altitude: 760 m
flexuous and
loosely
entangled
indistinct cylindrical
and partly
barrel
intercalary
and
terminal;
sub-
spherical
oblong
Nostoc sp. BTA-1027 Hydrophytes
Latitude: N24°30'23.6"
Longitude: E093°46'57.2"
Altitude: 760 m
loosely
entangled
distinct barrel and
partly
quadratic
spherical sub-
spherical
Nostoc sp. BTA-1034 Hydrophytes
Latitude: N24°31'14.7"
Longitude: E093°49'05.4"
Altitude: 807 m
flexuous diffluent barrel and
partly
cylindrical
sub-
spherical
oblong
53
Name of the strains Habitat of the
strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell
shape
Heterocyst Akinete
Nostoc spongiaeforme
Agardh ex Born. et
Flah. BTA-1035
Leaf of
hydrophytes
Latitude: N24°30'24.8"
Longitude: E093°47'11.0"
Altitude: 830 m
flexuous and
loosely
entangled
indistinct partly
cylindrical
and partly
barrel
sub-
spherical
ellipsoidal
Nostoc sp. BTA-1046 Hydrophytes Latitude: N24°30'24.8"
Longitude: E093°47'11.0"
Altitude: 830 m
flexuous diffluent barrel sub-
spherical
oblong
Nostoc sp. BTA-1055 Hydrophytes Latitude: N24°31'09.6"
Longitude: E093°47'56.0"
Altitude: 760 m
loosely
entangled
indistinct cylindrical sub-
spherical
ellipsoidal
Nostoc spongiaeforme
Agardh ex Born. et
Flah. BTA-1056
Hydrophytes
Latitude: N24°31'13.6"
Longitude: E093°49'07.2"
Altitude: 807 m
flexuous and
loosely
entangled
indistinct cylindrical sub-
spherical
ellipsoidal
Nostoc sp. BTA-1057 Leaf of
hydrophytes
Latitude: N24°30'33.3"
Longitude: E093°47'10.1"
Altitude: 765 m
flexuous and
entangled
hyaline barrel sub-
spherical
spherical
54
Comparative analysis of the genus-Anabaena
Name of the strains Habitat of the
strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell shape Heterocyst Akinete
Anabaena sp. BTA-69 Leaf of
hydrophytes
Latitude: N24°32'31.5"
Longitude: E093°43'32.4"
Altitude: 767 m
flexuous diffluent barrel intercalary;
sub-
spherical
oblong
Anabaena variabilis
Kutzing ex Born. et
Flah. BTA-72
Hydrophytes Latitude: N24°30'37.8"
Longitude: E093°47'36.0"
Altitude: 772 m
flexuous hyaline
and
colourless
barrel spherical oval
Anabaena iyengarii
Bharadwaja BTA-74
Attached on
surface of boat
Latitude: N24°30'37.8"
Longitude: E093°47'36.0"
Altitude: 772 m
curved;
rounded apex
distinct barrel; end
cell conical
barrel ellipsoidal
Anabaena circinalis
Rabenhorst ex Born.
et Flah. BTA-945
Stem of
hydrophytes
Latitude: N24°30'49.1"
Longitude: E024°30'49.1"
Altitude: 766 m
flexuous and
entangled
hyaline
and
indistinct
barrel or
spherical
sub-
spherical
cylindrical
Anabaena sp.
BTA-949
Leaf of
hydrophytes
Latitude: N24°31'09.6"
Longitude: E093°47'56.0"
Altitude: 760 m
flexuous thin and
hyaline
barrel; end
cell conical
spherical oblong
Anabaena sp. BTA-
958
Hydrophytes Latitude: N24°28'10.6"
Longitude: E093°45'49.2"
Altitude: 753 m
straight absent barrel spherical oblong
Anabaena sp. BTA-
964
Hydrophytes Latitude: N24°30'37.8"
Longitude: E093°47'36.0"
Altitude: 772 m
flexuous hyaline barrel spherical oval
55
Name of the strains Habitat of
the strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell shape Heterocyst Akinete
Anabaena sp. BTA-980 Leaf of
hydrophytes
Latitude: N24°30'39.6"
Longitude: E093°47'17.5"
Altitude: 822 m
bent absent barrel sub-spherical ellipsoidal
Anabaena ambigua
Rao, C. B. BTA-983
Moist soil
Latitude: N24°30'28.1"
Longitude: E093°47'03.0"
Altitude: 766 m
bent;
enclosed in a
mucilaginous
sheath
thin and
hyaline
barrel;
constriction
at the joints
spherical;
broader than
cells
ellipsoidal
Anabaena sp. BTA-988 Leaf of
hydrophytes
Latitude: N24°30'24.8"
Longitude: E093°47'11.0"
Altitude: 830 m
bent hyaline barrel;
granulated
oblong ellipsoidal
Anabaena sp. BTA-996 Lotus leaf
Latitude: N24°31'07.4"
Longitude: E093°48'56.4"
Altitude: 824 m
bent hyaline barrel; end
cell conical
sub-spherical oval
Anabaena sp. BTA-997 Hydrophytes
Latitude: N24°31'07.4"
Longitude: E093°48'56.4"
Altitude: 824 m
flexuous thin and
hyaline
barrel or
sub-
quadrate
spherical oblong
Anabaena sp.
BTA-1004
Submerged
root of
hydrophytes
Latitude: N24°31'13.6"
Longitude: E093°49'07.2"
Altitude: 807 m
slightly
curved
hyaline barrel oval oblong
56
Name of the strains Habitat of
the strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell shape Heterocyst Akinete
Anabaena sp.
BTA-1006
Leaf of lotus Latitude: N24°13'14.7"
Longitude: E093°49'05.4"
Altitude: 807 m
bent thin and
hyaline
barrel oval oblong
Anabaena anomala
Fritsch
BTA-1007
Leaf of
hydrophytes
Latitude: N24°30'49.9"
Longitude: E093°47'25.5"
Altitude: 779 m
bent diffluent barrel; apical
cell rounded
barrel ellipsoidal
Anabaena
ballyganglii Banerji
BTA-1009
Leaf of
hydrophytes
Latitude: N24°31'07.4"
Longitude: E093°48'56.4"
Altitude: 824 m
circinate hyaline barrel; long
and broad;
granular
contents
sub-
spherical
ellipsoidal
Anabaena circinalis
Rabenhorst ex Born.
et Flah. BTA-1010
Submerged
root of
hydrophytes
Latitude: N24°31'07.4"
Longitude: E093°48'56.4"
Altitude: 824 m
circinate;
seldom
straight
hyaline barrel sub-
spherical
oblong
Anabaena variabilis
Kutzing ex Born. et
Flah. BTA-1012
Moist soil Latitude: N24°31'09.6"
Longitude: E093°47'56.0"
Altitude: 760 m
straight;
slight bent
colourless barrel spherical or
oval
oblong
Anabaena sp.
BTA-1025
Hydrophytes Latitude: N24°30'33.3"
Longitude: E093°47'10.1"
Altitude: 765 m
bent thin and
hyaline
barrel to
quadratic
sub-
spherical
oval
Anabaena oryzae
Fritsch BTA-1026
Submerged
root of
hydrophytes
Latitude: N24°31'14.7"
Longitude: E093°49'05.4"
Altitude: 807 m
short to
straight;
aggregated
colourless barrel; longer
than broad
oval;
broader
than
vegetative
cells
ellipsoidal
57
Name of the strains Habitat of
the strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell shape Heterocyst Akinete
Anabaena variabilis
Kutzing ex Born. et
Flah. BTA-1030
Submerged
root of
hydrophytes
Latitude: N24°31'14.7"
Longitude: E093°49'05.4"
Altitude: 807 m
straight diffluent barrel, end
cells conical
spherical
or oval
oblong
Anabaena iyengarii
Bharadwaja
BTA-1033
Attached on
surface of
boat
Latitude: N24°33'15.6"
Longitude: E093°48'06.2"
Altitude: 780 m
curved; end
cell conical
hyaline barrel sub-
spherical
ellipsoidal
Anabaena iyengarii
Bharadwaja
BTA-1058
Leaf of
hydrophytes
Latitude: N24°28'06.2"
Longitude: E093°45'52.3"
Altitude: 764 m
curved; end
cell conical
diffluent barrel spherical ellipsoidal
Anabaena vaginicola
Fritsch et Rich
BTA-1074
Moist soil Latitude: N24°30'33.3"
Longitude: E093°47'10.1"
Altitude: 765 m
straight and
parallel
distinct sub-quadrate
to
cylindrical;
conical
apical cell
cylindrical oblong or
cylindrical
Anabaena variabilis
Kutzing ex Born. et
Flah. BTA-1075
Hydrophytes Latitude: N24°30'48.7"
Longitude: E093°47'31.1"
Altitude: 759 m
bent hyaline barrel spherical
or oval
oblong
58
Comparative analysis of the genus-Plectonema
Name of the strains Habitat of the
strains
GPS data of the strains Taxonomical enumeration
Filament/ Trichome Sheath Cell shape
Plectonema sp. BTA-65 Moist soil
Latitude: N24°30'37.8"
Longitude: E093°47'36.0"
Altitude: 772 m
curved and false
branched
firm and
hyaline
quadratic
Plectonema sp. BTA-79 Hydrophytes Latitude: N24°35'77.7"
Longitude: E093°57'78.0"
Altitude: 822 m
flexuous and false
branched
lamellated
and
colourless
as long as
broad
Comparative analysis of the genus-Microchaete
Name of the strains Habitat of
the strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell
shape
Heterocyst Akinete
Microchaete sp.
BTA-59
Epilithic Latitude: N24°30'37.8"
Longitude: E093°47'36.0"
Altitude: 772 m
straight and
constricted at
cross-walls
colourless long and
broad
basal and
spherical
absent
Microchaete uberrima
Carter, N. BTA-78
Wet surface
of rock
Latitude: N24°33'15.6"
Longitude: E093°48'06.2"
Altitude: 780 m
elongate firm sub-
quadrate
basal and
spherical
quadrate
to sub-
cylindrical
59
Name of the strains Habitat of the
strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell
shape
Heterocyst Akinete
Microchaete grisea
Thuret ex Born. et
Flah. BTA-1041
Submerged
root of
hydrophytes
Latitude: N24°31'14.7"
Longitude: E093°49'05.4"
Altitude: 807 m
broad and
bent
thin and
hyaline
long and
broad
basal,
intercalary
and spherical
absent
Microchaete uberrima
Carter, N. BTA-1043
Moist soil Latitude: N24°30'39.6"
Longitude: E093°47'17.5"
Altitude: 822 m
straight thin and
colourless
long and
broad
basal and
spherical
absent
Microchaete uberrima
Carter, N. BTA-1081
Wet surface of
rock
Latitude: N24°31'14.7"
Longitude: E093°49'05.4"
Altitude: 807 m
slightly bent thin and
hyaline
long and
broad
basal and
hemispherical
absent
Comparative analysis of the genus-Calothrix
Name of the strains Habitat of
the strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell
shape
Heterocyst Akinete
Calothrix ghosei
Bharadwaja BTA-57
Submerged
root of
hydrophytes
Latitude: N24°30'28.1"
Longitude: E093°47'03.0"
Altitude: 766 m
slightly bent hyaline
and
distinct
quadratic;
slightly
longer
than broad
basal and
spherical
next to
heterocyst;
cylindrical
Calothrix marchica
Lemmermann
BTA-70
Waterlogged
moist soil
Latitude: N24°32'35.3"
Longitude: E093°44'35.3"
Altitude: 770 m
slightly bent thin and
colourless
long and
broad
basal and
spherical
absent
60
Name of the strains Habitat of
the strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell
shape
Heterocyst Akinete
Calothrix sp. BTA-73 Epilithic
Latitude: N24°30'37.8"
Longitude: E093°47'36.0"
Altitude: 772 m
bent colourless barrel basal and
spherical
absent
Calothrix ghosei
Bharadwaja BTA-77
Moist soil Latitude: N24°30'37.8"
Longitude: E093°47'36.0"
Altitude: 772 m
slightly bent hyaline
and
distinct
quadratic basal and
spherical
cylindrical
Calothrix geitonos
Skuja BTA-998
Epilithic
Latitude: N24°31'07.4"
Longitude: E093°48'56.4"
Altitude: 824 m
bent;
constricted
at cross-
walls
thin and
colourless
long and
broad
basal and
spherical
absent
Calothrix clavata
West, G. S.
BTA-1002
Hydrophytes Latitude: N24°31'07.4"
Longitude: E093°48'56.4"
Altitude: 824 m
slightly bent thin and
colourless
long and
broad;
constrict
ed at the
cross
walls
basal and
hemispherical
absent
Calothrix marchica
Lemmermann
BTA-1014
Epilithic
Latitude: N24°30'58.9"
Longitude: E093°48’01.5"
Altitude: 820 m
straight thin and
colourless
long and
broad
basal and
spherical
absent
Calothrix ghosei
Bharadwaja
BTA-1019
Wet surface
of rock
Latitude: N24°31'14.7"
Longitude: E093°49'05.4"
Altitude: 807 m
bent hyaline
and
distinct
quadratic basal and
spherical
absent
61
Description of the genus-Hapalosiphon
Name of the strains Habitat of
the strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell
shape
Heterocyst Akinete
Hapalosiphon
welwitschii
W. et G.S.West
BTA-58
Epilithic Latitude: N24°30'37.8"
Longitude: E093°47'36.0"
Altitude: 772 m
flexuous;
lateral
branches short
as broad as the
main filament
hyaline
and
colourless
elongate;
longer
than broad
quadratic oblong
Comparative analysis of the genus-Westiellopsis
Name of the strains Habitat of
the strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell
shape
Heterocyst Akinete
Westiellopsis
prolifica Janet
BTA-51
Attached on
surface of
boat
Latitude: N24°36'29.5"
Longitude: E093°40'34.0"
Altitude: 770 m
erect; thin
and elongate;
true
branching
absent short
barrel
oblong-
cylindrical
spherical
Westiellopsis sp.
BTA-55
Epilithic Latitude: N24°33'31.4"
Longitude: E093°43'32.7"
Altitude: 773 m
thick primary
filament and
thin
secondary
filament; true
branching
absent barrel spherical spherical
62
Name of the strains Habitat of
the strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell
shape
Heterocyst Akinete
Westiellopsis sp.
BTA-68
Moist soil Latitude: N24°29'40.8"
Longitude: E093°47'36.0"
Altitude: 772 m
erect; thick
primary filament
and thin
secondary
filament; true
branching
hyaline elongate
and
cylindrical
oblong-
cylindrical
oblong
Westiellopsis sp.
BTA-76
Hydrophytes Latitude: N24°30'37.8"
Longitude: E093°44'33.5"
Altitude: 769 m
curved; true
branching
diffluent barrel oblong-
cylindrical
sub-
spherical
Comparative analysis of the genus-Limnothrix
Name of the strains Habitat of the
strains
GPS data of the strains Taxonomical enumeration
Filament/
Trichome
Sheath Cell shape
Limnothrix redekei (Van
Goor) Meffert BTA-987
Waterlogged soil Latitude: N24°30'58.9"
Longitude: E093°48'01.5"
Altitude: 820 m
straight hyaline isodiametric, two small
polar aerotypes at the
septa
Limnothrix vacuolifera
(Skuja) BTA-1051
Moist soil Latitude: N24°31'11.2"
Longitude: E093°48'01.9"
Altitude: 814 m
straight colourless long; granules scattered
and later aerotypes at
cross-walls
63
Photoplate-19 showed the processing unit and maintenance of all the cyanobacterial
strains of FWCMR, IBSD-DBT, Imphal, Manipur. The isolated cyanobacterial strains were
deposited to the fresh water cyanobacterial and microalgal repository of IBSD, Imphal,
Manipur, India with accession number for ready reference materials.
4.3.2. Seasonal variation of cyanobacterial strains
Seasonal collection of cyanobacterial and water samples was carried out in different
ecological sites of the lake in summer, rainy and winter seasons (table-4). During summer
season, a total of 16 strains, namely; Anabaena (4), Nostoc (4), Calothrix (2), Microchaete (2),
Westiellopsis (1), Phormidium (2) and Lyngbya (1) were encountered. During rainy season a
total of 21 strains, namely, Anabaena (1), Nostoc (2), Calothrix (1), Plectonema (2),
Phormidium (12), Lyngbya (2) and Limnothrix (1) and during winter season a total of 53
strains, namely; Anabaena (19), Nostoc (20), Calothrix (5), Microchaete (3), Cylindrospermum
(1), Westiellopsis (4) and Hapalosiphon (1) were established.
Table-4: Seasonal variation of cyanobacterial strains in Loktak lake, Manipur
Ana: Anabaena; Nos: Nostoc; Cal: Calothrix; Micro: Microchaete; Cylin: Cylindrospermum;
Wes: Westiellopsis; Hap: Hapalosiphon; Plec: Plectonema; Phor: Phormidium; Lyn: Lyngbya;
Limno: Limnothrix
4.3.3. Physico-chemical parameters of water samples
Seasonal variations in physico-chemical parameters of water samples from different
sites of Loktak lake were presented in table-5.
Different
seasons
Genera
Ana Nos Cal Micro Cylin Wes Hap Plec Phor Lyn Limno
Summer 4 4 2 2 0 1 0 0 2 1 0
Rainy 1 2 1 0 0 0 0 2 12 2 1
Winter 19 20 5 3 1 4 1 0 0 0 0
64
Table-5: Physico-chemical properties of water samples of Loktak lake, Manipur
4.3.4. Diversity indices
Data for relative abundance and diversity index (Shannon index, H and Simpson’s
index, 1-D) were presented (table-6 and 7). The highest cyanobacterial diversity, Shannon
index (H’) was observed for Nostoc (H’=2.16) and the lowest for Limnothrix (H’=0.86) (table-
7). Highest species dominance (Simpson’s index, 1-D) was showed by Nostoc, 0.89 and the
lowest found in Limnothrix, 0.5 (table-7).
Table-6: Cyanobacterial strains relative abundance (in %) of Loktak lake, Manipur
Ana: Anabaena; Nos: Nostoc; Cal: Calothrix; Micro: Microchaete; Cylin: Cyloindrospermum;
Wes: Westiellopsis; Hap: Hapalosiphon; Plec: Plectonema; Phor: Phormidium; Lyn: Lyngbya;
Limno: Limnothrix
Different
seasons pH
Temperature
(°C)
Nitrate
(mg/l)
Phosphate
(mg/l)
Total dissolved
solids (mg/l)
Summer 6.94-7.05 18.7-26.7 0.20-0.88 0.19-0.62 24.62-70.00
Rainy 6.97-7.24 21.4-29.5 0.24-0.97 0.24-0.86 40.51-110.23
Winter 6.88-6.98 9.80-19.7 0.21-0.90 0.21-0.70 35.72-89.32
Genera Ana Nos Cal Micro Cylin Wes Hap Plec Phor Lyn Limno
Relative
abundance 40.00 43.33 13.33 8.33 1.67 6.67 1.67 3.33 23.33 5.00 3.33
65
Table-7: Diversity indices of cyanobacterial strains of Loktak lake, Manipur
4.4. DISCUSSION
The north eastern region of India has been described as biodiversity hotspots
harbouring different kinds of flora and fauna unique to this region. The scattered information
was available on the diversity and cultural behaviour of cyanobacteria from the north eastern
region of India as reported by Devi et al. (2010); Singh et al. (2012); Akoijam and Singh
(2011). So far, little information is available about occurrences of cyanobacteria in Loktak lake
and adjoining areas (Tiwari and Singh, 2005). However, work and publications on this group of
microorganisms from north east India is sporadic despite the fact that this region falls within
Indo-Malayan biodiversity hotspots (Myres et al., 2000). The natural ecosystems such as soil,
freshwater bodies-streams, ponds and lakes of this region provide excellent habitats and
favourable environments for the luxuriant and diverse growth of cyanobacteria. The present
study described the distributional pattern and diversity of filamentous cyanobacteria strains of
Loktak lake. Out of the different habitats, hydrophytes (phumdis) were found to be supporting
Name of genus Shannon index, H (species
diversity)
Simpson’s index, 1-D (species
dominance)
Anabaena 2.13 0.86
Nostoc 2.16 0.86
Calothrix 1.32 0.72
Microchaete 1.05 0.64
Cylindrospermum 0 0
Westiellopsis 1.38 0.75
Hapalosiphon 0 0
Plectonema 0.69 0.5
Phormidium 1.44 0.72
Lyngbya 1.09 0.66
Limnothrix 0.69 0.5
66
maximum number of cyanobacterial species whereas waterlogged soil supported the least. This
might be because of nutrients availability in phumdis of the lake. Nostoc was found to be
dominant in all the samples collected from different sites and habitats followed by Anabaena
strains emphasizing their ability to adapt to a wide range of ecological niches.
The other genera were relatively infrequent in their occurrence (Phormidium, Calothrix,
Westiellopsis, Plectonema, Microchaete, Lyngbya, Limnothrix, Hapalosiphon and
Cylindrospermum) pointing to their limited ability to adapt to changes from their optimum
growth conditions. In the present observation it was observed that majority of Nostoc spp. were
associated with phumdis occurred mainly as epiphytic on the surfaces of the plant. Calothrix
species were found to occur near the shore of the lake on benthic habitat or as free floated near
the shore. Calothrix strains formed brown patches on soil or rock surfaces. Alternatively, they
were deep blue green in colour in their free floating forms. While growing on the soil surface it
forms patches which remain dark blue green in colour. As nutrient load increases, species that
can utilize increased nutrients efficiently took advantage and multiplied rapidly at the expense
of the less efficient species, which eventually were reduced numerically (Valsaraj and Rao,
1999).
Cyanobacteria being ubiquitous in nature possess a high potential of adaptation to
diverse environments (Garcia-Pichel et al., 2001). Loktak lake, being perennial ecosystem with
variable cyanobacterial diversity was composed of optimum level of light, water, temperature
and nutrient availability that provided a favourable environment for the luxuriant growth of
cyanobacteria. These factors played an important role in the distribution of cyanobacteria in
Loktak ecosystem. In the present study, the presence of diverse forms of cyanobacteria
indicates a good balancing of the ecosystem. In any ecosystem, not a single species grow
independently and indefinitely because all the species were interlinked and have cyclic
transformation of nutrients. The physico-chemical changes in the environment may affect
67
particular species and induce the growth and abundance of other species which leads to the
succession of several species in a course of time. Of the 90 strains of cyanobacteria recorded in
the present study, seven heterocystous genera namely; Anabaena (24), Nostoc (26), Calothrix
(08), Westiellopsis (04), Hapalosiphon (01), Microchaete (05) and Cylindrospermum (01) were
cultured. 04 genera belong to Phormidium (14), Plectonema (02), Lyngbya (03) and Limnothrix
(02) from non heterocystous filamentous group were also cultured successfully.
Unicellular forms of microalgae such as Aphanocapsa sp., Gloeocapsa sp.,
Glaucocystis sp., Gomphosphaeria sp. and Microcystis sp. were also observed but it could not
be able to isolate as unialgae and also not incorporated in present study. Hence, the present
study concluded in spite of the fact that the cyanobacteria were ubiquitous; their population
dynamics are often influenced by the available nutrients and the physico-chemical conditions
of the ecosystem. Low levels of nitrogen source in the environment were also eliminating non
heterocystous forms, since nitrogen free medium was commonly used for the isolation and
purification of heterocystous cyanobacteria. In the present study, the distributional pattern
showed that heterocystous filamentous cyanobacteria dominated in all the sampling sites. In
general, slightly acidic habitats harboured more of heterocystous filamentous forms than
unicellular or non heterocystous filamentous forms.
The interplay among the other environmental parameters such as temperature, moisture
content, availability of nutrients, etc. that are found in lake ecosystem also contributed towards
shaping the cyanobacterial diversity. Among the different environmental parameters, pH is one
of the most important factors responsible for proliferation of cyanobacteria (Nayak and
Prasanna, 2007). Since physico-geographically, Manipur consists of hilly terrains and during
heavy rainfall, these components get dissolved in the soil and water augmenting their acidic
properties. Majority of the dominant genera (heterocystous filamentous forms) were observed
in winter and summer season. It was followed by a drastic fall immediately in the next season
68
i.e. rainy season for non heterocystous filamentous forms. Anabaena, Nostoc and Calothrix
were observed to be consistent in all the seasons.
Chellappa et al. (2004) reported the collective dominance by the species of
cyanobacteria was due to their capacity to grow in turbid water and low light intensity to
maintain buoyancy. Diversity indices take into account both species richness and the relative
abundance of each species to quantify how well species are represented within a community.
The lower index value indicated the lack of species richness and degraded state of the
ecosystem. A high value of diversity index generally implies healthy ecosystem, while a low
value indicated degraded state (Manna et al., 2010). In the present study, it was observed that
cyanobacteria especially, Nostoc, Anabaena and Calothrix were observed as dominant and co-
dominant isolates. Nostoc sp. showed high Shannon (2.16) and Simpson’s (0.86) indices which
emphasizing the resilience and strong ability of Nostoc to adapt to variations in their
surroundings. Cylindrospermum and Hapalosiphon showed no Shannon and Simpson’s indices
which implies that they showed no diversity and species richness. Shannon’s indices are
strongly biased towards richness, as it is calculated from proportional abundances of the
species.
On the other hand, Simpson’s index is a measure of diversity, which takes into account
both richness and evenness, although it gives more weight to the more abundant species in a
sample. Generally, cyanobacteria increased the frequency of heterocysts during unfavourable
environment and nutrient deficiency. The abundance of heterocystous forms indicates suitable
environmental conditions for their growth. Nevertheless, good numbers of heterocystous
strains were observed during winter season which suggest the presence of some limiting factors
in heterocyst development. In Manipur, winter is characterized by low temperature, water and
light deficiency which may acts as limiting factors. Similar assumptions were made by several
workers and reported predominance of heterocystous form during dry periods (Roger and
69
Reynaud, 1976; Roger and Kulasooriya, 1980). Nutrient accumulation may have different
forces on the ecosystem at different periods (Glibert et al., 2007).
In the present study, the results on physico-chemical composition of sampling sites
revealed that they were nutrient rich environments in rainy season; in particular, total
phosphorous and nitrate concentrations which may have greatly supported the abundance of
different cyanobacterial morphotypes. Species diversity responds to changes in environmental
gradients and may characterize many interactions that can establish the intricate pattern of
community structure. Normally, it was found that any slight alteration in environmental status
can change diversity until there is no adaptation or gene flow from non-adaptive sources. Rainy
season characterized by high temperature and light intensity, supported maximum growth of
non heterocystous cyanobacteria. Low quantities of nitrates, phosphates, total dissolved solids,
acidity of water bodies in seasons enhance the growth of heterocystous cyanobacteria. Winter
season has shown maximum number of Nostoc isolates.
In this study, pH of 6.88-6.98 during winter season and the moist conditions just after
rainy season might have favoured the growth of Nostoc. There was a significant relation
between pH and heterocystous cyanobacterial dominance in winter season. This may be due to
the fact that some essential elements were bio available at certain required pH. A similar
phenomenon was found in lake Doirani (Temponeras et al., 2000) and lake Taihu (Chen et al.,
2003). It has been suggested that cyanobacteria are favoured in high pH environments, possibly
because of their ability to use bicarbonate ion as a carbon source when nitrogen or phosphorus
depletion occurs in summer (Shapiro, 1984; Dokulil and Teubner, 2000). In the present study,
non heterocystous cyanobacterial growth was observed during rainy season. The warm water
and high trophic levels favoured non heterocystous cyanobacterial growth also reported by the
previous workers (Dokulil and Teubner, 2000; Lei et al., 2005).
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Dominance of cyanobacteria in water bodies depended not only on factors such as light
and temperature but also on the nutrient load which affect the species composition (Riegman et
al., 1990). In this study, it was found that the availability of nitrate and phosphate in the rainy
and winter seasons were greater in comparison to the summer season. A high level of nitrogen
source in the environment was also eliminating heterocystous forms, since nitrogen free
medium was commonly used for the isolation and purification of heterocystous cyanobacteria.
This availability supported the abundant growth of cyanobacteria. The water of Loktak lake is
utilized for agriculture, fish culture and domestic purpose. Water temperature ranged from
18.7°C-26.7°C and 9.80°C-19.7°C which was highest during summer season and lowest during
winter. Higher temperature obtained from the lake could be due to increase in rate of chemical
reaction and nature of biological activities, since temperature is one of the factors that govern
the assimilative capacity of the aquatic system.
It is also because of the shallowness of the lake and consequently the volume of water
in contact with air, a close relationship exists between atmospheric temperature and water
temperature and as such the water is warmer during summer and rainy, colder during winter
also supported by previous investigators (Jawale and Patil, 2009; Narayana et al., 2008; Anita
et al., 2005). pH is one of the most important factors that serves as an index of the pollution.
Changes in the pH of water may be the result of various biological activities (Gupta et al.,
1996). The lake water was acidic to alkaline and lower value of pH was observed during winter
periods.
The pH was recorded highest (pH 7.24) during rainy and lowest (pH 6.88) during
winter season. Variations of pH over a higher range are often observed in the lakes due to
several factors such as interactions with suspended matter, influence of freshwater inputs,
pollution and photosynthesis. The highest pH value was recorded during rainy season, this
might be attributed high photosynthetic due to the abundance of the algal population and
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increase of carbonate (Reid, 1961). The lower pH during other season was evidently due to the
increased decomposition under low water depth. The high pH value is probably due to the
addition of hydroxyl, bicarbonate and carbonate anions (Zutshi et al., 1980). A large amount of
fertilizers residues are washed down the lake from the lake periphery paddy fields during the
rainy season and accelerate pollution in the lake.
Total dissolved solids were recorded in the range of 40.51-110.23 mg/l during the rainy
season and this may be attributed to influx of soil particles and the organic materials from the
catchment area due to rain. This variation may be due to the size of the water body, inflow of
water, consumption of salt by algae and other aquatic plants, and the rate of evaporation
(Lashari et al., 2009). Total dissolved solids had a cyclic of seasonal changes and maximum
during rainy season and minimum in summer. Similar results from Loktak lake was also
reported by Sharma and Sharma (2011). This indicated that the dissolved materials were of
allochthonous origin, which was brought into the lake system with surface runoff. Similarly,
Jawale and Patil (2009) and Salve and Hiware (2006) reported seasonal analysis and stated that
least total dissolved solids recorded in summer season while maximum value in rainy season.
Lake water quality is extremely influenced by the relative abundance of nutrients. High
concentration of nutrients in the water may be attributed to the fact that the lake receives huge
amount of domestic and municipal sewage and solid waste from the surface run-off and
effluents discharging from the catchment area and surrounding agricultural fields. The main
source of nitrate and phosphate is the run-off and decomposition of organic matter. The higher
inflow of water and consequent land drainage cause high value of nitrate (Thilanga et al.,
2005).
The seasonal variations of nitrates showed that there is a relative increase in the nitrates
during rainy season. Nitrates were maximum during rainy (0.24-0.97 mg/l) and minimum
during summer season (0.20-0.88 mg/l). This is mainly attributed to the oxidation of existing
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ammonia, yielding nitrate as intermediate state especially in abundant oxygen during winter
(Wetzel, 1983). During summer season, nitrates could be due to algal assimilation and other
the reduction in biochemical mechanism. Similar results have been reported by Gohram, 1961;
Rajashekhar et al., 2007. Phosphate-phosphorus concentrations fluctuate between 0.19 mg/l
and 0.86 mg/l at various sites of the lake. Overall high amount was observed during rainy
season. This may be attributed to the inflow of nutrients from the catchment area where
fertilizers are extremely used for agriculture. Also due to rain draining onto the lake with the
nutrient rich soil deposited from the catchment areas of the lake by its feeder streams and
rivers. Phosphate might be contaminated by the fertilizer used in nearby agricultural field and
detergent that are widely used. Similar results have been reported by the previous workers
(Chary, 2003; Rao, 2004).
At the same time, the maintenance of the isolated strains is fundamental for future
discoveries related to the broad potential application of cyanobacterial secondary metabolites
namely; pharmaceutical and bioengineering purposes (Abed et al., 2009). It has been reported
that cyanobacterial species with gas vacuoles can either move down to avoid the high
irradiance near the water surface or float up when underwater light environments are poor
(Ibelings et al., 1991; Brookes and Ganf, 2001). The significant relationship (s) between
cyanobacteria abundance and total dissolved solids and temperature is an indication of the
inter-dependance between these important water quality characteristics and the biota (Shehata
et al., 2009).
The maintenance of metabolically active cyanobacteria usually has one of the three
objectives: conservation of stock cultures, achievement of a specific morphological and
physiological status or mass culture. However, for stock cultures maintained by routine serial
subculture, it was often desirable to use sub-optimal temperature and light regimens; these
factors may be similar for different cyanobacteria. Freshly inoculated cultures were often
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incubated under optimal conditions for a short period to obtain sufficient biomass and refresh
the strain, after which the strain was maintained sub optimally. Culture conditions may
dramatically change with time in continuous culture even when the external environment
remains unchanged and the cyanobacterial culture has not exhausted the supply of any essential
nutrients.
Standard light intensities between 10-30 µmol photons/m2/s have proved appropriate in
combination with subdued temperatures for long-term culturing of most cyanobacteria. Over-
illumination is a widespread mistake in the perpetual maintenance of cultures. Not only
excessive light result in photo-oxidative stress in some cyanobacteria, localized heating may
also be problematic. Light and dark photoperiods are required for the maintenance of most
cultures. Inappropriate light: dark regimens may lead to unwanted photoperiodic effects.
Temperature is a major factor and should be carefully controlled; conditions may vary greatly
within a culture facility or laboratory and should be periodically monitored. Incubation
temperatures higher than 20°C should be combined with increased light intensities to prevent
photo-inhibition or damage. Therefore, temperatures of more than 20°C were mostly
inappropriate for maintaining stocks at extended transfer cycles. Furthermore, as maintenance
temperatures increase, evaporation increases. The evaporation of the medium effectively
determines the duration of their transfer cycles for many robust strains of cyanobacteria
(Lorenz et al., 2005).