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Characterization of cyanobacteria isolated frombiofilms on stone monuments at Santiniketan, IndiaNitin Keshari a & Siba Prasad Adhikary aa Department of Biotechnology , Institute of Science, Visva-Bharati , Santiniketan , IndiaPublished online: 16 May 2013.

To cite this article: Nitin Keshari & Siba Prasad Adhikary (2013): Characterization of cyanobacteria isolated from biofilms onstone monuments at Santiniketan, India, Biofouling: The Journal of Bioadhesion and Biofilm Research, 29:5, 525-536

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Characterization of cyanobacteria isolated from biofilms on stone monuments at Santiniketan,India

Nitin Keshari* and Siba Prasad Adhikary

Department of Biotechnology, Institute of Science, Visva-Bharati, Santiniketan, India

(Received 8 January 2013; final version received 4 April 2013)

Cyanobacterial biofilms occurring on the exterior of three stone monuments at Santiniketan, India were analyzed. Speciesof Scytonema and Tolypothrix were the major components of these biofilms. Identification was obtained by morphomet-ric procedures and 16S rRNA gene sequencing. Biofilms cultured for prolonged periods revealed the presence of severalother cyanobacteria belonging to 14 different genera. Cyanobacteria on stone in the tropical environment of India formeda distinct cluster that was quite different from that of cyanobacteria reported for a similar substratum in temperateregions. Absorption spectra of the organisms from Santiniketan showed a high quantity of scytonemin, mycosporine-likeamino acids, and carotenoids. All of the organisms survived in a desiccated state and rapidly revived after wetting. Theorganisms were heterocystous and nitrogenase activity was reactivated within 24 h of wetting by which time heterocystsin their filaments had also appeared.

Keywords: biofilm; cyanobacteria; stone monuments; molecular phylogeny; pigments; metabolic activity

Introduction

Stone monuments of cultural and archeological value aredisfigured in almost all parts of the globe due to coloni-zation by microorganisms. On exposed stone surfaces,phototrophic cyanobacteria, heterotrophic bacteria, proto-zoa, fungi, lichens etc form complex microbial biofilmsand their combined activity leads to weathering of thesubstratum (Büdel 1999; Gaylarde & Gaylarde 2000;Gaylarde et al. 2001; Crispim et al. 2003). These organ-isms secrete extracellular polymeric substances which actas a coating that also changes the physico-chemicalproperties and roughness of the exposed surfaces. Cya-nobacteria are the principal organisms colonizing thelight-exposed surfaces on stone monuments (Gaylarde &Morton 1999; Tomaselli et al. 2000; Videla et al. 2000;Crispim & Gaylarde 2005). The biodiversity of theseorganisms on monuments has been reported in tropical(Tripathy et al. 1997, 1999; Pattanaik & Adhikary 2002)as well as temperate regions (Tomaselli et al. 2000; Uher& Kováčik 2004; Albertano et al. 2005; Uher et al.2005; Lim & Lee 2008) of the globe. Monuments canbe made of different types of rock eg sandstone, granite,limestone, dolomite, basalt etc and certain specificcyanobacteria species colonize the exposed surfaces(Gaylarde & Morton 1999; Ramirez et al. 2010; Poloet al. 2012).

India is a tropical country which, for over 2000 years,has had a rich tradition of temple building using varioustypes of stones. Many of these structures are now

showing varying degrees of discoloration due to coloni-zation by phototropic cyanobacteria. Santiniketan, in theIndian state of West Bengal, is a heritage site with sev-eral stone and mortar monuments dating from the earlytwentieth century that are now heavily colonized bymicrobial biofilms. The cyanobacteria occurring in thebiofilms on the exterior of three important heritage stonemonuments at the Santiniketan site have been analyzedfor the first time using morphometric and molecularapproaches. The metabolic activity of the organisms inthe dried state and after wetting has been analyzed withrespect to sunscreen pigments and the appearance ofphotosynthetic and nitrogenase activities.

Material and methods

Study site

Three monuments of cultural importance in Santiniketan,West Bengal, India viz the Buddha statue (23° 40′ 49.3″N, 87° 40′ 56.6″ E), the Gandhi statue (23° 40′ 50.6″ N,087° 40′ 56.7″ E), and the elephant sculpture (23° 40′42.7″ N, 087° 40′ 58.1″ E) were chosen as the samplingsites. The monuments were built during the early twenti-eth century by the founder of the site, RabindranathTagore, the first Nobel laureate from Asia. The monu-ments were constructed primarily from stone and mortar.The temperature and light intensity at the sampling siteswere measured using a mercury thermometer and a Luxmeter, respectively. The relative humidity at the time of

*Corresponding author. Email: nitindna@gmail.com

Biofouling, 2013Vol. 29, No. 5, 525–536, http://dx.doi.org/10.1080/08927014.2013.794224

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sampling was obtained from the meteorological station,Sriniketan, West Bengal.

Collection, observation, culturing and identification

Blackish-brown biofilms were collected on several occa-sions soon after the monsoon rain in July 2011, usingsterile forceps to remove samples from the exposed sur-faces of the monuments. In other seasons, it was difficultto remove the biofilms from the stone surfaces, hencesamples were collected using a non-destructive methodemploying adhesive tape strips (La Cono & Urzi 2003).The biofilms were soaked in sterile distilled water andincubated under fluorescent light for up to 72 h beforeobserving microscopically. Since morphological featuresneeded for accurate identification were not distinct evenafter prolonged soaking (up to 7 days), a small amountof each sample was transferred to liquid BG 11 mediumwith and without nitrogen (Rippka et al. 1979) and toagar plates (1.2% w/v agar) of the same medium. Thesamples collected using adhesive tape were also cultured.Cultures were incubated at 25 ± 1 °C under continuouslight from fluorescent tubes at an intensity of 7.5Wm�2.All the organisms that appeared were isolated into unial-gal cultures and maintained. Measurement of the length,breadth, and diameter of the cells of each organism wascarried out using stage and ocular micrometers; micro-photographs were taken using an Olympus BX 41 micro-scope fitted with a Nikon Coolpix 4500 digital camera.The organisms were identified following Desikachary(1959), Komárek and Anagnostidis (1989, 1999), Anag-nostidis and Komárek (1990) and Hindák (2008). Thedominant organisms in the biofilms appeared within 72 hof wetting. These were isolated into pure culture andused for further experiments.

Viability staining

The biofilms were stained with a L7012 live/dead Bac-Light viability kit as per the manufacturer’s instructions(Invitrogen) and observed under an Olympus BX 41fluorescence microscope. The kit contained mixtures ofthe green-fluorescent stain SYTO 9 and the red-fluores-cent stain propidium iodide. These stains differ in theirspectral characteristics and in their ability to penetratehealthy cells. The excitation/emission maxima are 480/500 nm for SYTO 9 and 490/635 nm for propidiumiodide. Hence a blue filter (450–495 nm) was used toobserve the samples. Cells with intact cell membranesfluoresced green, whereas those with damaged mem-branes fluoresced red.

Extraction of genomic DNA, amplification andphylogenetic analysis

Genomic DNA was extracted from cyanobacterial cellsfollowing an isolation protocol for bacterial genomicDNA. Exponentially growing cells were pelleted by

centrifugation and resuspended in 567 μl lysis buffer(10mM Tris-HCl, pH 8.0, 1mM trisodium citrate and1.5% SDS) followed by incubation for 1 h at 37 °C afterthe addition of 30 μl of 10% SDS and 3 μl of 20mgml�1

proteinase K. Following this initial incubation, 100 μl of5M NaCl were added, followed by the addition of 80 μlCTAB/NaCl solution (10% CTAB/0.7M NaCl) and themixture was incubated for a further 10min at 65 °C. Thelysate was extracted with an equal volume of chloro-form:isoamyl alcohol (24:1) and then an equal volume ofphenol:chloroform:isoamyl alcohol (25:24:1). DNAwas pre-cipitated with 0.6 parts (v/v) of isopropyl alcohol (v/v),washed with 70% ethanol, air-dried, and resuspended in100 μl TE buffer. PCR amplification of 16S rRNA genesof the experimental organisms was carried out usingCYA359F and CYA781R (equimolar mixture ofCYA781R-a and CYA781R-b) primers specific for cya-nobacteria (Nübel et al. 1997). Template DNA (10 ng)was added to the reaction mixture in a total volume of50 μl containing 31 μl Milli Q water, 5 μl 10x buffer(15mM MgCl2), 2 μl dNTPs (10mM), 2.5 μl forwardprimer CYA359F, and 1.25 μl each of reverse primerCYA781R-a and CYA781R-b, 1 μl Taq polymerase, and1 μl BSA (20 μg μl�1). Amplification was performedusing the PCR system 9700 (Applied Biosystems). Theproducts were purified using a Qiagen gel extraction kitand sequenced (GCC Biotech Company, Kolkata). Thesequences of the 16S rRNA genes of the three majorcyanobacteria were deposited in the GenBank databaseunder accession numbers JX523941 (Scytonema millei),JX523942 (Tolypothrix campylonemoides), andJX523945 (Scytonema sp.). The 16S rRNA genesequences of other cyanobacterial species from sub-aerialhabitats were retrieved from GenBank and a consensustree was constructed following maximum parsimonyanalysis using Mega-4.0 software (Tamura et al. 2007).Bootstrap values at nodes were obtained from 1000replicates.

Extraction of pigments and spectral analysis

Pigments were extracted from 1 g of the dried biofilmscollected from each monument and the same amount ofthe pure cultures of S. millei, T. campylonemoides, andScytonema sp. isolated from the Buddha statue, theGandhi statue, and the elephant sculpture, respectively(absorbance of the homogenized culture suspension = 0.4at 750 nm). Chlorophyll a and carotenoid pigments wereextracted with 90% methanol (v/v). The absorbance ofchlorophyll a (665 nm) and carotenoids (475 nm), wasmeasured in a Shimadzu 1800 UV-visible spectropho-tometer and quantified following Mackinney (1941) andDavis (1976), respectively. Scytonemin pigments wereextracted using 100% acetone (v/v), the absorbance wasmeasured at 384 nm and the content determined accord-ing to Garcia-Pichel and Castenholz (1991). Absorption

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spectra of the 90% (v/v) methanolic extract of thebiofilm materials and the pure cultures of the organismsisolated from these biofilms were measured over a wave-length range of 250–750 nm using quartz cuvettes. Theratio of absorption of chlorophyll a at 665 nm withabsorbance measurements at 304, 320, 335, and 360 nmwhich are due to mycosporine amino acid-like sub-stances (MAAs), at 384 nm where absorbance is due toscytonemin, and 475 and 510 nm where carotenoids areresponsible for absorbance was also determined.

Measurement of photosynthesis, respiration andacetylene reduction activity (ARA)

The rate of photosynthesis, respiration, and nitrogenaseactivity of the biofilm samples from the exterior of themonuments were measured after different periods ofwetting. For measurement of photosynthetic oxygenevolution and respiratory oxygen uptake, a Clark-typeoxygen electrode (Hansatech, UK) was used. Homoge-nized suspensions of biofilms, and the major organisms

cultured from these samples, were transferred to thereaction vessel fitted with an outer jacket for watercirculation and temperature maintenance (25 °C). Thecell suspension was stirred with a magnetic stirrer toavoid reduction at the cathode and to maintain a stableoxygen gradient across the membrane. A Xenophot100W halogen lamp was used as the light source. Therate of photosynthesis and respiration was expressed interms of μmolO2 g

�1 dry material h�1. ARA wasmeasured in a AIMIL-Nucon 5765 gas chromatograph.Acetylene was injected into separate tubes containing thebiofilm samples soon after soaking as well as the corre-sponding major organisms from cultures. The tubes weretightened with rubber seals and incubated in the light for72 h. At different time intervals, 100 μl of the gas phasewere withdrawn from the tubes and activity was measuredusing a FID detector fitted with a Porapak-T column(oven temp. 75 °C; injector temp. 110 °C; detector temp.120 °C; flow of carrier N2 gas, 30mlmin�1) and the valuewas expressed as nmol C2H4 g

�1 dry material h�1.

Figure 1. Statues and sculpture of cultural heritage at Santiniketan, West Bengal, India showing profuse growth of blackish-brownbiofilms. (a–b): Buddha statue, (c–d): Gandhi statue, (e–f): elephant sculpture.

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Results and discussion

Santiniketan is a cultural heritage site with severalmonuments made up of sandstone and mortar. Manymonuments of cultural value are now disfigured by thecolonization of microorganisms that have produced black-ish-brown biofilms. Three of these monuments viz theBuddha statue, Gandhi statue, and elephant sculpture

(Figure 1) are located in close proximity to one another.The sides exposed to direct sunlight were sampled andthe organisms occurring in the biofilms were identified.The organisms in the biofilms survive in a desiccated statewhen the summer (March–June) temperature on theexposed surfaces exceeds 60 °C. Photomicrographs ofsamples of the biofilms stained using a BacLight viability

Figure 2. Photomicrograph of samples of biofilm stained with a BacLight viability kit viewed by epifluorescence microscopy.(a) Biofilm from the Buddha statue, (b) biofilm from the Gandhi statue, and (c) biofilm from the elephant sculpture. Scalebars = 30 μm.

Figure 3. Major species of cyanobacteria isolated from the exterior surface of the three different monuments in Santiniketan.Buddha statue: (a) within 72 h of wetting the biofilm; (b) Scytonema millei, which appeared in a culture of the same biofilm sample;Gandhi statue: (c) within 72 h of wetting the biofilm, (d) Tolypothrix campylonemoides, which appeared in a culture of the samebiofilm sample; Elephant sculpture: (e) within 72 h of wetting the biofilm; and (f) Scytonema sp., which appeared in a culture of thesame biofilm sample. Scale bars = 10 μm.

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kit showed green fluorescence indicating that some of thecells/filaments remained viable in the desiccated state(Figure 2) and they resumed growth soon after receivingmoisture from the monsoon rain (July–October). The bio-films sampled were composed mainly of two species ofScytonema and one species of Tolypothrix (Figure 3);however, each species was specific to a particularmonument and all three cyanobacteria did not colonizethe same stone structure. The cyanobacterial speciesrecorded from the exterior surface of the three stonemonuments are summarized in Table 1. S. millei,T. campylonemoides, and Scytonema sp. were the majororganisms on the Buddha statue, Gandhi statue, andelephant sculpture, respectively. Culturing of biofilmsfrom the three monuments revealed that after a prolongedperiod, three other species of the genus Scytonema, threespecies of the genus Nostoc, two species each of the gen-era Asterocapsa and Calothrix, and one species each ofthe genera Gloeocapsa, Aphanothece, Gloeothece, Aphan-ocapsa, Chroocococcus, Chroococcidiopsis, Tolypothrix,Aulosira, Cylindrospermum, and Westiellopsis appearedalong with the major organism, and hence were consid-ered as associated organisms (Figure 4). Two of theseassociated species, Nostoc punctiforme and Westiellopsisprolifica, were common to the Buddha and Gandhi stat-ues, which received a relatively high light intensity.Asterocapsa divina occurred on all three monuments and

Asterocapsa submersa was present on two. Furthermore,fewer cyanobacterial species occurred in biofilms on themonument receiving a lower intensity of light. Ecologicaldata for the sampling sites did not show marked variationin either temperature or relative humidity. However, thelight intensity falling on the monuments was quite distinct(Table 2). Variation in the micro-climate due to thealteration in the intensity of incident light is possibly themost important factor regulating the occurrence of a par-ticular species on these monuments.

The three major cyanobacteria viz S. millei from theBuddha statue, T. campylonemoides from the Gandhi sta-tue, and Scytonema sp. from the elephant sculpture,which were identified on the basis of their morphologywere also subjected to phylogenetic analysis. The 16SrRNA gene sequences of the species of cyanobacteriareported from similar sub-aerial habitats ie monuments,sculptures, and archeological sites worldwide (Billi et al.2001; Gugger & Hoffmann 2004; Casamatta et al. 2006;Marquardt & Palinska 2007; Bruno et al. 2009) wereretrieved from GenBank and used to make a consensustree (Table 3). The organisms belonged to the generaChroococcidiopsis, Leptolyngbya, Phormidium, Rexia,and Symphyonemopsis and had been isolated from stonesurfaces in Antarctica, Chile, Egypt, Israel, Italy, Mexico,Nepal, Spain, Switzerland, and the USA. However, thestone surfaces were located in temperate zones quite dif-

Table 1. Cyanobacterial species recorded from the exterior surface of three different stone monuments of Santiniketan, West Bengal,India.

Organism Buddha statue Gandhi statue Elephant sculpture

Major organism in the biofilmScytonema millei Bornet ex Bornet et Flahault +Scytonema sp. +Tolypothrix campylonemoides Ghose +

Associated cyanobacterial species which appeared in the culture of the biofilm containing the major organismGloeocapsa caldariorum Robenhorst +Aphanothece pallida (Kützing) Rabenhorst +Gloeothece rupestris (Lyngbye) Bornet in Wittrock Nordstedt +Aphanocapsa gravillei (Berkeley) Rabenhorst +Chroococcus turgidus (Kützing) Nägeli +Asterocapsa divina Komárek + + +Asterocapsa submersa Komárek + +Chroococcidiopsis cubana Komárek & Hindák +Cylindrospermum indicum Rao, CB Orth. mut. De Toni +Nostoc calcicola Brébisson ex Bornet et Flahault + +Nostoc commune Vaucher ex Bornet et Flahault +Nostoc punctiforme (Kützing) Hariot + +Calothrix elenkinii Kossinskaja +Calothrix sp. +Aulosira pseudoramosa Bharadwaja +Scytonema chiastum Geitler +Scytonema coactile Montagne ex Bornet et Flahault +Scytonema guyanense (Montagne) Bornet et Flahault +Tolypothrix sp. +Westiellopsis prolifica Janet + +

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ferent from the climatic regime found in Santiniketan.Maximum parsimony analysis showed the phylogeneticrelationships between the organisms isolated from thestone monuments at Santiniketan and the organisms fromsimilar types of surface based on 16S rRNA partial genesequences (Figure 5). Gloeobacter violaceus PCC 7421was used as the outgroup. The 16S rRNA genesequences of two other Scytonema species isolated fromsoil crusts (Yeager et al. 2007) were also used in the

construction of the tree to ascertain the monophyleticorigin of Scytonema under the order Nostocales. Theaccession numbers of the organisms obtained from aBLAST search are referred to in the dendrogram(Figure 5). Analysis of the resultant tree showed thatcyanobacteria in the genera Scytonema and Tolypothrix,ie the major components of biofilms on the monumentsin the tropics, were quite different from those occurringon similar surfaces in temperate zones. Furthermore,

Figure 4. Species of cyanobacteria that appeared as associated organisms in cultures of the major cyanobacterial biofilm speciesfrom the three monuments in Santiniketan. (a) Gloeocapsa caldariorum, (b) Aphanothece pallida, (c) Gloeothece rupestris, (d)Aphanocapsa gravillei, (e) Chroococcus turgidus, (f) Asterocapsa divina, (g) Asterocapsa submersa, (h) Chroococcidiopsis cubana,(i) Aulosira pseudoramosa, (j–k) Scytonema chiastum, (l) Scytonema coactile, (m–n) Scytonema guyanense, (o) Tolypothrix sp., (p)Calothrix elenkinii, (q) Calothrix sp., (r) Cylindrospermum indicum, (s) Nostoc calcicola, (t) Nostoc commune, (u) Nostocpunctiforme, and (v) Westiellopsis prolifica. Scale bars = 10 μm.

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these species of cyanobacteria colonizing stones in thetropical environment formed a distinct cluster quite differ-ent from species of Chroococcidiopsis, Leptolyngbya,and Phormidium reported from a similar substratum intemperate regions of the globe. However, both species ofScytonema from the stone monuments of Santiniketanwere clustered with two other Scytonema species isolatedfrom soil crusts originating from the USA, and were alsoclosely clustered with a Symphyonemopsis species froma cave in Spain (Gugger & Hoffmann 2004; Yeageret al. 2007).

Previous reports have shown that cyanobacterial spe-cies belonging to the genera Tolypothrix and Scytonemawere the dominant organisms in biofilms on severalstone temples and monuments in various localities of

India (Roy et al. 1997; Pattanaik & Adhikary 2002)along with associated species belonging to the generaNostoc, Calothrix, Gloeocapsa, Asterocapsa, Plectonema,and Lyngbya, all of which have either copious mucilageor a distinct sheath around their trichomes. In this study,the organisms occurring in the biofilms on stone and mor-tar monuments in Santiniketan in eastern India were alsosheath forming (ie species of Tolypothrix and Scytonema).There are reports that the extracellular sheath layers ofcyanobacteria are composed of polysaccharides that arecapable of retaining moisture, which enables them to sur-vive under extreme drought conditions (Gloaguen et al.1995; Adhikary 1998; Bertocchi et al. 1990; Cappitelliet al. 2012; Rossi et al. 2012). In addition, sheathedcyanobacteria are often colored due to the presence of the

Table 2. Location and local environmental conditions surrounding the stone monuments of Santiniketan, West Bengal, India duringthe sampling period.

Place of sampling GPS data

Temperature (°C)Relative

humidity (%) Light intensity (lux)

Max. Min. Max. Min. Max. Min.

Buddha statue, Kala Bhavana 23° 40′ 49.3″ N 34.5 26.9 95 84 12,800 12,500087° 40′ 56.6″ E

Gandhi statue, Kala Bhavana 23° 40′ 50.6″ N 34.5 26.9 95 84 6950 6800087° 40′ 56.7″ E

Elephant sculpture, Hatipukur 23° 40′ 42.7″ N 34.5 26.9 95 84 5100 4800087° 40′ 58.1″ E

Table 3. The detail of cyanobacteria isolated from monuments, stone surfaces, and biological soil crusts from several locations/regimes.

Organisms Accession no. Isolation source and place References

Leptolyngbya sp. AY769961 Catacombs, Italy Bruno et al. (2009)Leptolyngbya sp. DQ295209 Catacombs, Italy Bruno et al. (2009)Leptolyngbya sp. DQ295210 Catacombs, Italy Bruno et al. (2009)Leptolyngbya sp. DQ295207 Catacombs, Italy Bruno et al. (2009)Leptolyngbya sp. DQ295208 Catacombs, Italy Bruno et al. (2009)Chroococcidiopsis sp. AF279110 Beacon sandstone, Antartica Billi et al. (2001)Chroococcidiopsis sp. AF279107 Rock crust in Nubian sandstone, Israel Billi et al. (2001)Chroococcidiopsis sp. AF279108 Granite, Egypt Billi et al. (2001)Chroococcidiopsis sp. AF279109 Granite boulder, Chile Billi et al. (2001)Rexia erecta AY452533 Great Smoky Mountains National Park, USA Casamatta et al. (2006)Phormidium sp. AM398795 Rock surface, Switzerland Marquardt and Palinska (2007)Phormidium sp. AM398775 Rock surface, Italy Marquardt and Palinska (2007)Leptolyngbya sp. AM398803 Stone (granite), Nepal Marquardt and Palinska (2007)Phormidium tergestinum AM398776 Mortar, Spain Marquardt and Palinska (2007)Scytonema sp. DQ531698 Biological soil crust, USA Yeager et al. (2007)Scytonema sp. DQ531701 Biological soil crust, USA Yeager et al. (2007)Symphyonemopsis sp. AJ544085 Cave, Spain Gugger and Hoffmann (2004)Gloeobacter violaceus AF132790 PCC 7421 Turner et al. (1999)Scytonema millei JX523941 Mortar and stone, Buddha statue, Santiniketan, India Present workTolypothrix campylonemoides JX523942 Mortar and stone, Gandhi statue, Santiniketan, India Present workScytonema sp. JX523945 Mortar and stone, elephant sculpture Santiniketan, India Present work

Note: The 16S rRNA sequence accession number was retrieved from GenBank, NCBI for generating phylogenetic relationships with the organismsfrom the stone monuments of Santiniketan, West Bengal, India. The accession no. of Gloeobacter violaceus is also given as it is used as the outgroup.

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yellowish-brown scytonemin pigment, imparting distinctcoloration to the biofilms on the surface of rocks.

The content of chlorophyll a, carotenoids, andscytonemin pigments in the biofilms occurring on themonuments of Santiniketan is given in Table 4. Thequantity of these pigments was highest in the biofilmfrom the Buddha statue, which received a higher inten-sity of light. Lower quantities of pigment were found inbiofilms exposed to lower light intensities in their naturalhabitat (Table 4). All of the organisms in culture, as well

as in the natural biofilms, showed prominent absorptionat 665 nm due to chlorophyll a, carotenoids either at 475or 510 nm or both, as well as UV-A and UV-B absorbingpigments (scytonemin and MAAs) at wavelengthsbetween 304 and 384 nm (Tripathy et al. 1997).However, they differed depending on the absorptionmaxima of the MAAs and the amount of absorption at384 nm due to scytonemin. The sample of biofilm fromthe Buddha statue showed prominent absorption due toscytonemin, but did not possess MAAs absorbing in the

Figure 5. Consensus tree based on the 16S rRNA partial gene sequences from maximum parsimony analysis showing thephylogenetic relationships between the organisms isolated from monuments in Santiniketan and organisms from similar types ofsurfaces. Organisms isolated in this study are labeled with black circles. The number near the node represents the bootstrap value.The sequence accession numbers of the organisms obtained from GenBank are given in parenthesis.

Table 4. Pigment content of samples of biofilm taken from stone monuments.

Sample location Chlorophyll a (μg g�1) Carotenoids (μg g�1) Scytonemin (μg g�1)

Buddha statue 228.3 ± 3.4 15.8 ± 0.8 1288 ± 15.0Gandhi statue 166 ± 2.8 9 ± 0.4 699 ± 8.0Elephant sculpture 129.3 ± 1.5 8.2 ± 0.2 960 ± 12.0

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UV-B region. Furthermore, the pigments were retainedeven when the major biofilm organism was culturedunder fluorescent light. The biofilm from the Gandhistatue showed absorbance maxima at 304, 320, and335 nm due to MAAs; however, in culture, thecorresponding organism possessed MAAs absorbing onlyat 335 nm and the quantity of scytonemin was alsoreduced (Figure 6, Table 5). The sample of biofilm fromthe elephant sculpture showed prominent absorption at384 and 304 nm due to scytonemin and MAAs, respec-tively, as well as carotenoids that absorbed at 475 and510 nm. The major organism from the same biofilm inculture under fluorescent light retained all the carotenoidsand UV sunscreen pigments; however, the quantity of allof these was much reduced (Figure 6). The absorbanceratio due to carotenoids, scytonemin, and MAAs relativeto chlorophyll a was lower in the organisms isolatedfrom biofilms and grown in culture (Table 4). Further-more, similar to the samples of biofilms, the cyanobacte-ria occurring on the Buddha statue, which was exposedto a higher incidence of light, showed a higher quantityof scytonemin and MAAs than the same organisms fromthe other two monuments that were located in areasexposed to lower intensities of daytime light. There arereports that the UV-A sunscreen pigment scytonemin islocated primarily in the extracellular sheath of cyanobac-teria (Garcia-Pichel & Castenholz 1991, 1993; Büdelet al. 1997; Adhikary & Sahu 1998). The Scytonema andTolypothrix species occurring in the biofilms on thesemonuments had colored sheath layers due to scytonemin,thus ensuring protection from the high solar incidenceand the elevated UV component which is characteristicof the light in a tropical country such as India.

Recovery of the photosynthetic and respiratoryactivity of the biofilms after different durations of wetting(up to 72 h) is given in Table 6. Respiration of all threebiofilms started immediately after wetting. Photosyntheticactivity appeared in biofilms dominated by Scytonemaspecies after wetting for 4 h (Buddha statue) and 8 h(elephant sculpture). However, in the biofilms from theGandhi statue, with T. campylonemoides as the majorcomponent, photosynthetic as well as respiratory activitystarted immediately after rewetting. Recovery ofphotosynthetic activity in the terrestrial cyanobacteriumNostoc commune after rewetting has been reported tooccur in parallel with the first recovery phase of the PS IIreaction center activity (Satoh et al. 2002). In the presentcase, the rate of respiratory oxygen uptake of all the bio-films, from soon after wetting up to 24 h, was higher thanthat of their corresponding rate of photosynthesis. There-after, photosynthetic oxygen evolution increased, and after72 h the rate of both metabolic activities in all three

Figure 6. Absorption spectra of 90% (v/v) methanolicextracts: (a) the sample of biofilm from the Buddha statue (1)and the corresponding major organism in the biofilm,S. millei (2); (b) the sample of biofilm from the Gandhi statue(1) and the corresponding major organism in the biofilm,T. campylonemoides (2); and (c) the sample of biofilm from theelephant sculpture (1) and the corresponding major organism inthe biofilm, Scytonema sp. (2). Note the difference in scale ofy-axis in (a) cf (b) and (c).

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samples of biofilm remained almost constant with a netratio of photosynthesis: respiration ranging from 1.24 to1.0. The ratio of photosynthesis to respiration in all theorganisms isolated from biofilms and maintained in cul-ture also showed a ratio of 1.44–1.06. This showed thatthe energy balance between catabolic and anabolic activityof cells was very low; hence this is possibly the sole causeof the extremely slow growth of the biofilms formed fromsheathed cyanobacteria on the stone monuments.

Nitrogenase activity in the three different biofilmsafter various durations of wetting (up to 72 h) as well as

the corresponding organisms in culture is given inTable 7. Nitrogenase activity in the biofilms containingspecies of Scytonema was initiated after 24 h of rewettingand it increased progressively up to 72 h (Table 7). How-ever, biofilms with T. campylonemoides showednitrogenase activity within 16 h of rewetting. Micro-scopic examination of the filaments showed thatheterocysts started appearing after 16 h of wetting inT. campylonemoides and after 24 h for both species ofScytonema. All three cyanobacteria grown in cultureshowed ARA ranging from 108 to 116 nmol

Table 5. Absorbance of 90% (v/v) methanolic extracts of different biofilms.

Biofilms and the correspondingmajor organisms

Absorption of methanolic extract at variouswavelengths (nm) Ratio of absorbance

304 320 335 360 384 475 510 665 304 320 335 360 384 475 510 665

Biofilm of Buddha statue 0.58 0.67 0.67 0.27 0.17 0.14 4.1 4.8 4.8 1.9 1.2 1S. millei 0.1 0.2 0.07 0.05 2 4 1.4 1Biofilm of Gandhi statue 0.22 0.23 0.23 0.25 0.15 0.1 2.2 2.3 2.3 2.5 1.5 1T. campylonemoides 0.07 0.09 0.09 0.1 0.7 0.9 0.9 1Biofilm of elephant statue 0.25 0.28 0.14 0.08 0.08 3.1 3.5 1.7 1 1Scytonema sp. 0.12 0.17 0.11 0.06 0.12 1 1.4 0.9 0.5 1

Note: Left hand column details the source of the biofilm and the corresponding cyanobacterial species isolated from the respective biofilm in culture.

Table 6. Photosynthetic and respiratory activity (μmolO2 g�1material h�1) of biofilms after different durations of wetting and the

corresponding cyanobacteria in culture.

Biofilm from theexterior of the stone

Duration of wetting (h) Correspondingcyanobacteria in

culture0.5 1 4 8 16 24 36 48 72

Buddha statue S. milleiPhotosynthesis 0 0 2.1 ± 0.2 4.9 ± 0.4 13.4 ± 1.8 21.5 ± 2.3 39.3 ± 3.4 49.6 ± 5.9 54.7 ± 4.3 93.8 ± 5.2Respiration 5.8 ± 0.6 12.4 ± 0.9 18.3 ± 1.6 24.6 ± 1.7 38.2 ± 2.4 41.6 ± 2.8 43.8 ± 2.1 44.5 ± 2.3 44.1 ± 2.6 67.3 ± 4.9Gandhi statue T. campylonemoidesPhotosynthesis 1.2 ± 0.2 5.9 ± 0.8 16.4 ± 1.7 28.5 ± 2.4 42.5 ± 4.1 44.6 ± 2.3 42.9 ± 2.1 43.3 ± 3.6 41.4 ± 0.6 60.3 ± 4.7Respiration 9.3 ± 1.2 17.5 ± 2.3 25.9 ± 2.7 33.8 ± 2.3 44.5 ± 4.6 43.2 ± 2.8 41.1 ± 1.9 41.4 ± 2.3 41.2 ± 0.8 56.5 ± 3.5Elephant sculpture Scytonema sp.Photosynthesis 0 0 0 5.4 ± 0.9 16.5 ± 1.2 30.8 ± 4.1 41.4 ± 3.8 48.8 ± 2.7 51.1 ± 2.3 88.5 ± 6.6Respiration 2.6 ± 0.3 8.3 ± 0.5 11.6 ± 0.8 19.3 ± 1.1 26.1 ± 2.9 33.4 ± 3.2 37.5 ± 2.9 42.3 ± 3.3 41.8 ± 1.8 61.1 ± 5.4

Note: Values represent the mean of three independent determinations (±SD). Equal amounts of biofilm were used for each experiment.

Table 7. ARA (nmol C2H4. g�1 material h�1) of biofilms after different durations of wetting and the corresponding cyanobacteria in

culture.

Biofilm from the exteriorof the stone

Duration of wetting (h)

Corresponding cyanobacteria in culture8 16 24 36 48 72

Buddha statue 0 0 3.8 ± 0.9 8.6 ± 0.7 27.8 ± 2.1 53.2 ± 5.4 S. millei 116 ± 8.7Gandhi statue 0 3.2 ± 0.9 10.5 ± 1.2 48.4 ± 3.6 64.8 ± 6.1 73.7 ± 5.2 T. campylonemoides 123 ± 11.8Elephant sculpture 0 0 2.4 ± 0.7 7.6 ± 0.9 33.5 ± 1.2 67.7 ± 2.8 Scytonema sp. 108 ± 10.3

Note: Values represent mean of three independent determinations (±SD). Equal amounts of biofilm were used for each experiment.

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C2H4 g�1 dry material h�1. Thus, all the species of cya-

nobacteria occurring on the stone monuments were capa-ble of fixing nitrogen and they were able to survive onthe exterior of monuments even under the harsh environ-mental conditions of the tropics.

AcknowledgmentsThe authors are grateful to the Department of Science andTechnology (DST), the Government of India for financialassistance. Thanks are due to Mr SK Das for morphometricanalysis and help in the identification of the cyanobacterialspecies, and to the authorities of Visva-Bharati, Santiniketanfor providing laboratory facilities.

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