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International Biodeterioration & Biodegradation 90 (2014) 45e51

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Diversity of cyanobacteria on stone monuments and building facadesof India and their phylogenetic analysis

Nitin Keshari 1, Siba Prasad Adhikary*

Department of Biotechnology, Institute of Science, Visva-Bharati, Santiniketan 731235, West Bengal, India

a r t i c l e i n f o

Article history:Received 11 December 2013Received in revised form13 January 2014Accepted 22 January 2014Available online 6 March 2014

Keywords:BiofilmsStone monumentsBuilding facadesCyanobacteriaMolecular phylogeny

* Corresponding author. Tel.: þ91 3463261101.E-mail addresses: nitindna@gmail.com (N. Keshar

ac.in (S.P. Adhikary).1 Tel.: þ91 3463261101.

http://dx.doi.org/10.1016/j.ibiod.2014.01.0140964-8305/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Many archaeologically important stone temples, caves, mortar monuments with artistic expression aswell as building facades of India are now disfigured due to colonization of cyanobacterial biofilms leadingto weathering of the substratum. They are composed of species principally belonging to the generaHassallia, Tolypothrix, Scytonema, Lyngbya and Calothrix, which appeared soon after wetting of the bio-films. Several other species of genera Aulosira, Nostoc, Camptylonema, Dichothrix, Chlorogloeopsis andWestiellopsis occurred as associated organisms as they appeared upon prolonged culture of the biofilms.Molecular phylogenetic analysis based on 16S rRNA partial gene sequencing of all these 24 cyanobacteriaspecies under 11 genera isolated from the surfaces of monuments and building facades of India alongwith those of other species isolated from stone surfaces in subaerial habitats and hypogeal environmentsin different regions of the globe showed that those species from tropical regions were clustered together,quite different from cyanobacteria of the genera Chroococcidiopsis, Leptolyngbya, Phormidium, Nostoc,Rexia, Symphyonemopsis, Scytonema, Tolypothrix and Calothrix occurring in the temperate regions.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The exposed surfaces of stone temples, monuments as well asbuilding facades inmost regions of India look blackish colour due toformation of phototrophic biofilms. The principal species there-inare certain species of cyanobacteria (Roy et al., 1997; Tripathyet al., 1997). They could survive in the biofilms even during sum-mer months when the temperature on these structures exceeds60 �C coupled with high light intensity and extreme dryness(Tripathy et al., 1999; Pattanaik and Adhikary, 2002; Samad andAdhikary, 2008; Keshari and Adhikary, 2013). These organismssprout receiving moisture on the onset of monsoon and growutilizing the minerals leading to deterioration of monuments ofcultural value (Gaylarde and Morton, 1999; Gaylarde and Gaylarde,2000, 2001; Crispim et al., 2003). Certain species of cyanobacteriapossess well developed sheath layers around their trichome con-taining UV-sunscreen pigments like scytonemin andMAAs (Garcia-Pichel and Castenholz,1991,1993; Adhikary and Sahu,1998;Mandal

i), adhikarysp@visva-bharati.

and Rath, 2012) and secrete copious extracellular polymeric sub-stances (EPS) (Bertocchi et al., 1990; Gloaguen et al., 1995; Adhikary,1998; Rossi et al., 2012), all of which contribute to their protectionagainst desiccation and intense solar radiation. Due to their photo-trophic nature and many being nitrogen fixers, cyanobacteriacolonize easily on exposed surfaces that in due course lead to for-mation of patinas and incrustations causing aesthetic damage.

Cyanobacteria being important fromevolutionary and ecologicalpoint of view, themode of their diversity analysis and taxonomy arechanging with recent information and techniques. Very little in-formation is available on identification and phylogeny of cyano-bacteria species from Indian environments following molecularapproach (Sahu and Adhikary, 2012; Keshari and Adhikary, 2013).Organisms occurring in desiccated state on sub-aerial surfaces aredifficult to identify following available monographs as seldom themorphological features of the species can be seen even after prolongwetting of the natural material. Further, when adapted to extremeenvironments they undergo structural adaptation, thus identifica-tion and determining their evolutionary relationship is difficult.Hence 16S rRNA gene sequences of cyanobacteria species isolatedfrom stone temples and monuments as well as on building facadesfrom several locations of India were obtained and their molecularphylogeny was determined comparing the gene sequences of spe-cies from identical surfaces of other regions of the globe.

Table 1List of major and associated species of cyanobacteria isolated from the exterior of different stone monuments, caves and building facades of India.

Cyanobacteria Place of collection Substratum Majorspecies

Associatedspecies

Hassallia byssoidea Hassall (Fig. 2, a) Sun temple, Konark, Odisha Stone surface þDichothrix baueriana Bornet et Flahault (Fig. 2, b) Sun temple, Konark, Odisha Stone surface þTolypothrix scytonemoides (Gardner) Geitler (Fig. 2, c) Kedar-Gouri temple, Bhubaneswar, Odisha Stone surface þNostoc carneum Agardh ex Bornet et Flahault (Fig. 2, d) Kedar-Gouri temple, Bhubaneswar, Odisha Stone surface þTolypothrix distorta var. penicillata Kützing ex Bornet

et Flahault (Fig. 2, e)Jogin temple, Hirapur, Bhubaneswar, Odisha Stone surface þ

Nostoc commune Voucher ex Bornet et Flahault (Fig. 2, f) Lingaraj temple, Bhubaneswar, Odisha Stone surface þNostoc linckia (Roth) Bornet ex Bornet et Flahault (Fig. 2, g) Rameswar temple, Bhubaneswar, Odisha Stone surface þLyngbya aerugineo-coerulea (Kützing) Gomont (Fig. 2, h) Brahmeswar temple, Bhubaneswar, Odisha Stone surface þNostoc punctiforme (Kützing) Hariot (Fig. 2, i) Nrusinghanath temple, Paikamal, Odisha Stone surface þChlorogloeopsis fritschii Mitra et Pandey (Fig. 2, j) Vrihadeswar temple, Thanjavur, Tamil Nadu Stone surface þLyngbya kuetzingiana Kirchner (Fig. 2, k) Tiger cave, Khandagiri, Bhubaneswar, Odisha Stone surface þTolypothrix bouteillei (Brebissom et Desm.) Forti (Fig. 2, l) Ajanta caves, Maharashtra Stone surface þCalothrix gardneri De Toni (Fig. 2, m) Ajanta caves, Maharashtra Stone surface þScytonema coactile Montagne ex Bornet et Flahault (Fig. 2, n) Kutumsar cave, Chhattisgarh Stone surface þScytonema millei Bornet ex Bornet et Flahault (Fig. 2, o) Buddha statue, Santiniketan, West Bengal Stone and mortar þScytonema chiastum Geitler (Fig. 2, p) Buddha statue, Santiniketan, West Bengal Stone and mortar þWestiellopsis prolifica Janet (Fig. 2, q) Buddha and Gandhi statue, Santiniketan,

West BengalStone and mortar þ

Tolypothrix campylonemoides Ghose (Fig. 2, r) Gandhi statue, Santiniketan, West Bengal Stone and mortar þAulosira pseudoramosa Bharadwaja (Fig. 2, s) Gandhi statue, Santiniketan, West Bengal Stone and mortar þScytonema sp. (Fig. 2, t) Elephant sculpture, Santiniketan, West Bengal Stone and mortar þCalothrix marchica lemmermann (Fig. 2, u) Rabindra Bhavana and Siksha Bhavana,

Santiniketan, West BengalBuilding Facades þ

Scytonema rivulare Borzi ex Bornet et Flahault (Fig. 2, v) Patha Bhavana, P.M. hospital, Silpa Sadanand Santisree hostel, Santiniketan, West Bengal

Building Facades þ

Camptylonema indicum Schmidle (Fig. 2, w) Siksha Bhavana, Santiniketan, West Bengal Building Facades þTolypothrix rechingeri (Wille) Geitler (Fig. 2, x) Siksha Bhavana, Santiniketan, West Bengal Building Facades þ

N. Keshari, S.P. Adhikary / International Biodeterioration & Biodegradation 90 (2014) 45e5146

2. Material and methods

2.1. Description of sampling sites

Eight stone temples, three caves, three mortar monuments andsix building facades from different regions of India (Table 1, Fig. 1)were sampled for biofilms occurring on their exterior surfaces. Ofthese Sun temple of Konark; Kedar-Gouri, Jogin, Lingaraj, Rames-war and Brahmeswar temples of Bhubaneswar (19�800 to 20�240N,85�870 to 86�90E) and Nrusinghanath temple of Paikamal (20�680N,82�460E) were from Odisha and Vrihadeswar temple of Thanjavur(10�460N, 79�70E) from Tamil Nadu. These were built during 6th to16th A.D., hence important from archaeological point of view. Be-sides the Tiger cave of Khandagiri (20�200N, 85�440E) from Odisha,Ajanta caves (20�330N, 75�420E) famous for artistic expressions andfresco-paintings from Maharashtra, Kutumsar cave (19�070N,82�030E) from Chhattisgarh and the monuments made of mortarand the building facades of Santiniketan (23�680N, 87�680E), acultural heritage site in West Bengal were also sampled.

Fig. 1. Map of India showing sampling sites for cyanobacterial biofilms on exteriorsurfaces of temples andmonuments in different locations of India.1: Santiniketan (WestBengal), 2: Paikamal (Odisha), 3: Bhubaneswar (Odisha), 4: Konark (Odisha), 5:Kutumsarcave (Chhattisgarh), 6: Ajanta caves (Maharashtra) and 7: Thanjavur (Tamil Nadu).

2.2. Collection, observation, culturing and identification

Blackish-brown biofilms were collected from all the samplinglocations soon after monsoon rain and stored in pre-sterilizedsampling bottles. During the other seasons it was difficult toremove the biofilms from the stone surfaces, hence were collectedfollowing non-destructive method by employing adhesive tapestrips (La Cono and Urzi, 2003). The biofilms were soaked in steriledistilled water and incubated under fluorescent light for up to 72 hand observed microscopically. Since the morphological featuresneeded for identification were not distinct even after prolongedsoaking (up to 7 days), a small amount of each sample was trans-ferred to BG 11 medium with and without nitrogen (Rippka et al.,1979) and to agar plates (1.2% w/v agar in the same medium).

Fig. 2. Microphotographs of cyanobacteria isolated from the exterior of different stone monuments, caves and building facades of India. a. Hassallia byssoidea, b. Dichothrixbaueriana, c. Tolypothrix scytonemoides, d. Nostoc carneum, e. Tolypothrix distorta var. penicillata, f. Nostoc commune, g. Nostoc linckia, h. Lyngbya aerugineo-coerulea, i. Nostocpunctiforme, j. Chlorogloeopsis fritschii, k. Lyngbya kuetzingiana, l. Tolypothrix bouteillei, m. Calothrix gardneri, n. Scytonema coactile, o. Scytonema millei, p. Scytonema chiastum, q.Westiellopsis prolifica, r. Tolypothrix campylonemoides, s. Aulosira pseudoramosa, t. Scytonema sp., u. Calothrix marchica, v. Scytonema rivulare, w. Camptylonema indicum, x. Tolypothrixrechingeri. Scale bar: Fig. aex ¼ 10 mm.

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Cultures were incubated at 25 � 1 �C under continuous light fromfluorescent tubes at an intensity of 7.5 W m�2. The cyanobacteriaspecies appeared in the culturewere isolated andmaintained in thelaboratory. Microphotographs were taken using an Olympus BX 41microscope fitted with a Nikon Coolpix 4500 digital camera. Thesewere identified following standard monographs (Geitler, 1925;Desikachary, 1959; Hindák, 2008) and related articles (Komárekand Anagnostidis, 1989; Anagnostidis and Komárek, 1990; Doddset al., 1995).

2.3. Genomic DNA extraction, amplification of 16S rRNA gene andphylogenetic analysis

Genomic DNA was extracted from 24 different cyanobacteriaspecies isolated into culture (Fig. 2) following a bacterial genomicDNA isolation protocol. Exponentially growing cells were pelletedby centrifugation and resuspended in 567 ml lysis buffer (10 mMTriseHCl, pH 8.0, 1 mM trisodium citrate and 1.5% SDS) followed byincubation for 1 h at 37 �C after addition of 30 ml of 10% SDS and 3 ml

Table 2Cyanobacteria isolated from the exterior of monuments of different regions of India andnumber of other cyanobacteria from stone surfaces in subaerial habitats and hypogeaconstruction of Consensus tree.

Cyanobacteria Accession no Location

Leptolyngbya sp. AY769961 Catacombs, ItLeptolyngbya sp. DQ295209 Catacombs, ItLeptolyngbya sp. DQ295210 Catacombs, ItLeptolyngbya sp. DQ295207 Catacombs, ItLeptolyngbya sp. DQ295208 Catacombs, ItChroococcidiopsis sp. AF279110 Beacon sandsChroococcidiopsis sp. AF279107 Rock crust inChroococcidiopsis sp. AF279108 Granite, EgypChroococcidiopsis sp. AF279109 Granite bouldRexia erecta AY452533 Great SmokyPhormidium sp. AM398795 Rock surface,Phormidium sp. AM398775 Rock surface,Leptolyngbya sp. AM398803 Stone (GranitPhormidium tergestinum AM398776 Mortar, SpainNostoc cf. commune HQ877825 Building wallSymphyonemopsis sp. AJ544085 Cave, SpainScytonema sp. DQ531701 Biological soiTolypothrix sp. HM751852 Rock surfaceCalothrix sp. HM751844 Rock surfaceCalothrix sp. HM751845 Rock surfaceGloeobacter violaceus AF132790 PCC 7421Fischerella muscicola KC736951 Stone templeHassallia byssoidea KC736947 Stone templeDichothrix baueriana KC736961 Stone templeTolypothrix scytonemoides KC736948 Stone templeNostoc carneum KC736962 Stone templeTolypothrix distorta var. penicillata KC736949 Stone templeNostoc commune KC736958 Stone templeNostoc linckia KC736960 Stone templeLyngbya aerugineo-coerulea KC736956 Stone templeNostoc punctiforme KC736959 Stone templeChlorogloeopsis fritschii KC736950 Stone templeLyngbya kuetzingiana KC924443 Tiger cave, OdTolypothrix bouteillei KC736953 Ajanta caves,Calothrix gardneri KC736954 Ajanta caves,Scytonema coactile KC736955 Kutumsar cavScytonema millei JX523941 Stone and moScytonema chiastum JX523943 Stone and moWestiellopsis prolifica JX523946 Stone and moTolypothrix campylonemoides JX523942 Stone and moAulosira pseudoramosa JX523944 Stone and moScytonema sp. JX523945 Stone and moCalothrix marchica KC924438 Building facaScytonema rivulare KC924440 Building facaCamptylonema indicum KC924439 Building facaTolypothrix rechingeri KC924441 Building faca

of 20 mg ml�1 proteinase K. To this, 100 ml 5 M NaCl was added,followed by addition of 80 ml CTAB/NaCl solution (10% CTAB/0.7 MNaCl) and incubated for 10 min at 65 �C. The lysate was extractedwith equal volume of chloroform:isoamyl alcohol (24:1) and thenan equal volume of phenol:chloroform:isoamyl alcohol (25:24:1).DNA was precipitated with 0.6 volume isopropyl alcohol, washedwith 70% ethanol, air dried and resuspended in 100 ml TE buffer.PCR amplification of 16S rRNA genes of the experimental organismswas carried out using CYA359F and CYA781R (equimolar mixture ofCYA781R-a and CYA781R-b) primers specific for cyanobacteria(Nübel et al., 1997). Template DNA (10 ng) was added to the reac-tion mixture in a total volume of 50 ml containing 31 ml milli Qwater, 5 ml 10� Buffer (15 mM MgCl2), 2 ml dNTPs (10 mM), 2.5 mlforward primer CYA359F and 1.25 ml each of reverse primerCYA781R-a and CYA781R-b, 1 ml Taq polymerase and 1 ml BSA(20 mg ml�1). Amplification was performed using the PCR system9700 (Applied Biosystems). The products were purified using aQiagen gel extraction kit and sequenced (GCC Biotech Company,Kolkata). The sequences of 16S rRNA of all the species were

their accession number after deposition in NCBI GenBank and details of accessionl environments in different regions of the globe retrieved from GenBank used for

References

aly Bruno et al., 2009aly Bruno et al., 2009aly Bruno et al., 2009aly Bruno et al., 2009aly Bruno et al., 2009tone, Antartica Billi et al., 2001Nubian sandstone, Israel Billi et al., 2001t Billi et al., 2001er, Chile Billi et al., 2001Mountains National Park, USA Casamatta et al., 2006Switzerland Marquardt and Palinska, 2007Italy Marquardt and Palinska, 2007e), Nepal Marquardt and Palinska, 2007

Marquardt and Palinska, 2007s, Mexico Ramírez et al., 2011

Gugger and Hoffmann, 2004l crust, Cyanolands National Park, USA Yeager et al., 2007of siliceous stream, Madrid, Spain Berrendero et al., 2011of siliceous stream, Madrid, Spain Berrendero et al., 2011of siliceous stream, Madrid, Spain Berrendero et al., 2011

Turner et al., 1999, Nepal Unpublished, Odisha, India Present work, Odisha, India Present work, Odisha, India Present work, Odisha, India Present work, Odisha, India Present work, Odisha, India Present work, Odisha, India Present work, Odisha, India Present work, Odisha, India Present work, Tamil Nadu, India Present workisha, India Present workMaharashtra, India Present workMaharashtra, India Present worke, Chhattisgarh, India Present workrtar monument, West Bengal, India Present workrtar monument, West Bengal, India Present workrtar monument, West Bengal, India Present workrtar monument, West Bengal, India Present workrtar monument, West Bengal, India Present workrtar monument, West Bengal, India Present workdes, West Bengal, India Present workdes, West Bengal, India Present workdes, West Bengal, India Present workdes, West Bengal, India Present work

N. Keshari, S.P. Adhikary / International Biodeterioration & Biodegradation 90 (2014) 45e51 49

deposited in the GenBank database with accession no. JX523941to JX523946, KC736947 to KC736950, KC736953 to KC736956,KC736958 to KC736962, KC924438 to KC924441 and KC924443.The 16S rRNA sequences of other cyanobacterial species from sub-aerial habitats reported earlier were retrieved from GenBank for

Fig. 3. Consensus tree fromMaximum Parsimony showing the phylogenetic relationship betof India and the organisms from other regions of the globe based on the 16S rRNA partial gupon the types of monuments from where those were isolated (C: stone and mortar mon

construction of consensus tree following maximum parsimonyanalysis using Mega-4.0 software (Tamura et al., 2007). Gloeobacterviolaceus PCC 7421 was used as the outgroup (Turner et al., 1999).Number near node represents bootstrap value was obtained from1000 replicates.

ween the cyanobacteria isolated from biofilms on exterior surfaces in different locationsene sequences. Organisms from India were depicted with different symbols dependinguments, A: stone temples, -: caves, :: building facades).

N. Keshari, S.P. Adhikary / International Biodeterioration & Biodegradation 90 (2014) 45e5150

3. Results and discussion

The blackish brown biofilms on the exterior of monuments indifferent location of India were comprised of 24 species under 11genera (Table 1, Fig. 2). Hassallia byssoidea was the major cyano-bacterium on Sun temple, Konark, whereas species belonging toTolypothrix, Scytonema and Lyngbya were the major organisms onseveral temples of Bhubaneswar, caves of Maharashtra, Chhattis-garh and Odisha and also on the mortar monuments of Santinike-tan inWest Bengal. Other cyanobacteria under the same genera andin addition, species of Nostoc, Calothrix and Westiellopsis appearedalong with the major cyanobacteria upon prolonged culture, hencewere designated as associated organisms (Table 1). A species ofCalothrix, Calothrix marchica was the major organism on buildingfacades of Santiniketan and one species each of Tolypothrix andCamptylonema occurred as the associated components. In generalfilamentous cyanobacteria with thick sheath layer around theirtrichomes were the major species occurring on the exposed stoneand mortar surfaces forming biofilms in Indian environments(Tripathy et al., 1997, 1999; Pattanaik and Adhikary, 2002).

For molecular phylogeny analysis 16S rRNA partial gene se-quences of all these 24 species of cyanobacteria obtained in culturewere generated and deposited in GenBank (Table 2). The 16S rRNAsequences of cyanobacteria species from sub-aerial habitats re-ported so far from across the globe were retrieved from GenBankand used to make a consensus tree. These organisms were isolatedfrom Catacombs of Italy, stone/rock surfaces of Israel, Egypt, Chile,USA, Switzerland, Nepal, Spain, and Antarctica, caves of Spain,building facades of Mexico, biological soil crusts of USA as well asrock surface of siliceous stream of Spain covering the temperate aswell as tropical zones (Billi et al., 2001; Gugger and Hoffmann,2004; Casamatta et al., 2006; Marquardt and Palinska, 2007;Yeager et al., 2007; Bruno et al., 2009; Berrendero et al., 2011;Ramírez et al., 2011). Maximum parsimony analysis showingphylogenetic relationships between all these cyanobacteria speciesis given in Fig. 3. Sequence accession numbers of the organismsobtained from GenBank are given in parenthesis. Analysis of theresults showed that cyanobacteria under Nostocales and Stigone-matales from different localities of India formed a genera-wisemonophyletic clade i.e. Lyngbya kuetzingiana and Lyngbya aerugi-neo-coerulea (Fig. 3, clad-VII); Calothrix gardneri, Calothrix marchicaand Dichothrix baueriana (Fig. 3, clad-I); and Westiellopsis prolifica,Fischerella muscicola with Chlorogloeopsis fritschii (Fig. 3, clad-V).Likewise Hassallia byssoidea and species of Nostoc formed a sepa-rate clade (Fig. 3, clad-VI), species of Tolypothrix, Scytonema,Camptylonema almost clustered together (Fig. 3, clad-II, III, IV) whileAulosira pseudoramosa clustered with other heterocystous cyano-bacteria. Thus, phylogenetic clustering supported morphologicalcharacteristics in most cases. Further, the resultant tree showedthat the cyanobacteria in the biofilms on the exterior of stonemonuments in tropical climatic regime like India were clusteredquite differently from the species under genera Chroococcidiopsis(Fig. 3, clad-IX), Leptolyngbya (Fig. 3, clad-VIII), Nostoc, Rexia, Sym-phyonemopsis, Tolypothrix, Scytonema and Calothrix (Fig. 3, clad-X)with Phormidium in-between isolated from stone surfaces underdifferent climatic regime.

4. Conclusions

These results showed that different species of cyanobacteriaform blackish brown biofilms on stone surfaces of monuments aswell as building facades globally (Pattanaik and Adhikary, 2002;Crispim et al., 2004; Ortega-Morales et al., 2005; Pereira de Oli-veira et al., 2011) and are responsible for their biodeterioration(Ortega-Calvo et al., 1991; Gaylarde andMorton,1999; Gaylarde and

Gaylarde, 2000, 2001; Ramírez et al., 2010). The species that occuron such surfaces were not similar phylogenetically. Morphologicalas well as molecular techniques are essential to understand thecolonization pattern of cyanobacteria on exterior of stone monu-ments and building facades exposed to extreme heat and desicca-tion, and their role in biodeterioration.

Acknowledgements

We are grateful to the Department of Science and Technology(DST), Government of India for financial assistance through aCluster project on “Genomics and Proteomics of stress tolerantcyanobacteria”. We thank the authorities of Visva-Bharati, Santi-niketan for providing laboratory facilities.

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