Biodiversity and Molecular Evolution of Microalgae on Different Epiphytes and Substrates
M.I. Mohammed Ershath
An exploration of the microalgal biodiversity from different epiphytes and substrates of pool water in temple at Tiruchirappalli District was studied. Totally ten epiphytic forms were selected for this investigation. In that, totally 44 species of 30 genera belonging to 3 families of the Chlorophyceae, Cyanophyceae (heterocystous and non-heterocystous) and Bacillariophyceae were identified and recorded. The dominant species in this environment were Cyanobacteria (Chroococcus sp. and Oscillatoria sp., Phormidium sp.), Green algae (Tetradron sp. and Scenedesmus sp.) and Diatom (Fragilaria sp. and Navicula sp.) were documented. The molecular taxonomy of cyanobacteria were also analyzed, in this regards, DNA was extracted; 16S rDNA gene was amplified and sequenced. The evolutionary relationship was found in the epiphytic microalgae by Neighbour-Joining method by construction of phylogenetic tree.
Received: October 02, 2012;
Accepted: December 12, 2012;
Published: February 12, 2013
Microalgae are photoautotrophic oxygen evolving microorganisms which includes
(green algae, cyanobacteria and diatoms) often inhabit various types of substrate,
especially light and water resources and reservoirs. In addition to their morphological
diversity and extensive distribution, microalgae reflect a wide range of physiological
properties and are more tolerant to environmental stress (De
Marsac and Houmard, 1993). Microalgae are reported to be one of the principal
component involved in biofilm formation and are responsible for several problems
in many industrial cooling systems (Ludyansky, 1991;
Callow, 1993). In most of the environment, microalgae
are the primary producer at the base of the food web of the ecosystem and moreover,
these are symbionts of the variety of other organisms, viz. the marine diatom
Rhizosolenia, leaves of Azolla and the root of Cycas (Thajuddin
and Subramanian, 2005). Some algal forms can be useful indicator on which
major water management practice, pollution studies and water quality analysis
(Pandey et al., 1998). The nature of production,
distribution and relationship of the phytoplankton and zooplankton vary with
the prevailing environmental conditions. The major freshwater phytoplankton
groups of green algae, diatoms and cyanobacteria. In this investigation, we
studied the biodiversity of different substrate inhabiting microalgae in pool
water in Tiruchirappalli, Tamil Nadu (India) and 16S rDNA based molecular phylogeny
of isolated cyanobacteria.
MATERIALS AND METHODS
Sample collection: Collections were made from various epiphytic or substrate
in pool water of the Parthasarathy temple at Tiruchirappalli, Tamil Nadu, India.
Ten different epiphytic and substrate were selected for microalgal samples collection.
The methods used for the collection and studies of the same as described previously
(Anand, 1998). The microalgal samples were scrapped from
the substrate such as Tin (1), Plastic cup (2), plastic paper (3), mango seed
(4), ball (5), coconut (6), coins (7), cane cap (8), thermo coal (9) and pool
water (10) were transferred into BG 11 medium which contains both N+
and N-nutrient medium (Fig. 1). The physiochemical
parameter of water sample was analyzed by standard methods.
Biodiversity and morphological identification of microalgae: The specimens
were taxonomically determined with the help of standard literatures i.e., Desikachary
(1959) for cyanobacteria; Desikachary et al. (1987)
for bacillariophytes and Belcher and Swale (1978) and
Hortobagyi (1973); for chlorophytes and bacillariophytes.
|| Distribution of microalgae in different epiphytes and substrates,
(a) Tin, (b) Plastic cup, (c) Plastic paper, (d) Mango seed, (e) Ball, (f)
Coconut shell, (g) Coins, (h) Thermo coal and (i) Cane cap
|| Primers for the amplification of the cyanobacterial 16S rDNA
The sizes were measured and microphotography is documented using Light Microscopy
Molecular analysis: In DNA extraction, the genomic DNA was extracted
from the Phormidium and Oscillatoria sp., using the method of
Smoker and Barnum (1988). The extracted genomic DNA
was electrophoresed on 0.8% agarose against Tris-Acetic acid buffer (pH. 8.2).
In Polymerase Chain Reaction (PCR), amplification of 16s rRNA gene from
Phormidium animale and Oscillatoria acuminata were made using the
specific forward primer (CYA106) and the specific reverse primer (CYA781R).
Primer sequences and target regions within the 16S rRNA gene are listed in Table
1. In this reaction, 50 μL of reaction volume contains 25 μL of
premix (company), 1 μL of forward primer, 1 μL of reverse primer,
50 ng in 1 μL, of genomic DNA, sterile double distilled water 17 μL.
The PCR procedure consisted of denaturation at 93°C for 5 min followed by
35 cycles of the following: denaturation at 93°C for 1 min, annealing at
55°C for 1 min and extension at 72°C for 3 min and final extension was
72°C for 8 min. PCR products were electrophoresised on 1.2% agarose against
Tris-Acetic acid buffer (pH. 8.2). The amplified products were visualized in
Construction of phylogeny: The Sequencing was done using automatic sequencer.
The products were sequenced in both 5-3 direction and 3-5
directions. The evolutionary relationship was computed using neighbor-joining
method (Tamura et al., 2004) and is in the units
of the number of base substitutions per site and it was computed in MEGA 5 software.
The 16S rDNA gene sequences were analyzed with BLAST tool and submitted to GenBank
RESULTS AND DISCUSSION
In this present investigation, 44 species of microalgae has been distributed
in ten different epiphytes were recorded and tabulated (Table
|| Biodiversity of Microalgae (Chlorophyceae, Bacillariophyceae
and Cyanophyceae) from different epiphytes
|| Microphotograph of Euglena sp.
There are different morphological microalgae were documented, they are Cosmarium
granatum, Chlorococcum humicola, Pediastrum simplex, Scenedesmus
dimorphus, Coelastrum microsporum, Tetraedron minimum, Euglena
sp. (Fig. 2) in green algae (Fig. 3); Chroococcus
sp., Merismopedia sp., Oscillatoria sp., Anabaenopsis sp.,
Pseudoanabaena sp. in cyanobacteria (Fig. 4) and Navicula
sp., Nitzschia amphibia, Amphora ovalis in diatoms (Fig.
5). There are certain members of cyanophyceae which are tolerant to organic
pollution and resist environment stress caused by the pollutant. Such species
can be used as Marker species or indicator of particular habitat (Prasad
and Saxena, 1980).
Some microalgae such as Chlorella sp., Nitzschia sp. and Chroococcus
sp., adhered to hard substrates (Sekar et al., 2004).
The physiochemical parameters of the water were analyzed and tabulated (Table
3). The frequency of the some species and their survival may be due to the
micronutrients present in the water, the physiochemical changes in the environment
may affect particular species and induce the growth and abundance of other species
(Muthukumar et al., 2007). While studying the
bacterial attachment to surfaces (Wrangstadh et al.,
1996) found that the higher attachment on hydrophobic surfaces is mediated
by the water exclusion mechanism, whereas hydrophilic substrata water is poorly
excluded resulting in less attachment (Burchard et al.,
1990). In this study, the increased attachment observed by all the microalgae
both on hydrophilic and hydrophobic substrates (tin, plastic paper and thermo
coal) may be due to water exclusion mechanisms.
||Physiochemical analysis of water samples collected from temple
pool water at Tiruchirappalli
It has been reported the higher level of sulphide content is toxicity to the
heterocystous forms. It has been reported that the high values of BOD, COD,
Phosphates and nitrates with very DO favored the growth of cyanobacteria than
any other algae (Singh and Saxena, 1969; Venkateshwarlu,
1976). The continuous discharge of oils, flowers, rice and dhal to the water
may increase the concentration of sulphur and nitrogen. Increased amount of
nitrogen in the water may decrease the level of heterocystous forms. Even though
some microalgae inhabit unexpected substrates such as thermo coal and coins,
they might have derived its nutrients from these or adapted to that environment
for its survival.
It has been studied that the value of pH is at its lowest early in the morning
then it rises, at night it decreases again; it was daytime fluctuation is considerable.
This daily fluctuation of the pH value, in extreme cases, may even exceed 3
pH units (Hortobagyi, 1973). Some of the aerophytic algae
inhabit stony substrate (Hoffmann and Darienko, 2005).
It is well known that the molecular sequencing studies was confirmative procedure
in the current molecular taxonomy for species level identification, 16s rRNA
gene sequencing is a reliable methods for the identification of species. It
has been reported that the sequence analysis of genes encoding small subunit
ribosomal RNA is currently the most promising approach for the phylogenetic
classification of cyanobacteria (Wilmotte, 1994). There
are many workers have done with cyanobacterial gene sequencing for identification,
there were critical identification methods from sponges associated cyanobacteria
(Steindler et al., 2005), hot-spring inhabiting
cyanobacteria (Weller et al., 1991). PCR primer
is very important role for the amplification of target site; there is need specificity
to target sites for PCR with absolute primers. The alternative forward primer
CYA106F matches a numbers of published 16S rRNA sequences from prokaryotes with
various phylogenetic affiliations outside the phylum of the cyanobacteria and
also the amplification specificity checked by DGGE to investigate homogenecity
of the sequence of PCR products prior to sequence analysis (Nubel
et al., 1997).
|| Microphotograph of (a) Tetrdron sp., (b) Cosmarium
sp., (c) Chlorella sp., (d) Chlorella sp., (e) Cosmarium
sp., (f) Oocystis sp., (g) Scenedesmus sp., (h) Scenedesmus
sp., (i) Scenedesmus aureus, (j) Scenedesmus sp., (k) Astrococcus
sp., (l): Coalestrum sp., (m) Scenedesmus sp., (n) Scenedesmus
bijugatus., (o) Chlorococcum humicola., (p) Chlorococcum
sp., (q-r) Zygenema sp., (s) Pediastrum boyana., (t) Scenedesmus
|| Microphorgraph of (a) Gleocapsa ovale, (b) Gleocapsa
sp., (c) Calothrix braunii, (d) Anabaena sp., (e) Merismopedia
sp., (f) Nostoc sp.
|| Microphotograph of (a) Navicula sp., (b) Nitzchia
sp., (c) Fragilaria sp., (d-e) Fragilaria sp., (f) Stauronesis
||Evolutionary relationship of cyanobacterium, Oscillatoria
acuminata NTDM04 and Phormidium animale NTMP03
The genetic analysis can also be compared with hydrobiological and hydrochemical
analyses and the genetic abundance profiles may provide a foundation for separating
and quantifying genetically distinct groups of cyanobacteria in their natural
habitats (Rudi et al., 2000) (Fig.
Among the most popular molecular techniques, the sequence determination of
small subunit ribonucleic acids is widely employed. Vandamme
et al. (1996) reported that the genetic construction of the cyanobacteria
contributes significantly to the revision of their taxonomy and relevant classification
reflects the phylogenetic relationships. The integration of the phenotypic,
genotypic phylogenetic information renders possible a consensus type of taxonomy
known as polyphasic taxonomy. Ibraheem and Al-Sherif (2009)
reported that the flowering plants and algal were controlled by the edaphic
factors and physico-chemical characters of the soil. Pandiaraj
et al. (2012) also reported that the molecular characterization and
phylogeny of marine cyanobacteria using 16S rDNA sequencing. It was well characterizing
method to understand the taxonomical position of the isolates.
In this investigation micro algal biodiversity in the temple at Tiruchirappalli
showed total of 44 species of 30 genera belonging to 3 families of the Chlorophyceae,
Cyanophyceae and Bacillariophyceae, various types of species. Many works has
been reported that the cyanobacterial flora in temples in which walls, water
bodies and drainages. This may be the new report that the microalgal flora in
different epiphytes in the temple water pool. Representative nucleotide sequences
such as Oscillatoria acuminata and Phormidium animale have also
been submitted to GenBank under the following accession numbers: GU812859 and
The authors are grateful thanks to the Department of Biotechnology (DBT) Govt.
of India for their research grant.
1: Anand, N., 1998. Indian Freshwater Microalgae. Bishen Singh Mahendra Pal Singh Publication, Dehra Dun, India
2: Belcher, H. and E. Swale, 1978. A Beginners guide to Freshwater Algae. Her Majestys Stationery Office, London, pp: 1-47
3: Burchard, R.P., D. Rittschof and J. Bonaventura, 1990. Adhesion and motility of gliding bacteria on substrata with different surface free energies. Applied Environ. Microbiol., 56: 2529-2534.
PubMed | Direct Link |
4: Callow, M.E., 1993. A review of fouling in freshwaters. Biofouling, 7: 313-327.
5: Desikachary, T.V., 1959. Cyanophyta. 1st Edn., Indian Council of Agricultural Research, New Delhi, India, Pages: 686
6: Desikachary, T.V., S. Gowthaman and Y. Latha, 1987. Diatom Flora of Some Sediments from the Indian Ocean Region. In: Atlas of Diatoms, Desikachary, T.V. (Ed.). Madras Science Foundation, Madras, pp: 78-221
7: Hoffmann, L. and T. Darienko, 2005. Algal biodiversity on sandstone in Luxembourg. Ferrantia, 44: 99-101.
Direct Link |
8: Hortobagyi, T., 1973. The Microflora in the Settling and Subsoil Water Enriching Basins of the Budapest Waterworks. Akademiai kiado, Budapest, Pages: 612.
9: Ibraheem, I.B.M. and E.A. Al-Sherif, 2009. Distribution of flowering plants and cyanobacteria in relation to soil characters in Bahariya Oasis, Egypt. Int. J. Bot., 5: 36-46.
CrossRef | Direct Link |
10: Ludyansky, M.L., 1991. Algal fouling in the cooling system. Biofouling, 3: 13-21.
11: Muthukumar, C., G. Muralitharan, R. Vijayakumar, A. Panneerselvam and N. Thajuddin, 2007. Cyanobacterial biodiversity from different freshwater ponds of Thanjavur, Tamil Nadu (India). Acta Botanica Malacitana, 32: 17-25.
12: Nubel, U., F. Garcia-Pichel and G. Muyzer, 1997. PCR primer to amplify 16S rRNA gene from cyanobacteria. Applied Environ. Microbiol., 63: 3327-3332.
Direct Link |
13: Pandey, J., U. Pandey, H.R. Tyagi and N. Rai, 1998. Algal flora and physiochemical environment of fatel Sagar Lake. Phykos, 37: 29-39.
14: Pandiaraj, D., A.D. Mubarak, R.P. Kumar, S. Ravikumar and N. Thajuddin, 2012. Molecular characterization and phylogeny of Marine cyanobacteria from Palk Bay region of Tamil Nadu, India. Ecologia, 2: 23-30.
CrossRef | Direct Link |
15: Prasad, B.N. and M. Saxena, 1980. Ecological study of blue green algae in river Gomti. Ind. J. Environ. Health, 22: 151-166.
16: Rudi, K., O.M. Skulberg, R. Skulberg and K.S. Jakobsen, 2000. Application of sequence-specific labeled 16S rRNA gene oligonucleotide probes for genetic profiling of cyanobacterial abundance and diversity by array hybridization. Applied Environ. Microbiol., 66: 4004-4011.
17: Sekar, R., V.P. Venugopalan, K.K. Satpathy, K.V.K. Nair and V.N.R. Rao, 2004. Laboratory studies on adhesion of microalgae to hard substrates. Hydrobiologia, 512: 109-116.
18: Singh, V.P. and P.N. Saxena, 1969. Preliminary studies on algal succession in raw and stabilized sewage. Hydrobiologia, 34: 503-512.
19: Smoker, J.A. and S.R. Barnum, 1988. Rapid small-scale DNA isolation from filamentous cyanobacteria. FEMS Microbiol. Lett., 56: 119-122.
CrossRef | Direct Link |
20: Steindler, L., D. Huchon, A. Avni and M. Ilan, 2005. 16S rRNA phylogeny of sponge-associated cyanobacteria. Applied Environ. Microbiol., 71: 4127-4131.
CrossRef | Direct Link |
21: Tamura, K., M. Nei and S. Kumar, 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc. Natl. Acad. Sci. USA., 101: 11030-11035.
CrossRef | Direct Link |
22: De Marsac, N.T. and J. Houmard, 1993. Adaptation of cyanobacteria to environmental stimuli: New steps towards molecular mechanisms. FEMS Microbiol. Lett., 104: 119-189.
CrossRef | Direct Link |
23: Thajuddin, N. and G. Subramanian, 2005. Cyanobacterial biodiversity and potential applications in biotechnology. Curr. Sci., 89: 50-57.
Direct Link |
24: Vandamme, P., B. Pot, M. Gillis, P. de Vos, K. Kersters and J. Swings, 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol. Mol. Biol. Rev., 60: 407-438.
Direct Link |
25: Venkateshwarlu, V., 1976. Taxomony and ecology of algae in the river Moosi, Hyderabad India. Nova Hedwigia, 27: 661-676.
26: Weller, R., J.W. Weller and D.M. Ward, 1991. 16S rRNA sequences of uncultivated hot spring cyanobacterial mat inhabitants retrieved as randomly primed cDNA. Applied Environ. Microbiol., 57: 1146-1151.
27: Wilmotte, A., 1994. Molecular Evolution and Taxonomy of the Cyanobacteria. In: The Molecular Biology of Cyanobacteria, Bryant, D.A. (Ed.). Kluwer Academic Publishers, New York, USA., pp: 1-25
28: Wrangstadh, M., P.L. Conway and S. Kjelleberg, 1996. The release and production of extracellular polysaccharaides during starvation of marine Pseudomonas sp. and the effect thereof on the adhesion. Arch. Microbiol., 145: 220-227.