III. ISOLATION, IDENTIFICATION, AXENIC
CULTURE AND BIOMASS
PRODUCTION
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
31
3.1. INTRODUCTION
In recent past, a number of cyanobacteria have been screened and recognized as a
rich but not yet extensively examined source of food, feed and pharmacological as well as
structurally interesting secondary metabolites (Ugwu et al. 2008). Cyanobacteria
characterize by their blue green color and can be distinguish from other organism. Further
classification of them is based on microscopic and molecular characterization. These
organisms are susceptible and their growth is affected by sudden physical and chemical
fluctuation of environmental conditions such as light, salinity, temperature and nutrient
limitations (Tomaselli and Giovannetti 1993; Oliveira et al. 1999). In literature it is
mentioned, cyanobacteria can grow where moisture is available but in actual experience
the cyanobacterial behavior in nature and in laboratory culture is differ (Chlipala et al.
2011). There is a need to observe the behavior in nature and growth requirements for
individual cyanobacterial species and to apply it in laboratory to scale up the biomass
production.
In the early 1950’s, the increase in the world’s population and predictions of an
insufficient protein supply led to a search for new alternative and unconventional protein
sources. Algal biomass appeared to be a good candidate for this purpose (Becker 2004;
Cornet 1998; Spolaore 2006). Commercial large-scale culture of algae started in the early
1960’s especially using the algal genera Chlorella, Scenedesmus and Dunaniella (Iwamoto
2004; Borowitzka 1999). The Spirulina has been traditionally consume by tribal people in
Central Africa and Mexico therefore in 1970’s, for cyanobacteria Spirulina, the culturing
and harvesting facility established at Lake Texcoco by Sosa Texcoco S.A. (Borowitzka
1999; Muller-Feuga 1996). Among the green and blue green algal species, Spirulina have
received greater attention as a source of human food and poultry feed. Now most species
of Spirulina are mass cultivated globally. The growth of Spirulina is faster and become
possible to obtain the large scale biomass in open and closed culture systems.
However, till date little industrial and economic success is achieved in utilization
of other cyanobacteria. Except Spirulina meager information is available on biomass
production of cyanobacteria. There are evidence that almost a billion year earlier
cyanobacterial forms present in stromalites (Charpy et al. 2012) and they are the pioneer
oxygen producing phtotosynthetic organisms. Additionally, new methods need to be
developed to allow the cultivation of previously ‘uncultivable’ strains.
Axenic cultures are important in genetic, biochemical, physiological and
taxonomic studies. Although numerous methods such as single cell isolation,
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
32
centrifugation cleaning (Vaara et al. 1979; Bolch and Blackburm 1996), UV irradiation or
gamma irradiation (Kraus MP 1966), differential filtration (Meffert and Chang 1978) and
bactericidal chemical treatment, lysozyme treatment, antibiotic (Pinter and Provasoli,
1958; Rippka R 1988; Vaara et al. 1979; Carmichael et al. 1974; Anderson 2005) have
been suggested to produce the axenic culture of cyanobacteria. Since cyanobacteria are
grows in moist and watery conditions, exhibiting enormous variation in growth,
morphology, possess complex multilayer envelop, filaments are tightly aggregated and
metabolic capabilities, application of any particular approach cannot guarantee success of
their purification. Cyanobacteria and bacteria have similar prokaryotic cell organization
showed the more or less similar responses to selective procedure such as antibiotic
treatment.
Therefore in this chapter, the attempts were made for isolation, identification and
establishment of culture and purification of culture. Further the emphasis was given for
large scale biomass production of some cyanobacteria.
3.2. MATERIALS AND METHODS
3.2.1 Source of cyanobacteria
A total of 840 soil and water samples were collected randomly from paddy field, water
bodies and moist rock from different locations belonging to Pune and adjoining area of
Ahmednagar, and Satara district of Maharashtra state, India during 2009 and 2010 (June –
October) (Table 3.2; Fig. 3.1). The Ahmednagar, Pune and Satara districts are situated at
19° 8′ N 74° 48′ E, 18° 31′ N, 73° 51′ E, 17° 42′ N 74° 02′ E, respectively on a plateau
which is about 43187.89 km2 in area.
Fig. III. 1 District location of study area under Maharashtra (India).
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
33
The collection of sample and isolation of cyanobacterial strains was carried out by
following the method described by Rippka et al. (1979); Anand et al (1990) and protocol
described by National Facility for Blue Green algae, IARI, New Delhi (Singh et al.
2001).The visualize blue-green filamentous forms were collected from water bodies and
moist soil and rock surface.
The samples were collected in the sterilized culture bottle (0.3 L capacity) and
brought to the laboratory. Some portions of algal samples were preserved in 4% formalin
for microscopic study.
3.2.2. Isolation and establishment of cultures
The soil sample (1g), scrape from moist rock (1g), few filaments from water samples and
Nostoc ball were suspended separately in 100 ml sterile BG-11 medium. The suspension
was shake thoroughly and 10ml suspension was diluted serially using sterile BG-11
medium as: 100, 10
-1, 10
-2, 10
-3 up to 10
-9. Each dilution was separately streaked on BG-11
agar plate. The filamentous forms exhibited radial spreading growth on the agar surface.
The filaments at the peripheral portions were picked up with the nichrome loop and
washed in the sterilized distilled water. Then the filaments were inoculated in the liquid
medium. This procedure was repeated till the unialgal cultures were established. At
weekly interval the cultures were observed under the research microscope for
contamination if any. Then the unialgal cultures were transferred to the culture bottles
with 50 mL of the liquid culture media. The cultures were incubated in culture room at
25±2oC temperature and 50-60% humidity. The cultures were exposed to 8 h light and 16
h dark cycle (30-40µmolm-2
S-1
light intensity).
3.2.3. Identification and enumeration of the cyanobacterial isolates:
The algal biomass was analyzed for chlorophyll and phycobilliprotein pigments using
paper chromatography (Jeffery 1961). After confirmation of cyanobacterial nature on the
basis of pigments the forms were observed for microscopic character. The dimension of
cell and filaments were measured using ocular and stage micrometer under the
microscope. The morphological characters of the cyanobacterial isolates were compared
with the characters enlisted in the monographs, research paper and keys published by
Prescott (1951), Desikachary (1959), Anand (1990, 1993), Kumar (1999), Graham and
Wilcox (2000), Trivedi (2001), Hoek et al. (2002), Anderson (2005), Komarek (2006),
Adhikary (2006), Lee (2008), Amsler (2008), Markou and Georgakakis (2011), Komarek
and Mares (2012). For some of the forms the recorded observations were verified and
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
34
confirmed with the help of experts from Krishnamurthy Institute of Algology, Chennai.
Identification of some of the cyanobacterial isolates was confirmed with 16s rRNA (Nubel
1997; Shih et al. 2013). The identified isolates were classified and arranged as per the
system of classification described by Desikachary (1959), Komarek and Mares (2012).
The relative abundance and percent of cyanobacterial species were determined
following the method of Devi et al. (1999). The relative abundance was calculated using
the formula: Relative abundance = (Y/X) × 100, where X is the total number of samples
collected and Y is number of samples from which a cyanobacterial strain was isolated.
The percentage of cyanobacterial strains in the soil samples was calculated as: The
percentage of cyanobacterial species (z) = (p/q), where q is the total number of
cyanobacterial species and p is number of particular species of cyanobacteria.
3.2.4. Nutrient media
The recommended nutrient media namely BG-11 (Stanier et al. 1971), Fogg’s medium
(Fogg’s 1949; Jacobson 1951), Allen and Arnon’s medium (Allen and Arnon 1955),
Zarrouk’s medium (Zarrouk 1966), CFTRI medium (Venkataraman and Becker 1984)
were used for establishment and growth studies of cyanobacterial isolates (Table 3.1).
3.2.4.1. Glasswares and plastic wares
The experiments were carried out using borosil make glasswares. The cultures were
grown in autoclavable glass culture bottles with polypropylene caps (0.3L, 5L, 10L, 20L).
For cleaning, the glasswares were soaked overnight in liquid soap and washed in running
tap water thoroughly and placed in an hot air oven at 80oC for 2 hours for drying. The
plastic semi-transparent culture trays (22.5 x 15 x 5.5 cm) were used for growing the
cyanobacterial isolates. After cleaning, the trays were sterilized by soaking in 4 %
formalin overnight. After drying the trays were rinsed with sterilized distilled water.
3.2.4.2. Preparation of stock solutions and nutrient media
The nutrient salts and other chemicals used for preparation of different media were
obtained preferably from HiMedia, Qualigens, SRL, E-Merck and Sigma Company. Glass
distilled water was used for the preparation of stock solutions of nutrient constituents.
Milli Q water (Millipore) was used for the preparation of the nutrient media. The nutrient
constituents and the concentrations recommended for different media are presented in
Table 3.1. The desired quantity of nutrient constituents for 10X stocks were weighed
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
35
accurately on Contech make single pan digital balance and dissolved in 75ml of distilled
water individually. The final volume (100 mL) was made by adding distilled water in
volumetric flask. The stock solution of the micronutrient was made by adding 100 X
concentration of each nutrient separately in 10 ml of water and finally all the dissolved
nutrients were mixed to make final volume of 100 mL with distilled water. From this stock
solution the desired quantities were picked up for preparation of particular volume of
medium. All stock solutions were stored at 0-4oC in refrigerator and used within a period
of month.
3.2.4.3. BG-11 (Stanier et al. 1971)
The required stock solutions of nutrient components for BG-11 medium were
added so as to maintain the recommended concentration of nutrient constituents as
described in Table 3.1. The stock solutions of were added in distilled water (volume less
by 10 mL than that of final volume). The pH of the medium was adjusted to 7.1 with 0.1 N
NaOH or 0.1 N HCl and final volume made with distilled water.
3.2.4.4. Fogg’s Medium (Fogg 1949; Jacobson 1951)
Required quantity of macro nutrients and micro nutrient stock solutions were
added in distilled water (volume less by 10 mL than that of final volume) as given in Table
3.1. The stock of Fe-EDTA was prepared separately; 26.1 gm of EDTA in 268 ml of 1 N
KOH was taken and 24.9 gm FeSO4. 7H2O was added to it and contents were dissolved by
stirring. The solution was aerated for 16-18 hours and Fe-EDTA was stored in amber
colored reagent bottle (Jacobson 1951). The pH of the medium was adjusted to 7.5 by drop
wise addition of 0.1 N NaOH or 0.1 N HCl.
3.3.4.5. Allen and Arnon medium (Allen and Arnon 1955)
Allen and Arnon medium was prepared as described by Allen and Arnon (1955).
The required stock solutions of nutrient components of Allen and Arnon medium were
added as described in Table 3.1. The stock solutions of different minerals were added in
distilled water (volume less by 10 mL than that of final volume). The pH of the medium
was adjusted to 8.2 with 0.1 N NaOH or 0.1 N HCl.
3.2.4.6. Zarrouk’s medium (Zarrouk 1966)
The required stock solutions of nutrient components of Zarrouk’s medium were
added as described in Table 3.1. The stock solutions of different minerals were added in
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
36
Table 3.1: Composition of nutrient media.
Constituent BG-11
medium
(g L-1
)
Fogg’s
Medium
(g L-1
)
Allen and
Arnon
medium
(g L-1
)
Zarrouk's
Medium
(g L-1
)
CFTRI
(g L-1
)
NaNO3 1.5 - - 2.5 1.5
K2HPO4.3H2O 0.04 0.20 0.456 1.0 0.5
MgSO4.7H2O 0.075 0.20 0.246 0.02 2.0
CaCl2.2H2O 0.036 0.10 0.074 0.04 0.04
Citric acid 0.006 - - - -
Ferric ammonium
citrate
0.006 - - 0.01 -
EDTA 0.001 - 0.004 0.01 -
Na2CO3 0.002 - - - -
KNO3 - - 2.02 - -
NaCl - - 0.232 1.0 1.0
K2SO4 - - - 1.0 1.0
NaHCO3 - - - - 4.5
FeSO4 - - - - 0.01
Micro nutrients mg/L mg/L mg/L mg/L mg/L
H3BO3 2.86 2.86 2.86 2.86 2.86
MnCl2.4H2O 1.81 1.81 1.81 1.81 1.81
ZnSO4.7H2O 0.222 0.222 0.222 0.222 0.222
Na2MoO4.2H2O 0.390 - - - 0.390
CuSO4.5H2O 0.080 0.079 0.079 0.079 0.080
CO(NO3)2.6H2O 0.040 - - - 0.040
MoO3 - 0.0177 0.0177 0.0177 -
pH 7.1 7.5 7.8 9.2 10.0
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
37
distilled water (volume less by 10 mL than that of final volume). The pH of the medium
was adjusted to 9.2 with 0.1 N NaOH or 0.1 N HCl.
3.2.4.7. CFTRI medium (Venkataraman and Becker 1984)
The required stock solutions of nutrient components of CFTRI medium were added
as described in Table 3.1. The stock solutions of different minerals were added in distilled
water (volume less by 10 mL than that of final volume). The pH of the medium was
adjusted to 10.0 with 0.1 N NaOH or 0.1 N HCl.
3.2.5. Preparation of solid medium
The desired quantities of liquid media were prepared and after adjusting recommended pH
2% w/v of agar-agar (Himedia, India) powder was added. The flask was kept in
microwave oven to dissolve agar. After well mixing, pour the medium in culture vessels
and sterilized by autoclaving or otherwise for petri-plate preparation the media was
sterilized in conical flask and poured in sterilized petri-plate in an aseptic condition under
laminar air flow.
3.2.6. Sterilization of media, glasswares and equipments
The clump of 7 or 10 plugged culture tubes containing medium was wrapped by
paper partially (above half portion) to prevent wetting of plugs during autoclaving. The
medium was sterilized at 105 kPa pressure and 121oC for 12 minutes. The required
glasswares and equipments such as scalpels, forceps, scissors, petri dishes, and surgical
blade holders were wrapped in the paper and sterilized in an autoclave. Distilled water was
sterilized in the conical flask. As per the recommendations for larger volumes of media or
water, the sterilization time was increased 12 min for 20ml; 20 min upto 100ml; 25 min up
to 2 lit and 35 min up to 5lit and more (Barsanti and Gualtieri 2006).
3.2.7. Inoculation
All the operations were carried out under laminar airflow fitted with ultra violet
(UV) tube and HEPA filter. The sterilized culture tubes, flasks, petri dishes, forceps and
surgical blades were kept in the laminar air flow and exposed to UV for 20 minutes to
sterilize surfaces of all glassware and instruments prior to use for transfer. 1g inoculum of
fresh biomass of cyanobacterial isolates was used for each culture vessel.
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
38
3.2.8. Culture conditions
All the cultures were maintained in the culture room at temperature 25±2°C under
8-h light/16-h dark photoperiod with a photosynthetic photon flux density of 40 µmoles-2
S-
1 provided by cool white fluorescent tube lights (Philips, India).
The growth of cyanobacterial isolates was observed in different culture media
namely BG-11 (Stanier et al. 1971); Fogg’s medium (Fogg 1949; Jacobson 1951); Allen
and Arnon’s medium (Allen and Arnon 1955), Zarrouk’s medium (Zarrouk 1966) and
CFTRI medium (Venkataraman and Becker 1984).
3.2.9. Purification of cyanobacterial isolates with antibiotics
By using centrifugation method (1000 x for 2 min), the filaments of unialgal isolates
(Table 3.3), were washed 7 times with sterile BG-11 medium. The filaments were
homogenized in BG-11 media using sterile homogenizer. The density of suspension of
was adjusted to 0.5 OD at 540 nm using sterile BG-11 medium. Inoculum size maintained
as 15-20% (v/v) of medium. For antibiotic treatment, the cyanobacterial suspensions were
initially starved for 24 h in dark at 300C. After starvation, sterile Luria Broth media
(Sambrook and Russell 2001) was added to the suspension and incubated for 24 h in dark
at 300C on rotary shaker at 180rpm. After incubation, the culture was centrifuge at
5000rpm for 1 min and washed 3 times with sterile distilled water. The pellet was
suspended in BG-11 medium containing gentamycin, penicillin, streptomycin, pipericillin,
carbenicillin, ampicillin, kanamycin, cefotaxim, imipenem and cephalosporin (100 µg/ml)
individually and incubated at 300C in dark for 24 h. Then the suspensions were washed
with sterile distilled water for 3 times and suspended in sterile BG-11 medium. These
antibiotics were used along with the cyclohexamide (100µg/ml) to avoid the fungal
contamination.
The above treatments of antibiotics were not effective for establishment of axenic
cultures of Leptolyngbya foveolarum. Therefore by following above method the
suspension of Leptolyngbya foveolarum was treated with lysozyme (0.5 mg mL-1
for 10
min), ppm solutions (Plant preservative mixture) and antibiotics namely Meropenem,
Augmentin and Amikacin. The lysozyme treatment was given by following the method of
Sarchizian and Ardelean (2010).
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
39
3.2.10. Confirmation of axenic culture
After antibiotic treatment and washing with sterile distilled water, the algal suspensions
were inoculated in culture tube containing 10 ml BG-11 medium and incubated at 22±2 °C
for 7 days with 8h light: 16 h dark cycle. To detect the presence of bacteria in the cultures,
1ml of algal suspensions were inoculated with 4ml of broth media enriched with 0.05%
peptone and 0.01% yeast extract and incubated for 24 h. The contaminants were detected
by observing the turbidity in the medium. The cultures were also streaked on the nutrient
agar plate to observed growth of bacteria. To check the purity of the cyanobacterial
strains, the aliquots were examined critically under the phase contrast microscope and oil
immersion microscopy.
3.2.11. Molecular identification
Among 18 cyanobacterial isolates, the morphological characters of Leptolyngbya sp. did
not match with species recorded in literature therefore the attempts were made to identify
this genus with help of 16 s rRNA. For this identification the 16 s rRNA analysis was
carried out with the help of Genome bio Pvt. Ltd Pune.
3.2.12. Extraction of genomic DNA
The total genomic DNA was isolated from the fresh material by the following methods:
600µl of TNES buffer (10mM Tris, pH 7.5, 400 mM NaCl, 100mM EDTA, 0.6% SDS)
and 35µl of Proteinase-K (20mg/ml) was added to the sample and mixed well by inverting
the tube several times. The sample was incubated overnight at 500C after that 166.7 µl of
6M NaCl was added to it. The sample shakes vigorously for 20 seconds and microfuge at
14,000 rpm for 5 min at room temperature. Equal volume of ethanol was added and gently
mixed by inverting the tube a couple of times. The sample was then centrifuged at 14,000
rpm for 10 min at 40C. DNA pellet was washed with 200-700 µl of 100% ethanol followed
by 70% ethanol. The DNA sample was air dried and the pellet was resuspended in Tris-
EDTA.
3.2.13. Amplification of 16s rDNA primers
The PCR amplification of the bacterial 16S rDNA was performed using two bacterial
universal primers, CYA 106F (5’-CGGACGGGTGAGTAACGCGTGA-3’) and
CYA781R (GACTACTGGGGTATCTAATCCCATT-3’), with temperature cycler. DNA
was amplified in a 100 µL mixture composed of fifty picomole of each primer, 25nmol of
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
40
each dNTP, 200 µg of bovin serum albumin, 10 µl of 10X PCR buffer (100 mM Tris –
HCl [pH9.0], 15mM MgCl, 500mM KCl, 1% [v/v] Triton X-100, 0.1% [w/v] gelatin), and
10ng of template DNA and sterilized ddH2O. To minimize non specific annealing of the
primers to nontarget DNA, 0.5 U of super Taq DNA polymerase (make) was added to the
reaction mixture after the initial denaturation step (5min 1t 940C), at 80
0C.
After PCR is completed, the PCR products were checked on 2% Agarose by
Agarose Gel Electrophoresis and amplicons size was compared using reference Ladder.
2% agarose gel spiked with Ethidium bromide at a final concentration of 0.5 µg/ml was
prepared using Agarose (LE, Analytical Grade, Promega Corp., Madison, WI 53711 USA)
in 0.5X TBE buffer. 5.0 µl of PCR product was mixed with 1 µl of 6X Gel tracking dye.
5µl of gScale 100bp size standard (geneOmbio technologies, Pune; India) was loaded in
one lane for confirmation of size of the amplicon using reference ladder. The DNA
molecules were resolved at 5V/cm until the tracking dye is 2/3 distance away from the
lane within the gel. Bands were detected under a UV Trans illuminator. Gel images were
recorded using BIO-RAD GelDocXR gel documentation system. The PCR product of size
675 bp was generated through reaction using primers CYA106F/ CYA781R1 and 422 bp
product was generated using primer pair CYA359F/ CYA781R
The thermal cycler profile was as follows: initial denaturation at 94°C for 3 min,
followed by 35 cycles of denaturing at 94°C for 1 min, annealing at 60°C for 1 min, and
extension at 72°C for 1 min, and a final extension at 72°C for 10 min.
3.2.14. Sequence analysis and data processing
After this amplification, products were purified by using a geneO-spin PCR product
Purification kit (geneOmbio technologies, Pune; India) and were directly sequenced using
an ABI PRISM BigDye Terminator V3.1 kit (Applied Biosystems, USA) on an automated
DNA sequencing machine (3130 Genetic Analyzer). DNA sequencing was performed
using primer CYA106F and CYA781R1.
The sequences were analyzed using Sequencing Analysis 5.2 software. BLAST (Basic
local alignment search tool) analysis was performed at BlastN site at NCBI (National
center for biotechnology information) server (http://www.ncbi.nlm.nih.gov/BLAST).
Guiding phylogenetic tree was drawn using cluster algorithm with first five hits in NCBI
nucleotide sequence database. Tree generated using TreeTop – Phylogenetic analysis tool
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
41
(http://www.genebee.msu.su/services/phtree_reduced.html). The distance matrix was also
generated using TreeTop phylogenetic analysis tool.
3.2.15. Large scale biomass production:
For large scale biomass production, the unialgal cultures were grown in 0.3 L, 5lit,
10 lit and 20 lit capacity bottles containing 150 ml, 2.5, 5.0 and 10 lit medium
respectively. For establishment of tray culture system the culture rack unit was design (2.6
m H X 1.35 m L X 0.9m W) which can accommodate 160 trays (22.5 x 15 x 5.5 cm) in 11
compartments. The rack system was equipped with drip system to add the media as per
requirements. The unit was covered with highly transparent polythene. The racks were
placed in a shade net (50%). The experiments for large scale biomass production were
carried out at ambient conditions (light 80-120 µmol m-2
s-1
, temp 23±90 C. humidity 80-
90%). The inoculum of cyanobacterial isolates was prepared in 300 ml bottles. In each
tray or bottle 1g (approx.) of fresh inoculum was added.
3.2.16. Harvesting and measurement of growth:
The biomass from culture bottles and trays was harvested at the end of fifth week
of culture by filtration through double layer of fine nylon net. The harvested biomass was
washed 3 times with sterile distilled water and then air dried in desiccator for 5 min to
remove the surface water. The resultant biomass was weighed and fresh weight was
recorded. The biomass was dried on application of air blower in shade at room
temperature till constant weight achieved. The growth recorded on the basis of fresh
weight and dry weight.
3.2.17. Experimental design and statistical analysis
The experimetns were carried out in completely randomized design and repeated at
least thrice. Results were represented as analysis of variance (ANOVA) followed by
Duncan’s Multiple Range Test (DMRT) at 5% probability level. Otherwise variability in
data was shown as mean ± standard error.
3.3. RESULTS
3.3.1. Cyanobacterial diversity in study area
In the present investigation, cyanobacterial samples were collected from Western ghat
region especially from the locations of Pune, Ahmednagar and Satara district of
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
42
Maharashtra state, India (Table 3.2). The Western Ghats better known as Sahyadri range is
a hilly range running parallel to the coast, at an average elevation of 1,200 m, is one of the
hot spots of biodiversity in India. The area chosen for this study is represented by
prominent peaks like Kalsubai (1646 m), old Mahabaleshwar (1326 m), Bhor ghat (1285
m), Purandar fort (1313 m), Rajgad (1103 m). Kalsubai is the highest peak of the Sahaydri
ranges. The samples were randomly collected from 35 different localities, collection site,
latitude; longitude and altitude are shown in Table 3.2.
Total 840 samples were collected from 35 localities, out of which 378 were
identified as cyanobacteria and were described based on their morphology. Eighteen
cyanobacteria taxa belonging to 12 genera were recorded; among them twelve were
heterocystous. These identified cyanobacteria belong to 2 order and 5 families. The
number of species found per family was in order Oscillatoriaceae > Nostocaceae >
Scytonmeataceae > Rivulariaceae > Stigonematacae. Among the identified species, 7
species belong to the family Oscillatoriaceae and 6 to Nostocaceae. However only single
species was recorded from the Rivulariaceae and Stigonemataceae. The most densely
populated genus was Nostoc punctiforme followed by Nostoc ellipsosporum. However, the
genus Leptolyngbya fovelolarum found only in 4 habitats. Total 18 species (Table 3.3)
were identified with the help of morphological characters described in the monographs
and keys as per the references mentioned in material and method section.
Recently, the identification and classification of cyanobacteria is based on the
polyphasic approach (Johansen and Casamatta 2005; Komark and Mares 2012) which is a
combination of molecular and traditional morphological characters. In the present
investigation, the morphological characters of the cyanobacterial isolates were compared
with the characters enlisted in the monographs, research paper and keys published by
Prescott (1951), Desikachary (1959), Anand (1990, 1993), Kumar (1999), Graham and
Wilcox (2000), Trivedi (2001), Hoek et al (2002), Anderson (2005), Komarek (2006),
Adhikary (2006), Lee (2008), Amsler (2008), Markou and Georgakakis (2011); Komarek
and Mares (2012). For some of the forms the recorded observations were verified and
confirmed with the help of experts from Krishnamurthy Institute of Algology, Chennai.
Identification of Leptolyngbya foveolarum and Leptolyngbya sp. of the cyanobacterial
isolates was confirmed with 16s rRNA (Nubel 1997; Shih et al. 2013) The identified
isolates were classified and arranged as per the system of classification described by
Desikachary (1959), Anand (1989), and recommendation of Graham and Wilcox (2000),
Bergys (2002), Komarek 2006, Komarek and Mares (2012).
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
43
Table 3.2 Collection sites of the cyanobacterial samples from the state of Maharashtra
(India)
District Location Latitude N
Longitude E
Altitude
(meters)
Pune Pune University campus 18o 32' 59.21” 73
o 49' 31.75” 583
Pashan Lake 18o 31' 56.64” 73
o 74' 23.96” 592
NCL ground 18o 32' 22.13” 73
o 84' 28.14” 589
Bhimashankar 19o 04' 15.16” 73
o 32' 09.15” 933
Pirangut Ghat 18o 30' 03.25” 73
o 42' 33.08” 699
Paud 18o 31' 34.92” 73
o 36' 58.29” 584
Paud-Sus Road 18o 33' 42.19” 73
o 40' 22.38” 606
Mulshi 18o 31' 47.98” 73
o 31' 28.71” 577
Hinjewadi 18o 34' 53.97” 73
o 43' 28.57” 572
Katraj Ghat 18o 23' 42.62” 73
o 51' 13.60” 915
Nasarapur 18o 15' 21.29” 73
o 53' 23.79” 663
Shirwal 18o 08' 03.47” 73
o 58' 53.23” 595
Bhor Ghat, Bhor 18o 01' 44.50” 73
o 51' 06.75” 1285
Dive Ghat 18o 24' 55.63” 74
o 00' 22.07” 846
Purandar-Saswad Road 18o 18' 28.50” 73
o 58' 48.70” 861
Purandar Fort, Purandar 18o 16' 36.40” 73
o 58' 18.56” 1312
Paud Mulshi Road 18o 31' 41.46” 73
o 36' 40.47” 568
Mulshi Road 18o 34' 26.61” 73
o 32' 36.75” 590
Mulshi area 18o 31' 52.97” 73
o 33' 03.89” 574
Mulshi Dam 18o 28' 53.70” 73
o 29' 53.00” 633
Shivneri 19o 11' 39.66” 73
o 51' 29.76” 918
Ahmednagar
Arangao 19o 01' 15.96” 74
o 43' 19.23” 655
Bhandardara 19o 32' 45.25” 73
o 47' 05.14” 702
Akole 19o 32' 12.82” 73
o 59' 01.38” 601
Sangamner 19o 35' 10.52” 74
o 19' 06.61” 576
Rahata 19o 42' 03.91” 74
o 29' 06.05” 523
Shrirampur 19o 33' 37.25” 74
o 38' 33.02” 503
Rahuri 19o 22' 55.70” 74
o 38' 54.82” 509
Newase 19o 26' 29.84” 75
o 04' 16.40” 497
Shevgaon 19o 20' 39.20” 75
o 12' 54.59” 491
Pathardi 19o 07' 42.36” 74
o 58' 30.60” 647
Satara
Khandala 18o 02' 57.72” 74
o 00' 54.01” 665
Khambataki Ghat 18o 00' 48.87” 74
o 49' 46.95” 920
Pachgani 17o 55' 55.56” 73
o 51' 04.33” 952
Old Mahabaleshwar 17o 57' 43.42” 73
o 39' 45.19” 1326
Lingmala 17o 55' 21.00” 73
o 42' 17.36” 1272
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
44
Table 3.3: Cyanobacterial diversity from Pune, Ahmednagar and Satara district
Order, Family, Genus and
Species
No. of samples
in which
particular
cyanobacteria
observed
Relative
abundance
(%)
% distribution
of
Cyanobacterial
species
Nostocales
Oscillatoriaceae
Spirullina platensis (Nordst.)
Geitler 07
0.83 1.85
Oscillatoria chalybea 39 4.64 10.3
Phormidium fragile(Meneghini)
Gomont 19
2.26 5.02
Leptolyngbya sp. 01 0.11 0.26
Leptolyngbya fovelarum 04 0.47 1.05
Lyngbya bipunctata Lemm. 32 3.80 8.46
Microcoleus lacustris (Rabenh.)
Farlow 28
3.33 7.40
Nostocaceae
Nostoc punctiforme Born. Et Flah. 68 8.09 17.9
Nostoc entophytum Born. Et Flah. 32 3.80 8.46
Nostoc ellipsosporum (Desm.)
Rabenh. Ex Born. et Flah. 41
4.88 10.8
Nostoc calcicola Brebsson ex Born.
et Flah. 17
2.02 4.49
Nostoc muscorum Ag. ex. Born. at
Flah. 13
1.54 3.43
Anabaena subcylindrica. 18 2.14 4.76
Scytonemataceae
Scytonema tolypothrichoides
Kützing ex Born. et Flah 14
1.66 3.70
Scytonema mirabile (Dillw.) Born. 08 0.95 2.11
Tolypothrix fragilis (Gardner)
Geitler 11
1.30 2.91
Rivulariaceae
Calothrix javanica de Wilde 23 2.73 6.08
Stigonematales
Stigonemataceae
Westiellopsis prolifica Janet 03 0.35 0.79
Total no of cyanobacterial form 378
Total no. of sample collected 840
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
45
3.3.1.1. Taxonomic enumeration of the species isolated (Plate-III-1&2)
1. Spirulina platensis (Nordst.) Geitler (Desikachary 1959: 190) (Plate-III-1E) Trichome
spirally coiled, filamentous, Multicellular, unbranched, 6-8 µm broad not attenuated at
the ends, spirals 20-30 µm broad, 3-8 µm long; end cells broadly rounded, Cross wall
of cells not distinct, composed of cylindrical cells.
2. Oscillatoria chalybea (Mertens) Gomont (Desikachary 1959: 218) (Plate-III-1J)
Thallus dark blue green, trichome straight, attenuated at the apex and somewhat bent,
8-13 µ broad, cells broad, quadrangular, without calyptra, trichome single forming flat
or thallus sheath, motile end cells showing typical oscillatory movements unbranched,
hormogones present.
3. Phormidium fragile (Meneghini) Gomont (Desikachary 1959: 253) (Plate-III-1K)
Filaments parallel many forming a sheet like thallus, attached to soil, sheath diffluent,
thallus yellowish blue-green, mucilaginous, lamellated; trichomes more or less
flexuous, entangled, constricted at the cross walls, 1-3 µm broad; cells nearly quadrate,
attenuated at the ends; end cell conical, calyptras absent.
4. Lyngbya bipunctata Lemm. (Desikachary 1959: 290) (Plate-III-1E). Filament free or
entangled forming expanded thallus, trichome solitary, free floating; sheath narrow,
colorless cells, 1-2 µm broad, 4-5 µm long, not constricted at the cross walls, end cell
rounded not attenuated.
5. Microcoleus lacustris (Rabenh.) Farlow (Desikachary 1959: 345) (Plate-III-2D).
Filamentous, many trichomes in bundles inside the firm or gelatinizing sheath,
contorted like rope, thallus blackish blue-green; sheath colorless, slimy, trichome
distinctly constricted at the cross-walls, 4-5 µm broad, 6-12 µm long; end cell
rounded, conical not capitate.
6. Leptolyngbya kmn-1(KC589411) (Plate-III-1D): New species recorded in the
present investigation
Filament in cluster and mats, enveloping in thin, firm and colourless sheaths opened at
the apical end. It is immotile with rounded apical cells, usually constricted at the cross
walls, and very rarely false branching. Cells is pale blue-green or olive-green
cylindrical-shaped, 0.5-3 µm wide, length is longer than wide up to several times,
rarely with prominent granules, without heterocytes and akinetes.
7. Leptolyngbya foveolarum (Montagne ex Gomont) Anagnostidis & Komarek 1988
(Plate-III-1C): filamentous, long, thin, bright blue green trichome, cell content
homogenous or sparsely granulated, cells cylindrical, shorter, rarely longer than wide,
0.9 µm -2.3 µm wide, 1-4 µm long, constricted at ungranulated cross wall, sheath often
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
46
extend beyond end of thrichome, firm, thin, colorless, diffluent, variously curved,
sometimes straight filaments and in parallel arranged, often tangled together, pseudo-
branching rare.
8. Nostoc punctiforme Born. Et Flah. (Desikachary 1959: 374) (Plate-III-1G). Thallus
without firm outer layer, soft formless, trichome densely coiled hardly seen, trichome
1-3 µm broad, filament densely entangled, sub-globose, blue green, 2 mm in diameter,
scattered, attached, sheath delicate, hyaline, mucilaginous; cells barrel-shaped;
heterocyst 2-5 µm broad.
9. Nostoc entophytum Born. et Flah. (Desikachary 1959: 375) (Plate-III-1H). Thallus
macroscopic, blue-green, small, trichome densely entangled with distinct hyaline
sheath; trichome 2-3 µm broad; cells short, 3.8 µ broad, barrel shaped; heterocyst
broader than vegetative cells.
10. Nostoc ellipsosporum (Desm.) Rabenh. Ex Born. et Flah. (Desikachary 1959: 383)
(Plate-III-1F). Thallus irregularly expanded, gelationous; filaments flexuous, loosely
entangled; cells 3-4 µm wide, 5-8 µm long; heterocysts subspherical, 4-7 µm wide, 4-
10 µm long; akinetes ellipsoidal, wall smooth, colorless. Spore ellipsoidal to
cylindrical, 5.8-7.8 µ X 7.8-11.7 µ.
11. Nostoc calcicola Brebsson ex Born. et Flah. (Desikachary 1959: 384) (Plate-III-1B).
Thallus olive grey, up to 2-3 cm in diameter, mucilaginous, slightly diffluent; filament
loosely entangled; sheath indistinct, only at the periphery of the thallus, colorless;
trichome blue-green, 2 µm broad; cells subspherical, longer than broad; heterocysts
subspherical, 2-4 µm broad. Spores spherical, 4.5-5.8 µ diameter.
12. Nostoc muscorum Ag. ex. Born. et Flah. (Desikachary 1959: 385) (Plate-III-1I).
Colony expanded, 3 cm in diameter, olive green; filaments thickly entangled; sheath
distinct only at the periphery of the colony, yellow brown; trichome 4 µm broad; cells
short barrel shaped; heterocysts subspherical, 6 µm wide; akinete oblong, 3-6 µm
broad, 7-10 µm long, wall smooth, yellow. Trichome 5 µ broad, cells barrel shaped,
shorter or longer than broad.
13. Anabaena subcylindrica Borge 1921 (Plate III-1A): Trichome straight, often
aggregated to form blue green colony, Mucilage thin and usually colorless, cells
subspherical or ellipsoidal, 3-4 µ wide, 5-7 µm long, heterocyst 4.5-5.5 µm wide, 5.5 -
8 µm long. Akinetes cylindrical 5-8 µm wide, 21-30 µm long, wall smooth colorless.
14. Scytonema tolypothrichoides Kutzing ex Born. et Flah (Desikachary 1959: 479)
(Plate-III-2B). Thallus dense, brownish green; filaments 8-10 µm broad, 2-3 mm long,
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
47
repeatedly false branched, false branches very similar to main filaments; sheath
lamellated; trichome 6-8 µm broad; cells longer, densely granulated; heterocyst varied.
15. Scytonema mirabile (Dillw.) Born. (Desikachary 1959: 483) (Plate-III-2A) Colony
bushy, blackish brown; filaments entangled, 2-12 mm long, mostly false branched;
cells 8-12 µm wide, cylindrical, at the end of trichome disc-shaped; sheath blue green;
heterocyst rounded.
16. Tolypothrix fragilis (Gardner) Geitler (Desikachary 1959: 500) (Plate-III-2F) Colonies
dense, blue-green; filament heteropolar, united, free apical ends, falsely branched,
solitary lateral branches; sheath distinct, colorless; trichome with basal cylindrical
heterocyst; cells cylindrical.
17. Calothrix javanica de Wilde (Desikachary 1959: 525) (Plate-III-2C) Filaments
heteropolar, simple, lateral false branches, 30-40 µm broad; trichome with widened
basal, constricted at the cross walls; sheath present, thick, yellow-brownish colored;
heterocyst basal; cells cylindrical. Spores spherical upto 6.5 µ diameter.
18. Westiellopsis prolifica Janet (Desikachary 1959: 596) (Plate-III-1L) Thallus
filamentous, filament loosely entangled, lateral branches clearly seen, cells of main
filament short, 5.0 -9.1 µ broad, 3.5 -7.5 µ long, barrel shaped, cylindrical, lateral
filament 5.5 – 9.8 µ broad, 4.5 -11.5 µ long, heterocyst intercalary, solitary,
cylindrical, true-branched; filament of two types, primary thicker, creeping, torulous,
two seriate, intensely constricted at cross walls, secondary branches thinner, composed
of rounded cells, two seriate, pseudohormocyst.
3.3.2. Purification of Cyanobacteria
The serial dilution and streak plate method were effective to minimize the bacterial and
fungal contaminants associated with cyanobacterial isolates (Plate III-3). Precautiously the
filaments of isolates were picked up and subculture by maintaining all possible aseptic
conditions but the bacterial and fungal contaminants was not removed completely.
Therefore different antibiotics were used to obtain the axenic culture of 18 cyanobacterial
isolates. The data presented in Table 3.4 showed that streptomycin, penicillin, ampicillin
and pipericillin were ineffective to control the bacteria present in the culture of 18
cyanobacterial isolates. Among the different antibiotics, the treatment of 100 µg/ml of
cefotaxim was found to be effective to inhibit the growth of bacteria in the culture of
Oscillatoria chalybea, Scytonema mirabile, Nostoc calcicola, Nostoc entophytum,
Scytonema tolypothrichoides and Calothrix javanica (Plate III-4).
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
48
Table 3.4: Effect of antibiotic on growth of bacteria associated with the
cyanobacterial filaments.
Antibiotics 100µg/ml
Cyanobacterial isolates 1 2 3 4 5 6 7 8 9 10
Lyngbya bipunctata +++ +++ +++ ++ +++ - - - - -
Phormidium fragile +++ +++ +++ ++ +++ - - - - -
Spirulina platensis +++ +++ +++ +++ +++ + ++ + - -
Oscillatoria chalybea +++ +++ +++ ++ - + - - - -
Leptolyngbya sp. +++ +++ +++ ++ ++ + ++ + - -
Scytonema mirabile +++ +++ +++ + - + - - ++ - Nostoc muscorum +++ +++ +++ ++ ++ + - + - -
Anabaena
subcylindrica
+++ +++ +++ +++ +++ - - + + -
Nostoc calcicola +++ +++ +++ + - - - ++ + -
Nostoc ellipsosporum +++ +++ +++ + + + - + - -
Nostoc punctiforme +++ +++ +++ ++ + - + + - -
Nostoc entophytum +++ +++ +++ +++ - - - + + - Scytonema
tolypothrichoides
+++ +++ +++ ++ - - - - - -
Tolypothrix fragilis +++ +++ +++ +++ + - - + - -
Westellopsis prolifica +++ +++ +++ + + - - + + -
Leptolyngbya
foveolarum
+++ +++ +++ +++ +++ +++ +++ +++ +++ +++
Calothrix javanica +++ +++ +++ ++ - + - + - -
Microcoleus lacustris +++ +++ +++ +++ + - - + - -
1- Streptomycin , 2- Penicillin 3 – Ampicillin, 4- Pipericillin, 5- Cefotaxim, 6-
Cephalosporin, 7- Gentamicin, 8- Carbenicillin, 9-Kanamycin, 10-Imipenem
The antibiotic cephalosporin, gentamycin, carbenicillin, kanamycin and imipenem
were effective to control the growth of bacteria present in cultures of Lyngbya bipunctata
and Phormidium fragile. The bacteria present in the culture of Spirulina platensis were
sensitive to 100 µg/ml of kanamycin or 100 µg/ml of imipenem.
Gentamicin was effective for inhibiting the growth of bacteria present in culture of
Oscillatoria chalybea, Scytonema mirabile, Nostoc muscorum, Anabaena subcylindrica,
Nostoc calcicola, Nostoc ellipsosporum, Nostoc entophytum, Scytonema tolypothrichoides,
Tolypothrix fragilis, Westellopsis prolifica, Calothrix javanica and Microcoleus lacustris.
But at the same time it resulted in decolorization of the filament.
Among the antibiotics used (Table 3.4) imipenem was found to be effective to
check the growth of bacteria present in the culture of all the cyanobacterial isolates except
Leptolyngbya foveolarum. The treatment of this antibiotic did not affect the growth of
cyanobacterial isolates, the filaments were healthy and dark blue green in color.
The treatments of ampicillin, penicillin, streptomycin, pipericillin, kanamycin,
carbenicillin, cefotaxim, gentamicin, cephalosporin, individually and in combinations
were not effective in controlling the bacterial growth completely in cultures of
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
49
Leptolyngbya foveolarum. The aliquots staining of Leptolyngbya foveolarum showed the
bacteria firmly attached to the filaments (fig 3.2) Therefore the lysozyme, commercial
plant preservative mixture (ppm) solution, meropenem, amikacin and augmentin were
used to control the bacteria. The lysozyme treatment (500µg/ml) and ppm solution (10-
100µl) were observed to be ineffective for preparation of axenic culture. The bacterial
growth observed immediately after 24h on streaking the treated algal suspension on
nutrient agar. The higher concentration of imipenem and meropenem (7 mg/ml) required
to control the growth of bacteria present in the culture of Leptolyngbya foveolarum. 25
mg/ml of amikacin was effective for controlling the growth of bacteria. At the same time,
very low concentration of augmentin (500 µg/ml) (Table 3.5) was highly effective to
control the bacterial growth and finally to eliminate the bacteria from the cultures of L.
foveolarum.
Fig 3.2: Bacterial association with the filaments of Leptolyngbya foveolarum
Table 3.5 Effect of antibiotic on purification of L. foveolarum
Concentration Imipenem Augmentin Meropenem Amikacin
200µg + + + +
250µg + + + + 500µg + - + +
750 µg + - + +
1mg + - + +
1.5mg + - + +
2mg + - + +
3mg + - + +
4mg + - + +
5mg + - + +
6mg + - + +
6.5mg - - - +
7mg - - - +
10mg - - - +
15mg - - - +
20mg - - - +
25mg - -
+ = bacteria present, - = bacteria absent after 72 h incubation at 300 C
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
50
3.3.3. Identification of Leptolyngbya foveolarum and Leptolyngbya sp by 16 s rRNA
Phylogenetic relationships of the 16S rDNA sequences from the isolated strains were
compared with other cyanobacterial sequences retrieved from GenBank. Molecular
characterization showed that the isolated cyanobacterium was Leptolyngbya foveolarum
and all result inferred from 16s rRNA sequence analysis were in agreement (Table 3.6).
The isolates morphological feature also suggested that the isolate was L. foveolarum
(Plate-III-1C)
Table 3.6. Result from the blast searches using 16s rRNA of Leptolyngbya foveolarum
and Leptolyngbya sp.
Marker gene Length
(bp)
Closest math
(Gene bank Accession
number)
Overlap % Sequence
similarity %
Taxanomic
affinity
16SrRNA 611 Leptolyngbya
foveolarum VP1-08
(FR798945)ab
100% 100% Leptolyngbya
foveolarum
16SrRNA 562 Leptolyngbya kmn-1.
(KC589411)
95% 95% Leptolyngbya
sp.
a=the closest culture match b=isolated from concrete-made fountain with stagnant water,
gray dry crust, Italy
Fig 3.3: Analysis of PCR products from the cyanobacterial 16s rRNA
gene related to genus-specific sequence signatures
Lane 1: 200-1000bp DNA Molecular Marker; Lane 1: Leptolyngbya sp. 1 PCR with CYA106F/
CYA781R1; Lane 2: Leptolyngbya foveolarum PCR with CYA106F/ CYA781R1; Lane 3: Sample
1 PCR with CYA359F/ CYA781R1; Lane 4: Negative control reaction CYA106F/ CYA781R1;
Lane 5: Negative control reaction CYA359F/ CYA781R1.
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
51
Fig 3.4 Phylogenetic tree based on 16srRNA Phylogenetic Tree (Guiding tree) Guiding phylogenetic tree drawn using cluster algorithm with first five
hits in NCBI nucleotide sequence database. Tree generated using TreeTop – Phylogenetic analysis tool. (B-
128 Leptolyngbya sp, DQ431002 - Leptolyngbya sp,, EU068733 - Leptolyngbya sp, GU186906 – Calothrix
sp., FJ839352 – Leptolyngbya sp., AY493575-Leptolyngbya frigida.
3.3.3.1. 16S rRNA gene sequence of the Leptolyngbya kmn-1(KC589411)
1 accgctaaga ccccatatgc cggaaggtga aatagttttc tgcctgagga tgagctcgcg
61 tccgattagc tagttggtgg ggtaagagcc taccaaggcg acgatcggta gctggtctg
121 gaggatgatc agccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt
181 ggggaatttt ccgcaatggg cgaaagcctg acggagcaag accgcgtgag ggaagaaggt
241 ctgtggattg taaacctctt ttgattggga agaagcactg acggtaccaa tcgaatcagc
301 ctcggctaac tccgtgccag cagccgcggt aatacggagg aggcaagcgt tatccggaat
361 tattgggcgt aaagcgtccg taggtggttt gtcaagtctt ctgtcaaagc gcggagctta
421 actccgtaaa ggcagaggaa actgacaggc tagagtgcga taggggcaag gggaattccc
481 agtgtagcgg tgaaatgcgt agatattggg aagaacaccg gtggcgaaag cgccttgctg
541 ggtctgcact gacactgagg ga
3.3.4. Culture media and growth of isolates
Total 18 species were identified in the laboratory and were studied for the growth and
biomass production. These 18 species were grown in BG-11, Fogg’s, Allen and Arnon,
Zarrouk's Medium and CFTRI media and the growth was recorded on the basis of dry
weight (Table 3.4). The growth of Leptolyngbya foveolarum, Phormidium fragile,
Tolypothrix fragile, Microcoleus lacustris, Scytonema mirabile, Scytonema
tolypothrichoides, Calothrix javanica and Westellopsis prolifica was higher in BG-11
medium as compared to other media (Table 3.4). The obtained dry weight in the BG-11
medium for these different isolates ranges between 12 to 95 mg per bottle. Among these
the growth of Phormidium fragile was higher (95 mg). The dry weight obtained of these
isolates was comparatively very low in Fogg’s and Allen and Arnon medium. The isolates
Nostoc entophytum, N. ellipsosporum, N. muscorum, N. punctiforme, N. calcicola and
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
52
Anabaena subcylindrica showed maximum growth in Fogg’s media. The dry weight of
these forms remains in the range of 13 to 34 mg per culture. The maximum 34mg dry
weight/ bottle was observed in Nostoc calcicola. The Spirulina platensis showed
maximum 18 mg dry weight/ bottle in CFTRI medium.
Table 3.7. Growth of cyanobacterial isolates in different nutrient medium.
Sr.
No.
Cyanobacteria Growth in media in terms of dry wt (mg/culture)
BG-11 Allen &
Arnon
Fogg's
Medium
Zarrouk's
Medium CFTRI
1 Phormidium fragile 95±2.4a 35±0.9a 22±1.9b 21±2.3a 18±0.5a
2 Lyngbya bipunctata 84±1.5b 34±1.7a 12±1.7cd 15±1.8bc 11±2.1b
3 Microcoleus lacustris 25±1.9d 13±1.2c 12±0.5cd 09±0.4c 10±2.3b
4 Nostoc punctiforme 12±1.6f 13±0.8c 16±2.3c 04±0.2d 05±0.6d
5 Nostoc entophytum 12±0.9f 07±0.5de 13±2.5cd 10±0.4c 08±0.3c
6 Tolypothrix fragile 12±1.4f 08±0.2d 07±0.4d 10±1.2c 05±0.2d 7 Anabaena subcylindrica 12±0.5f 11±0.4c 14±2.9cd 05±0.5d 10±0.3b
8 Nostoc ellipsosporum 14±0.2f 10±1.2cd 17±1.7c 12±1.4bc 10±0.2b
9 Nostoc calcicola 21±1.8d 13±1.7b 34±1.9a 18±1.7b 12±0.9b
10 Nostoc muscorum 12±2.4f 11±1.7c 16±1.4c 09±0.7c 07±0.4c
11 Leptolyngbya sp. 22±1.2d 06±1.2e 09±0.2d 05±0.2d 02±0.5e
12 Leptolyngbya foveolarum 45±2.8c 12±1.3c 15±1.9c 19±1.7b 02±0.5e
13 Scytonema
tolypothrichoides
29±3.6d 18±1.4b 12±1.6cd 13±1.3bc 09±0.2bc
14 Westellopsis prolifica. 11±4.5f 13±1.9c 12±1.3cd 11±1.8c 10±0.2b
15 Calothrix javanica. 22±1.6d 15±1.8bc 13±1.2cd 19±0.9b 10±0.4b
16 Scytonema mirabile 18±2.9e 12±0.9c 09±0.5d 04±0.05 07±1.8c
17 Spirulina platensis 12±2.3f 09±0.6d 12±0.2cd 13±0.1bc 18±1.2a
18 Oscillatoria chalybea 17±1.5e 09±0.3d 03±0.01e 06±0.7d 02±0.6e
Values are Mean± SE of three independent experiments. Means followed by the same
letters are not significantly different at p=0.05 by Duncan’s Multiple Range Test
3.3.5. Biomass production
In pilot experiments the BG-11 medium supported the maximum growth of Leptolyngbya
foveolarum, Phormidium fragile, Tolypothrix fragile, Microcoleus lacustris, Scytonema
mirabile, Scytonema tolypothrichoides, Calothrix javanica and Westellopsis prolifica.
While Fogg’s media supported the growth of Nostoc entophytum, N. ellipsosporum, N.
muscorum, N. punctiforme, N. calcicola and Anabaena subcylindrica. CFTRI medium
was best for the growth of Spirulina platensis. Therefore for the large scale biomass
production of these isolates in 0.3 L (Plate III-5), 5 L, 10 L and 20 L bottle and
polypropylene trays (22.5 x 15 x 5.5 cm) (Plate III-6) the respective media were used and
data on biomass production is depicted in Table 3.5. The biomass of Spirulina platensis in
0.3 L, 5L, 10L, 20L bottle and tray was 18, 28, 59, 116 and 213 mg respectively (Table
3.8). Similar trend was observed for biomass production in other isolates of cyanobacteria.
For all isolates of cyanobacteria, the maximum growth in terms of dry weight was
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
53
Table 3.8. Biomass production of cyanobacterial isolates in BG-11 medium in different culture vessel.
Values are Mean± SE of three independent experiments. Means followed by the same letters are not significantly different at p=0.05 by
Duncan’s Multiple Range Test.
Sr.
No.
Cyanobacterial
isolates
Type of culture vessel
Biomass production in terms of dry weight (mg/vessel)
0.3 L 5 Lit 10 lit 20 Lit Tray
1 Phormidium fragile 95±2.4a 190±2.8a 294±4.5a 528±1.6a 743±0.8d
2 Lyngbya bipunctata 84±1.5a 183±1.4b 273±3.7a 481±2.7b 635±0.4e
3 Microcoleus lacustris 25±1.9cd 94±2.4f 141±3.2c 234±1.2g 327±0.5l
4 Nostoc punctiforme 16±2.3ef 75±1.9g 118±2.6e 286±1.6f 394±0.3j
5 Nostoc entophytum 13±2.5fg 36±1.5j 139±2.2de 342±1.4e 649±0.2e
6 Tolypothrix fragile 12±1.4fg 42±2.4i 98±3.8f 181±1.8h 405±0.9h
7 Anabaena subcylindrica 14±2.9ef 53±1.2h 87±4.5fg 194±2.7h 389±0.7i
8 Nostoc ellipsosporum 17±1.7de 93±1.7f 129±1.7e 197±1.2h 412±0.2h
9 Nostoc calcicola 34±1.9c 127±1.4e 219±5.8b 376±1.9d 839±0.1c
10 Nostoc muscorum 16±1.4ef 91±1.2f 143±9.5c 329±2.2e 525±0.2f
11 Leptolyngbya sp. 22±1.2d 40±2.3i 148±3.7c 312±2.3e 729±0.2d
12 Leptolyngbya foveolarum 45±2.8b 136±4.2d 217±4.8b 475±3.2b 982±0.1a
13 Scytonema tolypothrichoides 29±3.6cd 143±1.8c 223±4.3b 435±2.2c 862±0.4b
14 Westellopsis prolifica. 11±4.5fg 35±3.2j 74±3.2g 199±2.5h 373±0.5k
15 Calothrix javanica. 22±1.6d 73±1.9g 155±2.7c 236±3.1g 531±0.8f
16 Scytonema mirabile 18±2.9de 51±2.3h 137±3.5d 219±2.4g 453±0.2g
17 Spirulina platensis 18±2.3de 28±2.7k 59±3.6h 116±3.1j 213±0.1n
18 Oscillatoria chalybea 17±1.5de 27±1.7k 43±3.8i 138±2.2i 298±0.4m
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
54
Table 3.9. Biomass production of cyanobacterial isolates in BG-11 medium in culture rack unit
Sr.
no.
Cyanobacterial isolates Biomass production in culture rack unit
Fresh
weight (g)
Dry weight (g) Moisture content
%
1 Spirulina platensis 803.5±3.6h 70.8±2.7g 91.2
2 Scytonema tolypothrichoides 1587.6±2.4c 138.9±1.9c 91.3
3 Leptolyngbya foveolarum 1675.2±2.7b 162.7±1.4a 90.3
4 Nostoc calcicola 2032.3±3.4a 149.0±1.5b 92.6
5 Tolypothrix fragilis 896.4±4.5g 65.7±1.3h 92.6
6 Westellopsis prolifica. 678.7±5.6j 59.9±1.9h 91.9
7 Calothrix javanica. 1009.4±2.3e 86.4±2.4f 91.4
8 Phormidium fragile 1446.3±2.5d 112.1±3.2d 92.2
9 Lyngbya bipunctata 995.4±5.2f 98.3±2.9d 90.1
10 Nostoc entophytum 985.2±3.5f 102.5±3.5e 89.5
11 Scytonema mirabile 824.8±4.8h 72.92±2.4g 91.1
12 Microcoleus lacustris 557.8±0.8k 45.8±1.2i 91.7
13 Nostoc punctiforme 732.2±1.4i 59.6±2.4h 93.7
14 Anabaena subcylindrica 787.6±1.7h 57.4±2.9h 92.7
15 Nostoc ellipsosporum 895.7±1.8g 53.6±2.4h 94.0
16 Oscillatoria chalybea 865.3±2.4h 51.2±1.9h 94.0
17 Nostoc muscorum 978.5±2.6f 49.8±1.7hi 94.9
18 Leptolyngbya sp. 1088.6±2.8e 42.2±1.5i 96.1 Values are Mean± SE of three independent experiments. Means followed by the same letters are not
significantly different at p=0.05 by Duncan’s Multiple Range Test.
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
55
observed in trays with one liter medium compare to bottle cultures. Therefore the biomass
production was studied in trays.
Considering these factors the culture rack unit was designed (size) which can
accommodate 160 trays in 11 compartments. The result of the present investigation
suggested that cyanobacterial biomass production is possible by using the culture rack
covered with polythene sheet and application of 50% shed net at ambient conditions where
diffuse sunlight is available (light 80-120 µmol m-2
s-1
, temp 23±90 C humidity 80-90%).
The use of transparent plastic trays increase the area for the growth and therefore the
developed method was found to be suitable as compare to other containers for maximum
biomass production in static conditions. The arrangement of drip system was convenient
for timely addition of nutrient solution and water in each culture tray (Plate III-7). This
helps to reduce the time and labor cost for filling the tray with nutrient solutions. In tray-
rack system, the visible growth was observed within 5 to 7 days after initiation of culture
which was evident by the formation of thin film of biomass at the surface of the medium.
Maximum growth in terms of dry weight was observed at the end of 5th
week of culture.
The growth was not increased in the sixth week of culture and the filaments start turning
to yellowish-whitish in color. The maximum average dry weight of Leptolyngbya
foveolarum in tray-rack unit was 162.7±1.4 g less to it was of Nostoc calcicola >
Scytonema tolypothrichoides> Phormidium fragile> Nostoc entophytum> Lyngbya
bipunctata > Calothrix javanica> Spirulina platensis>Scytonema mirabile> Tolypothrix
fragilis> Westellopsis prolifica (Table 3.9, Plate III-8,9).
3.4. Discussion
3.4.1. Cyanobacterial diversity in study area
Cyanobacteria are prokaryotes and lacks sexual fusion of gametes. Therefore taxonomist
historically used morphological features to define cyanobacterial taxa including variation
in cyanobacterial thallus structure i.e. occurrence as unicells, colonies, unbranched
filaments or branched filaments, false or true branches, presence or absence of exospores,
endospores or akinets, heterocyst, mucilaginous sheath, trichome, harmogonia, separation
disc (Graham and Wilcox 2000). However, for the modern taxonomic classification of
cyanobacteria, molecular sequence comparisons together with morphological features,
ecophysiological characters and other biochemical and molecular marker is essential
(Komarek and Mares 2012).
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
56
In the present investigation, total 840 soils and water samples were analyzed and
existence of cyanobacteria was observed in 378 samples. On the basis of morphological
and molecular characters 18 cyanobacterial species were identified belonging to 12
genera, 5 families and 2 orders (Table 3.2, 3.3). Order Nostocales was dominant and at the
family level, Nostocaceae and Oscillatoriaceae showed a higher percent frequency of
distribution. The results on relative abundance of genera depicted in Table 3.3 shows that
Nostoc punctiforme Born. Et Flah (8.09 %) was dominant. Other prominent and common
cyanobacteria were Nostoc ellipsosporum (Desm.) Rabenh. Ex Born. et Flah. (4.88 %),
Nostoc entophytum Born. Et Flah and Lyngbya bipunctata Lemm (3.8 %). Leptolyngbya
foveolarum was observed in seven soil samples. However, Leptolyngbya sp. showed least
relative abundance (Table 3.3). Previous studies on the cyanobacterial diversity from other
Indian states revealed that Nostoc sp. was dominant in Asam, Hariyana, Kerala,
Tamilnadu, West Bengal (Venkatramn 1975), Orisa (Sahu et al. 1996) and Manipur (Devi
et al. 1999). A limited study from the arable domain of the Pravara area of Ahmednagar
district Maharashtra revealed that, in addition to Nostoc, the most common other genera
were Oscillatoria, Westellopsis, Scytonema, Microchaete, Anabaena and Tolypothrix were
the most common genera (Auti and Pingle 2010). The genus Westellopsis showed
restricted distribution in the rice field soils of Manipur (Devi et al. 1999). The genera
Scytonema and Oscillatoria were represented by maximum number of species in paddy
fields of Western Maharashtra (Patil and Chaugule 2009). Scytonema was most abundant
in salt-affected soils of Kolhapur district (Madane and Shinde 1993). In the area of the
present investigation the relative abundance of Scytonema was only 1-2%, which is very
low compared to Madane and Shinde’s (1993) study which reported that Scytonema was
most abundant in saline soils of Kolhapur district, Maharashtra state. This difference could
be because our study was not specific to saline soils. Cyanobacteria are the pioneer
oxygenic phototrophs on the earth whose distribution around the world is surpassed only
by bacteria (Adams 2000). Until past few decades of research cyanobacterial were of
academic interest and were mostly ignored as nuisance but now are proved as potential
organism for food and utilizable molecules (Thajuddin and Subramanian 2005). Literature
suggest that cyanobacteria comprising more than 150 genera and 2000 species. On
verification and exploitation of rest of the area the number may increase. (Gupta et al
2013).
In this context, identifying and cataloguing the diversity of cyanobacterial species
from different regions including the Sahyadri ranges, a biodiversity hot spot of India in
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
57
Western Ghats, will be very beneficial in exploiting them for source for producing
metabolites with novel biological activity.
3.4.2. Purification of cyanobacteria
Purification of cyanobacteria from bacteria is tiresome and time demanding process (Singh
et al. 2001). In the present investigation also difficulty encountered in the isolation of
axenic cultures of cyanobacterial isolates. In the present investigation series of attempts
were made including serial dilution, sonication, repeated streaking, centrifugation cleaning
and various antibiotic treatments. However, the methods of centrifugation cleaning and
treatment of some antibiotics were effective in purification of cyanobacterial isolates.
Except Leptolyngbya foveolarum, the treatment of 100 µg/ml of imipenem was best for
elimination of bacteria and purification of 17 isolates of cyanobacteria (Lyngbya
bipunctata, Phormidium fragile, Spirulina platensis, Oscillatoria chalybea, Leptolyngbya
sp., Scytonema mirabile, Nostoc muscorum Anabaena subcylindrica, Nostoc calcicola,
Nostoc ellipsosporum, Nostoc punctiforme, Nostoc entophytum, Scytonema
tolypothrichoides, Tolypothrix fragilis, Westellopsis prolifica, Calothrix javanica and
Microcoleus lacustris). Antibiotics are widely used to remove bacteria associated with
cyanobacteria (Han et al. 2010). In this study, thirteen broad-spectrum antibiotics, namely,
imipenem, meropenem, augmentin, penicillin, streptomycin, pipericillin, carbenicillin,
ampicillin, gentamycin, kanamycin, amikacin, cephalosporin, cefotaxim were used. The
variable results were obtained to control the growth of bacteria and purification of the
cyanobacterial isolates. The results indicate that Imipenem was good for 17 cyanobacterial
isolates. Among the used antibiotics some antibiotics were not effective and some others
were effective for elimination of bacteria from the cultures (Table 3.4). One of the reason
is the cyanobacterial isolates were filamentous and most of them possess mucilaginous
sheath which helps to adhere the heterotrophic bacteria. Earlier reports in Phormidium
animalis, even selective antibiotic treatment have proven to be ineffective, as
mucilaginous coats surrounding the algal filaments harbor and protect bacteria from
antibiotics and long filaments make it practically impossible to eliminate all the bacteria
associated along the whole extension of the filament (Varquez-Martinez et al. 2004).
Axenic cultures of Arthospira platensis SAG 21.99 were obtained with the
combination of a washing step and a consecutive treatment with antibiotics imipenem,
neomycin, and cyclohexamide (Choi et al. 2008). The treatment of 1 g/L of lysozyme for
establishment of axenic culture of Anabena flos-aquae and Aphanothece nidulans (Kim et
al. 1999). Bolch and Blackburn (1996) were succeeded in purifying 12 of the 17 strains of
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
58
Microcystis aeruginosa on application of centrifugation cleaning, sulfide gradient
selection and antibiotic imipenem treatment. Axenic cultures of Phormidium anamalis
was obtained by combination of washing in a series of centrifugation and resuspension in
liquid medium followed by vacuum driven filtration through 8 µm pore size membrane
and treatment with carbenicillin followed by streptomycin then chloramphenicol, and
finally with kanamycin (Varquez-Martinez et al. 2004). Hong et al. (2010) succeeded in
purifying the filament of Nodularia spumigena KNUA005 after three purification step;
centrifugation, antibiotic treatment and streaking. He used antibiotic imipenem, the
bacteria still survived after treatment of imipenem which was further eliminated by
kanamycin.
Ppm is commercially available formulation which is used to avoid contamination
in plant tissue culture. In the present study the ppm was not effective to control the growth
of bacteria. Earlier report suggests that imipenem is broad spectrum beta lactum antibiotics
which kill the most of the bacteria (Pinter and Provasoli 1958; Rippka 1988; Bolch and
Blacburn 1996; Ferris and Hirsch 1991). However, in the present investigation for L.
foveolarum the antibiotic imipenem was ineffective up to 7mg/ml to eliminate the bacteria
from the culture. Similar results were also observed in Microcystis aeruginosa (Han et al.
2010).
Augmentin was the most effective antibiotic of this pool to eliminate the bacteria
from the cultures of L. foveolarum. This might be possible being augmentin a broad-
spectrum beta-lactam antibiotic which inhibits peptidoglycan biosynthesis and making it
superior to the other antibiotics used i.e. imipenem, meropenem, amikacin, gentamicin for
reducing the number of heterotrophic bacterial contaminants associated with L.
foveolarum culture.
3.4.3. Identification of the cyanobacterium
Literature suggest that cyanobacteria comprising more than 150 genera and 2000
species (Gupta et al. 2013). Members of the cyanobacteria are generally classified into five
orders: Chroococcales, Nostocales, Oscillatoriales, Pleurocapsales, and Stigonematales
(Anagnostidis and Komarek, 1988). Members of the order Oscillatoriales are filamentous
and lack heterocysts and akinetes (Albertano and Kovacik 1994; Turner 1997). The order
Oscillatoriales includes the families Borziacaea, Homoeotrichaceae, Oscillatoriaceae,
Phormidiaceae, Pseudanabaenaceae, and Schizothrichaceae as discussed in Anagnostidis
and Komarek (1988).
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
59
Many of the species within the genera Oscillatoria, Lyngbya, Phormidium,
Schizothrix, and Plectonema were originally classified by Gomont (1892) based on sheath
characteristics and the presence or absence of false branching. However, the sheath
characteristics frequently used in identifying and classifying members of this group are
influenced by culturing and environmental conditions (Albertano and Kovacik 1994). This
led to the eventual transfer of a number of strains within the Oscillatoriales to a new group
designated by Rippka et al. (1979) as LPP-group B (Lyngbya, Phormidium, and
Plectonema).
The identification of microorganisms including cyanobacteria in the natural
ecosystem is difficult. Traditional taxonomic criteria developed over a century ago were
based on erratic characters such as false branching and sheath characteristics. The
morphology of cyanobacteria is severely influenced by environmental factors which
induced phenotypic plasticity was not observed or well understood by early taxonomists.
The problem in using botanical criteria to classify cyanobacteria is that culturing
conditions and environmental plasticity often induce morphological changes (Burja et al.
2001). The most plastic characters observed in cyanobacteria include sheath color, sheath
thickness, granulation, false branching, and cell length (Anagnostidis and Komarek 1985).
Less variable characters include pigmentation, cell length to width ratios, tapering,
trichome width, and the ability to form calyptra (Anagnostidis and Komarek 1985).
Furthermore, morphological characters such as type of cell division and thylakoid
structure are constant, and are not influenced by variations in environmental or culturing
conditions (Anagnostidis and Komarek 1985). Culture and environmentally induced
morphological changes among cyanobacteria often lead to inaccurate identification and
classification taxa (Nelissen et al. 1992; Nubel et al. 1997). Morphological changes can be
problematic in establishing species definitions in that they are usually defined based on
cell dimensions and ecology, such that distinct separations are often not evident (Nubel et
al. 1997).
Anagnostidis and Komarek (1988) reassigned many different taxa from the LPP-B
group into a new genus Leptolyngbya, thus creating over 75 new combinations (Albertano
and Kovacik 1994; Turner 1997). This newly established group is typified by the presence
of a number of distinguishing morphological characteristics such as thin sheaths,
immobility of filaments, thin uniseriate trichomes, arrangement of thylakoids, and cell
wall constrictions (Albertano and Kovacik 1994; Turner 1997).
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
60
In recent years, molecular techniques have provided excellent complementary data
to address the limitations of taxonomic, physiological and biochemical approaches in
biodiversity assessment (Graham and Wilcox 2000; Bursanti and Gualteri 2006;
Srivastava et al. 2007; Lin et al. 2012).
Sequencing of DNA play an important role in the reconstruction of evolutionary
relationships among organism’s leads to new genetic classifications which confirm or
conflict with traditional taxonomy. Molecular techniques used to amplify some portions
of the genome in order to characterize and deduce phylogenetic relationships of
cyanobacteria has increased considerably in the recent years (Neilan et al. 1995; Garcia-
Pichel et al. 1996; Orcutt et al. 2002; Taton et al. 2003; Premanandh et al. 2006). The
rRNA genes are the most widely used markers for the identification of bacteria and
cyanobacteria due to their conserved function and universal presence. Several researchers
have exploited the conserved regions of the 16S rRNA gene for phylogenetic analysis of
cyanobacteria (Nubel et al. 1997; Crosbie et al. 2003; Salomon et al. 2003).
The 16S rRNA is an effective tool in inferring phylogenetic relationships between
different genera within orders proposed by Komarek and Anagnostidis (Turner 1997).
Additionally, it has been useful in identifying and classifying strains that belong to a single
clade (Palinska et al. 1996; Otsuka et al. 1998). Nelissen et al. (1992) found that the 16S
rRNA sequences of five strains of Pseudanabaena were nearly identical, and therefore
concluded that Pseudanabaena was a single monophyletic taxon. In a more recent study,
Honda et al. (1998) found that trees constructed using 16S rRNA sequence data resulted in
the clustering of members of the genus Synechococcus, indicating that these strains were
closely related.
Leptolyngbya belongs to the most taxonomically problematic and poorly defined
genus (McGregor and Rasmussen 2008). To clarify this genus, further study of samples
from various environments is necessary (Lin et al. 2012). The heterogeneous Leptolyngbya
is one such cyano prokaryotic genus, which needs further study (Komarek 2007).
Leptolyngbya were created as new genera to include large number of oscillatorean species
with trichome up to 3µm wide (Anagnostidis and Komarek 1988).
In the present investigation, morphological characters of two cyanobacterial
isolates matches with the description provided by Anagnostidis and Komarek (1988) and
were classified in the genus Leptolyngbya. One of the isolate further classified with the
similarities in characters as L. foveolarum while the other isolate in the genus
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
61
Leptolyngbya did not matches with any of the species characters listed in the literature
(Anagnostidis and Komarek 1988; Singh et al. 2001; Komarek and Mares 2012). On the
basis of Bioguided assay L. foveolarum noted to be source of potent cytotoxic metabolites.
While another Leptolyngbya isolate also showed biological activity (antibacterial,
antifungal and cytotoxic). Therefore in the present investigation the two isolates of the
genus identified as Leptolyngbya foveolarum and Leptolyngbya sp. were confirmed by 16s
rRNA sequence.
The highest similarity for 16s rRNA was to Leptolyngbya foveolarum FM7988945
(100% identity), Leptolyngbya sp. HM 776036 (100%), Leptolyngbya sp. EF08833.1,
Leptolyngbya sp. AF132785 and Leptolyngbya boryana (100%). Using BLAST we
searched the NCBI database for the sequence that was most closely related and its closest
hits were Leptolyngbya foveolarum. Thus on the basis of morphological and 16 s rRNA
characterization the isolate is identified as Leptolyngbya foveolarum.
The sequence of Leptolyngbya sp. was not matches with the available sequence in
the NCBI library therefore it was identified as novel species. The sequence (562 bp) was
submitted to NCBI for accession number, Leptolyngbya sp. kmn-1(KC589411). The basic
local alignment search tool (BLAST) of the national center for biotechnology information
(NCBI) was utilized for locating the isolate sequences. Sequences of this Leptolyngbya sp.
have been deposited in GenBank under accession numbers KC589411.
Leptolyngbya foveolarum falls within the clade of the genus Leptolyngbya in all
analysis conducted using 16 s rRNA. The PCR amplified product of 16 s rRNA of another
isolate of Leptolyngbya on sequencing and pairwise alignment showed 95 % homology
with the 16 s rRNA of Leptolyngbya sp. (DQ134002), 93 % homology with Leptolyngbya
sp. (EUO68733), 92 % with Leptolyngbya sp. (EUO68733), Leptolyngbya frigidia
(AY493575), Calothrix sp. (GU186906) and Leptolyngbya sp. (FJ839352) available in
the NCBI database.
3.4.4. Growth of cyanobacteria in different culture media
The distribution of cyanobacterial species depends on chemo-physical environment and
organism ability to grow in a particular environment. This has led to development of
various culture media and was used for isolation and cultivation. The methods of
microalgal culture and basic culture media formulation were developed in the late 1800s
and early 1900s. Many of these methods and media formulations are in used today
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
62
(Anderson 2005). In the present investigation, the growth of cyanobacterial isolates
obtained in recommended nutrient media namely BG-11 (Stanier et al. 1971); Fogg’s
medium 1949; Jacobson, 1951); Allen and Arnon’s medium (Allen and Arnon 1955),
Zarrouk’s medium (Zarrouk 1966), CFTRI medium (Venkataraman and Becker 1984) is
depicted in Table 3.4. It was observed that the growth increase up to 4th
and 5th
week of
culture. In the 6th
week of culture the cyanobacterial filaments become yellowish whitish
and not showing any sign of growth. Decrease in the growth after certain time is a natural
phenomenon for any algal culture (Barsanti and Gualtieri 2006). Colla et al. (2007)
reported similar results in Spirulina platensis. Once the stationary growth phase is started,
the biochemical composition is changed (Vonshak 1997), which results in decrease in the
growth. The cultures of cyanobacteria pass through different growth phases. These growth
phases are lag phase, log phase, stationary phase and death phase (Becker 1994). Due to
different phases of growth of cyanobacteria, it is impossible to attain uniform growth
throughout the time of experiments. Similar observations have been recorded in our study
on cyanobacterial isolates. Initially the growth was very slow which increased
progressively up to the time of harvesting. Initially the culture mat was very thin hence
shading effect was not observed. Thickness of culture mat increased with time and it
resulted in self-shading effect because of which the inside cells did not receive sufficient
light and these cells might have undergone respiration (Vonshak 1983). Similar types of
results were obtained by Ramirez et al. (2000) in Calothrix sp. and Costa et al. (2001) in
Spirulina platensis.
The growth of Oscillatoria chalybea, Leptolyngbya foveolarum, Leptolyngbya sp.,
Microcoleus lacustris, Calothrix javanica, Phormidium fragile, Lyngbya bipunctata,
Westellopsis prolifica, Tolypothrix fragile, Scytonema mirabile and Scytonema
tolypothrichoides was more in BG-11 medium than in other media. However, Nostoc
entophytum, N. ellipsosporum, N. punctiforme, N. muscorum, N. calcicola and Anabaena
subcylindrica showed maximum growth in Fogg’s medium. CFTRI was the best medium
for the growth of Spirulina platensis. The variability in growth of cyanobacterial isolates
was attributed to the differences in recommended mineral salts and their concentrations in
the nutrient medium. The cyanobacterial growth also depends on the adaptation to the
chemo-physical environment (Anderson 2005). Spirulina sp. occurs in environments with
higher nutrient levels and varying temperature and salinity with pH 9.0– 10.0 and grows
well at 11.5 pH but not at 7.0 pH (Marquez et al. 1993, Vonshak and Tomaseli 2000,
Quasim et al. 2012). This might be the reason for less growth of Spirulina platensis in BG-
11, Fogg’s and Allen and Arnon media. While Anabaena sp. displays optimal growth at
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
63
pH 7.4–8.4 and its productivity decreases significantly at pH values higher than 9
(Marquez et al. 1993).
For optimum growth of cyanobacteria, appropriate Ka+: Na
+ ratio is required in
the cytoplasm (Barsanti and Gualtieri 2006). BG-11 medium consists of moderate
concentration of Na+ and in Allen and Arnon medium, Zarrouk’s medium and CFTRI
medium there is high concentration of Na+ while in Fogg’s medium; there is no Na
+
source. This might be the one of the reason for the variability in growth in cyanobacterial
isolates.
3.4.5. Large scale biomass production
Cultivation of green algae, Dunaliella and Chlorella and cyanobacteria Spirulina
in open and closed system has been extensively studied in the past few years and very
good results have been achieved for their biomass production (Vonshak 1997; Borowitzka
1999). However monoculture of these microalgae is possible only due to their specific
growth conditions such as Dunaliella grows in high salinity, Chlorella in high nutrition
and Spirulina in high alkalinity where with these condition growth and survival of other
microorganism is difficult (Lee 2001). For maximizing productivity in mass algal culture
system various growth conditions, physiological and technological approaches have been
proposed and investigated (Chaumont 1993; Richmond 1996; 2000; Lee 2001; Grobbelaar
2000; Anderson 2005; Wang et al. 2013).
In recent past, a number of cyanobacteria have been screened and recognized as a
rich but not yet extensively examined source of food, feed and pharmacological as well as
structurally interesting secondary metabolites (Becker 2004; Spolaore 2006). The major
constraint is of their biomass production in closed and open system is difficult. Therefore
till date little industrial and economic success is achieved (Ugwu et al. 2008; Wang et al.
2013). Closed culture system allow control over illumination, temperature, nutrient level,
contamination with predators and competing algae, whereas open culture system though
cheaper make it very difficult to grow specific cyanobacterial cultures for extended
periods and are more readily contaminated (Wang et al. 2013). It is also possible axenic
cultivation but it is too expensive and difficult because it requires a strict sterilization of all
glass wares, culture media and vessels to avoid contamination. These constrains make it
impractical and very expensive for commercial operation. On the other hand non axenic
cultivation cheaper and less laborious but associated with less predictable and inconsistent
quality (Lee 2001).
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
64
In the present investigation on use of tray - rack culture system it become possible
to achieve the biomass production of Spirulina platensis, Scytonema tolypothrichoides,
Leptolyngbya foveolarum, Nostoc calcicola, Tolypothrix fragilis, Westellopsis prolifica,
Calothrix javanica, Phormidium fragile, Lyngbya bipunctata, Nostoc entophytum and
Scytonema mirabile. Among these studied form maximum biomass production (162 g dry
weight/ rack) was observed for Leptolyngbya foveolarum. While minimum observed for
Westellopsis prolifica (59.9 g dry weight /rack) the other forms produced the biomass in
range of the 69-149 g/rack (Table 3.6). Under similar conditions the different forms
showed the variation for biomass production. This indicates that the requirement for
growth might vary with the cyanobacterial forms. Variation in biomass production was
also observed in the cyanobacterial forms grown in 0.3 L, 5 L, 10 L and 20 L bottles.
However, the produced biomass was low comparative to tray-rack culture system.
Although the height of nutrient medium level is more in bottle but in static condition the
growth of cyanobacterial forms was observed at the surface of the medium and some
forms also showed the growth at the wall of the bottle. This indicates that the buoyancy
characteristic and phototactic properties possess in cyanobacteria. This might limits the
growth and biomass production in rest of the volume of the medium in bottle culture of
system in static condition.
In tray-rack culture system on inoculation of less biomass (0.5 g FW /tray) not
resulted in proper growth and biomass formation. In this situation the growth of other
microorganisms (contaminant) was observed together with the inoculated cyanobacteria.
The proper growth and biomass formation and prevention of growth of other contaminant
organism was observed on addition of higher inoculum biomass (1 g FW /tray). According
to Lee (1986), Anderson (2005), Barsanti and Gualtieri (2006), high cell concentration is
necessary to achieve higher biomass productivity, and the need to maintain monoculture
for microalgae that grow in mild culture conditions.
As per the convenience and response of algal organism different types of culture
system have been developed the most routinely adapted include batch, continuous, semi-
continuous, ponds and photo-bioreactors. In the present investigation the tray-rack culture
system was developed (2.6 m H X 1.35 m L X 0.9m W) which can accommodate 160
trays (22.5 x 15 x 5.5 cm) in 11 compartments. This is a partially closed system. It is
equipped with drip system for addition of nutrient solution as per the requirement. On
addition of inoculum in trays with 1 lit medium within 1-3 days the inoculum floats and at
the surface forms a thin mat. This is the first sign of healthier growth of desirable
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
65
organism. In subsequent period, the cyanobacterial filament density increases constantly
and resulted in dark green cyanobacterial filament mat formation.
The nutrient medium components and water level decrease over time. Therefore at
two week period the fresh half strength nutrient medium was added to trays through drip
system. The evaporation of water is very high in summer season, showed the requirement
of addition of fresh medium and water at weekly interval. Maintenance of 2.5 to 3 cm
level of nutrient medium was suitable for proper growth and dark green filamentous mat
formation. Addition of more nutrient medium and presence of 4 to 5 cm level of nutrient
medium hamper the growth and filamentous mat formation in tray-rack culture system.
Application of drip system prevents the labor involved in physical addition of
nutrient medium. Provision of transparent plastic cover to the tray-rack culture system
helps to maintain the requirement of high humidity (80-90%) and being transparent allows
the passage of sufficient light for the growth (80-120 µmol m-2
s-1
). The tray culture unit
was placed under 50% category shade-net. Application of 50% of shade net was best to
filter the excess light intensity and also helps to prevent the increase of temperature.
Otherwise in open condition the culture becomes yellowish and finally whitish and did not
show any sign of growth.
The growth of cyanobacteria in tray –rack culture system showed a typical pattern
of growth which follows the sigmoid pattern, consisting of a succession of six phases,
characterized by variation in the growth rate. After the inoculation, 24 to 72 h no growth
or very slow growth was observed (i.e. the lag phase of growth curve). This is possible as
the cells may not be in condition to divide immediately or required time period to adapt to
the newer situation and start growing. In this situation the cell has to prepare with increase
in the level of enzymes and metabolites involved in cell division and carbon fixation
(Barsanti and Gualtieri 2006). After 72 h the mat formation started becoming dark-blue
green showed the good sign of growth. This continues for about four to five weeks after
inoculation. During the fifth and sixth week of culture the cells become yellowish in color
at the bottom. This might be due to shading of cells and not availability of sufficient light
and limiting of other factors such as nutrient, pH, carbon dioxide and other physical and
chemical factors (Anderson 2005). Similar trend in growth of Calothrix sp. was observed
in 12 days batch mass culture experiment with 8 g inoculum in 8 Lit disposal polythene
containers (Ramirez-Olvera et al. 2000). Similar results for different microalgal mass
culture system are difficult because of different geographic locations, culture strategies,
algae sp. etc. (Chaumont 1993). Biomass production in open pond system is
Chapter III………………Isolation, Identification, Axenic culture and Biomass production
66
technologically simple but not necessary cheap due to the high down stream processing
cost and the products from such system could be marketed as value added health food
supplement, specially feed and alternative product for research (Lee 2001). In open pond
system the growth of desirable microalgae is affected by zooplankton, bacteria, virus
(Wang et al. 2013).
Despite that a great deal of work has been done to develop large scale microalgal
cultivation system, more efforts are still required to improve the technologies and know-
how of algal culture (Ugwu et al. 2008; Wang et al. 2013). In this context the attempts
made in present investigation in design, development and utilization of tray-rack culture
system is one of the good steps towards the improvement in large scale biomass
production of cyanobacteria.
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