38 ISOLATION, IDENTIFICATION AND MAINTENANCE...

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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

39

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

40

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

43

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



Altitude: 772 m

straight and

slightly

constricted at

cross-walls

diffluent longer than broad

Phormidium corium (Ag.)

Gomont BTA-64

Hydrophytes



Altitude: 782 m

flexuous thin quadrate

Phormidium sp. BTA-71 Waterlogged soil Latitude: N24°28'31.5"


Altitude: 767 m

straight thick and

colourless

longer than broad

Phormidium sp. BTA-75 Hydrophytes Latitude: N24°36'41.8"


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



Altitude: 816 m

flexuous;

constricted at

cross-walls

diffluent quadrate; conical

end cell

48


strains


Filament/

Trichome

Sheath Cell shape


Gomont BTA-1045

Hydrophytes



Altitude: 779 m

straight thin and diffluent longer than

broad

Phormidium sp. (Ag.)

Gomont BTA-1048

Waterlogged soil Latitude: N24°30'58.9"


Altitude: 820 m

slightly bent thin and diffluent quadrate

Phormidium fragile

(Meneghini) Gomont

BTA-1052

Hydrophytes



Altitude: 760 m

flexuous;

constricted at

cross-walls

diffluent quadrate

Phormidium corium (Ag.)

Gomont

BTA-1065

Moist soil Latitude: N24°31'08.2"


Altitude: 825 m

long; straight

ends

thin quadrate


Gomont

BTA-1073



Altitude: 769 m

slightly bent diffluent longer than

broad


Gomont

BTA-1076



Altitude: 766 m

slightly bent;

blunt end

thin longer than

broad

49

Comparative analysis of the genus-Lyngbya


strains


Filament/

Trichome

Sheath Cell shape

Lyngbya aestuarii Liebm. ex Gomont

BTA-66



Altitude: 772 m

straight; flat

end cells

firm and

lamellated

short and

granulated

Lyngbya putealis Mont. ex Gomont

BTA-1013



Altitude: 820 m

curved thin constricted at

cross-walls

and granulated

Lyngbya birgei Smith G. M.

BTA-1080

Hydrophytes



Altitude: 825 m

straight firm and

colourless

short; rounded

ends

Description of the genus-Cylindrospermum

Name of the strains Habitat of

the strains


Filament/

Trichome

Sheath Cell

shape

Heterocyst Akinete

Cylindrospermum

muscicola Kutzing ex

Born. et Flah. BTA-963

Waterlogged

soil



Altitude: 760 m

broad;

constricted

at cross-

walls

hyaline quadrate

to

cylindrical

oblong oval

50

Comparative analysis of the genus-Nostoc


strains


Filament/

Trichome

Sheath Cell

shape

Heterocyst Akinete

Nostoc sp. BTA-53 Hydrophytes Latitude: N24°30'37.8"


Altitude: 772 m

flexuous;

curved

diffluent barrel sub-spherical oblong

Nostoc paludosum Kutzing

ex Born. et Flah. BTA-56



Altitude: 770 m

entangled distinct

and

colourless

barrel to

cylindrical

broader than

vegetative

cells

oval

Nostoc sp.

BTA-60

Waterlogged

soil



Altitude: 769 m

flexuous hyaline barrel or

spherical

both

intercalary

and terminal;

sub-spherical

spherical

Nostoc sp.

BTA-61



Altitude: 832 m

flexuous thin and

hyaline

quadratic spherical oblong

Nostoc commune Vaucher

ex Born. et Flah.

BTA-67



Altitude: 778 m

flexuous

and

entangled

distinct barrel spherical oblong

Nostoc sp.

BTA-80



Altitude: 768 m

flexuous diffluent quadratic spherical spherical

Nostoc muscorum

BTA-950

Waterlogged

soil



Altitude: 765 m

flexuous hyaline barrel spherical oblong

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strains


Filament/

Trichome

Sheath Cell

shape

Heterocyst Akinete



Altitude: 770 m

flexuous distinct barrel spherical ellipsoidal

Nostoc sp. BTA-979 Hydrophytes



Altitude: 760 m

flexuous

and loosely

entangled

indistinct barrel spherical sub-

spherical

Nostoc calcicola

Brebisson ex Born. et

Flah. BTA-984



Altitude: 766 m

loosely

entangled

hyaline barrel sub-

spherical

sub-

spherical

Nostoc sp. BTA-995 Wet surface of

wall



Altitude: 830 m

flexuous

and loosely

entangled

indistinct cylindrical sub-

spherical

oval

Nostoc ellipsosporum

(Desm.) Rabenh. ex

Born. et Flah.

BTA-999

Waterlogged

soil



Altitude: 822 m

flexuous

and loosely

entangled


spherical

ellipsoidal

Nostoc muscorum Ag.

ex Born.et Flah.

BTA-1001

Waterlogged

soil



Altitude: 761 m

flexuous

and loosely

entangled

distinct barrel spherical oblong

Nostoc sp. BTA-1003 Leaf of

hydrophytes



Altitude: 766 m

flexuous indistinct barrel oval ellipsoidal

52


strains


Filament/

Trichome

Sheath Cell

shape

Heterocyst Akinete

Nostoc spongiaeforme

Agardh ex Born. et

Flah. BTA-1005

Root of

hydrophytes



Altitude: 807 m

flexuous and

loosely

entangled

distinct short and

barrel

sub-

spherical

oblong

Nostoc sp. BTA-1008 Waterlogged

soil



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



Altitude: 760 m

flexuous and

loosely

entangled


spherical or

oblong

ellipsoidal

to oblong


Agardh ex Born. et

Flah. BTA-1018

Hydrophytes



Altitude: 760 m

flexuous and

loosely

entangled

indistinct cylindrical

and partly

barrel

intercalary

and

terminal;

sub-

spherical

oblong




Altitude: 760 m

loosely

entangled

distinct barrel and

partly

quadratic

spherical sub-

spherical




Altitude: 807 m

flexuous diffluent barrel and

partly

cylindrical

sub-

spherical

oblong

53


strains


Filament/

Trichome

Sheath Cell

shape

Heterocyst Akinete


Agardh ex Born. et

Flah. BTA-1035

Leaf of

hydrophytes



Altitude: 830 m

flexuous and

loosely

entangled

indistinct partly

cylindrical

and partly

barrel

sub-

spherical

ellipsoidal



Altitude: 830 m

flexuous diffluent barrel sub-

spherical

oblong



Altitude: 760 m

loosely

entangled


spherical

ellipsoidal


Agardh ex Born. et

Flah. BTA-1056

Hydrophytes



Altitude: 807 m

flexuous and

loosely

entangled


spherical

ellipsoidal

Nostoc sp. BTA-1057 Leaf of

hydrophytes



Altitude: 765 m

flexuous and

entangled

hyaline barrel sub-

spherical

spherical

54

Comparative analysis of the genus-Anabaena


strains


Filament/

Trichome

Sheath Cell shape Heterocyst Akinete

Anabaena sp. BTA-69 Leaf of

hydrophytes



Altitude: 767 m

flexuous diffluent barrel intercalary;

sub-

spherical

oblong

Anabaena variabilis

Kutzing ex Born. et

Flah. BTA-72



Altitude: 772 m

flexuous hyaline

and

colourless

barrel spherical oval

Anabaena iyengarii

Bharadwaja BTA-74

Attached on

surface of boat



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



Altitude: 766 m

flexuous and

entangled

hyaline

and

indistinct

barrel or

spherical

sub-

spherical

cylindrical

Anabaena sp.

BTA-949

Leaf of

hydrophytes



Altitude: 760 m

flexuous thin and

hyaline

barrel; end

cell conical

spherical oblong

Anabaena sp. BTA-

958



Altitude: 753 m

straight absent barrel spherical oblong

Anabaena sp. BTA-

964



Altitude: 772 m

flexuous hyaline barrel spherical oval

55


the strains


Filament/

Trichome



hydrophytes



Altitude: 822 m

bent absent barrel sub-spherical ellipsoidal

Anabaena ambigua

Rao, C. B. BTA-983

Moist soil



Altitude: 766 m

bent;

enclosed in a

mucilaginous

sheath

thin and

hyaline

barrel;

constriction

at the joints

spherical;

broader than

cells

ellipsoidal


hydrophytes



Altitude: 830 m

bent hyaline barrel;

granulated

oblong ellipsoidal

Anabaena sp. BTA-996 Lotus leaf



Altitude: 824 m

bent hyaline barrel; end

cell conical

sub-spherical oval

Anabaena sp. BTA-997 Hydrophytes



Altitude: 824 m

flexuous thin and

hyaline

barrel or

sub-

quadrate

spherical oblong

Anabaena sp.

BTA-1004

Submerged

root of

hydrophytes



Altitude: 807 m

slightly

curved

hyaline barrel oval oblong

56


the strains


Filament/

Trichome


Anabaena sp.

BTA-1006

Leaf of lotus Latitude: N24°13'14.7"


Altitude: 807 m

bent thin and

hyaline

barrel oval oblong

Anabaena anomala

Fritsch

BTA-1007

Leaf of

hydrophytes



Altitude: 779 m

bent diffluent barrel; apical

cell rounded

barrel ellipsoidal

Anabaena

ballyganglii Banerji

BTA-1009

Leaf of

hydrophytes



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



Altitude: 824 m

circinate;

seldom

straight

hyaline barrel sub-

spherical

oblong

Anabaena variabilis

Kutzing ex Born. et

Flah. BTA-1012



Altitude: 760 m

straight;

slight bent

colourless barrel spherical or

oval

oblong

Anabaena sp.

BTA-1025



Altitude: 765 m

bent thin and

hyaline

barrel to

quadratic

sub-

spherical

oval

Anabaena oryzae

Fritsch BTA-1026

Submerged

root of

hydrophytes



Altitude: 807 m

short to

straight;

aggregated

colourless barrel; longer

than broad

oval;

broader

than

vegetative

cells

ellipsoidal

57


the strains


Filament/

Trichome


Anabaena variabilis

Kutzing ex Born. et

Flah. BTA-1030

Submerged

root of

hydrophytes



Altitude: 807 m

straight diffluent barrel, end

cells conical

spherical

or oval

oblong

Anabaena iyengarii

Bharadwaja

BTA-1033

Attached on

surface of

boat



Altitude: 780 m

curved; end

cell conical

hyaline barrel sub-

spherical

ellipsoidal

Anabaena iyengarii

Bharadwaja

BTA-1058

Leaf of

hydrophytes



Altitude: 764 m

curved; end

cell conical

diffluent barrel spherical ellipsoidal

Anabaena vaginicola

Fritsch et Rich

BTA-1074



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



Altitude: 759 m

bent hyaline barrel spherical

or oval

oblong

58

Comparative analysis of the genus-Plectonema


strains


Filament/ Trichome Sheath Cell shape

Plectonema sp. BTA-65 Moist soil



Altitude: 772 m

curved and false

branched

firm and

hyaline

quadratic

Plectonema sp. BTA-79 Hydrophytes Latitude: N24°35'77.7"


Altitude: 822 m

flexuous and false

branched

lamellated

and

colourless

as long as

broad

Comparative analysis of the genus-Microchaete


the strains


Filament/

Trichome

Sheath Cell

shape

Heterocyst Akinete

Microchaete sp.

BTA-59

Epilithic Latitude: N24°30'37.8"


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



Altitude: 780 m

elongate firm sub-

quadrate

basal and

spherical

quadrate

to sub-

cylindrical

59


strains


Filament/

Trichome

Sheath Cell

shape

Heterocyst Akinete

Microchaete grisea

Thuret ex Born. et

Flah. BTA-1041

Submerged

root of

hydrophytes



Altitude: 807 m

broad and

bent

thin and

hyaline

long and

broad

basal,

intercalary

and spherical

absent


Carter, N. BTA-1043



Altitude: 822 m

straight thin and

colourless

long and

broad

basal and

spherical

absent


Carter, N. BTA-1081

Wet surface of

rock



Altitude: 807 m

slightly bent thin and

hyaline

long and

broad

basal and

hemispherical

absent

Comparative analysis of the genus-Calothrix


the strains


Filament/

Trichome

Sheath Cell

shape

Heterocyst Akinete

Calothrix ghosei

Bharadwaja BTA-57

Submerged

root of

hydrophytes



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



Altitude: 770 m


colourless

long and

broad

basal and

spherical

absent

60


the strains


Filament/

Trichome

Sheath Cell

shape

Heterocyst Akinete

Calothrix sp. BTA-73 Epilithic



Altitude: 772 m

bent colourless barrel basal and

spherical

absent

Calothrix ghosei

Bharadwaja BTA-77



Altitude: 772 m

slightly bent hyaline

and

distinct

quadratic basal and

spherical

cylindrical

Calothrix geitonos

Skuja BTA-998

Epilithic



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



Altitude: 824 m


colourless

long and

broad;

constrict

ed at the

cross

walls

basal and

hemispherical

absent

Calothrix marchica

Lemmermann

BTA-1014

Epilithic



Altitude: 820 m

straight thin and

colourless

long and

broad

basal and

spherical

absent

Calothrix ghosei

Bharadwaja

BTA-1019

Wet surface

of rock



Altitude: 807 m

bent hyaline

and

distinct

quadratic basal and

spherical

absent

61

Description of the genus-Hapalosiphon


the strains


Filament/

Trichome

Sheath Cell

shape

Heterocyst Akinete

Hapalosiphon

welwitschii

W. et G.S.West

BTA-58



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


the strains


Filament/

Trichome

Sheath Cell

shape

Heterocyst Akinete

Westiellopsis

prolifica Janet

BTA-51

Attached on

surface of

boat



Altitude: 770 m

erect; thin

and elongate;

true

branching

absent short

barrel

oblong-

cylindrical

spherical

Westiellopsis sp.

BTA-55



Altitude: 773 m

thick primary

filament and

thin

secondary

filament; true

branching

absent barrel spherical spherical

62


the strains


Filament/

Trichome

Sheath Cell

shape

Heterocyst Akinete

Westiellopsis sp.

BTA-68



Altitude: 772 m

erect; thick

primary filament

and thin

secondary

filament; true

branching

hyaline elongate

and

cylindrical

oblong-

cylindrical

oblong

Westiellopsis sp.

BTA-76



Altitude: 769 m

curved; true

branching

diffluent barrel oblong-

cylindrical

sub-

spherical

Comparative analysis of the genus-Limnothrix


strains


Filament/

Trichome

Sheath Cell shape

Limnothrix redekei (Van

Goor) Meffert BTA-987



Altitude: 820 m

straight hyaline isodiametric, two small

polar aerotypes at the

septa

Limnothrix vacuolifera

(Skuja) BTA-1051



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).

70

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

71

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

72

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

73

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).

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