orientation affects trentopohia dominated biofilms on maya monuments

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International Biodeterioration & Biodegradation

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Orientation affects Trentepohlia-dominated biofilms on Mayan monumentsof the Rio Bec style

B.O. Ortega-Morales a,*, C. Gaylarde b, A. Anaya-Hernandez c, M.J. Chan-Bacab a, S.C. De la Rosa-García a,D. Arano-Recio d, J. Montero-M e

aCentro de Microbiología Ambiental y Biotecnología, Universidad Autónoma de Campeche, Av. Agustín Melgar s/n, Col. Buenavista, C.P. 24039 Campeche, MexicobMicrobiology Research Laboratory, School of Pharmacy and Biomedical Sciences, University of Portsmouth, St. Michael’s Building, White Swan Rd., Portsmouth PO1 2DT, UKcCentro de Investigaciones Históricas y Sociales, Universidad Autónoma de Campeche, Av. Agustín Melgar s/n, Col. Buenavista, C.P. 24039 Campeche, MexicodCentro INAH Campeche, Calle 59 No. 36 entre la calle 14 y 16, Col. Centro, Campeche, Campeche C.P. 24000, MexicoeDepartamento de Recursos del Mar, CINVESTAV Unidad Mérida, Carretera antigua a Progreso No. 6, Cordemex, 97310 Mérida, Yucatán, Mexico

a r t i c l e i n f o

Article history:Received 30 January 2012Received in revised form24 July 2012Accepted 25 July 2012Available online xxx

Keywords:Building orientationMayan heritageBiofilmsTrentepohliaGeographic information Systems

* Corresponding author. Tel.: þ52 981819818119800x52100.

E-mail addresses: [email protected], benotttomail.uacam.mx (B.O. Ortega-Morales).

0964-8305/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.ibiod.2012.07.014

Please cite this article in press as: Ortega-MoRio Bec style, International Biodeterioration

a b s t r a c t

The microbial composition of red and black patinas associated with representative buildings from theMayan sites of Becán, Chicanná and Hormiguero (Campeche, México) was studied. Algae and cyano-bacteria were the prevalent microbial groups; the alga Trentepohlia and the coccoid cyanobacteriumGloeocapsa were dominant in terms of biomass. Orientation of buildings played a key role in the spatialdistribution of these biological patinas. Irrespective of the site, red patinas, whose coloration was due tothe presence of carotenoids (beta carotene and luteine), as evidenced by HPLC, were predominantlyassociated with North- and East-facing walls, or on otherwise orientated sites if under the influence oftree canopies or monument architecture. In contrast, extensive black patinas were mainly observed onSouth and West-facing walls. A GIS-based assessment of total solar irradiance for the year 2010 at the siteof Becán showed that in general North and East facing walls of monuments received significantly lowerlevels of radiation, suggesting that the major impact of orientation is to affect incident levels of damagingsolar radiation, ensuring higher availability of water for Trentepohlia colonization in low-irradiance areas.Dehydration- and UV-resistant cyanobacteria form dark patinas in more exposed regions. The influenceof orientation on biofilm formation on limestone at these sites is briefly discussed.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The archeological sites of Hormiguero (18�24010.9600N,89�2900.4500W), Chicanná (18�30016.9500N, 89�29009.8600W) andBecán (18�31002.7800N, 89�27050.4900W) are located in the Mexicanstate of Campeche, at approximately 105 km from the Guatemalanborder, bordering the national reserve of Calakmul, the secondlargest tropical rainforest in America. They are typical examples ofthe Rio Bec style of architecture, which contributed enormously tothe later, more famous Puuc style seen in sites such as Chichén-Itzáand Uxmal. Typical of the Rio Bec style is the presence of false non-functional towers topped by temples, resembling the twin-towercomplex of Tikal in Guatemala. The lack of inscribed stelae makesthese buildings difficult to date, but it is considered that, although

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the sites were occupied around 600 BC, the typical Rio Bec styleheld sway only between 700 and 1200 AD.

The ruins at the three sites investigated here have not under-gone much restoration, owing to financial restrictions anddiscrepant criteria about restoration schemes. Most of thesemonuments, built with limestone, were originally coated witha stucco layer, which is amixture of burnt limestone, sand and plantextracts (Littmann, 1959), conveying protection and aestheticappeal on the underlying stone. Nowadays, most of these monu-ments have lost their stucco coating, leaving the bare stone exposedfor surface patina formation.

The word “patina” is often used to refer to surface changes onworks of art. This term also implies not only surface discolorationbut other forms of physical alterations, along with chemicalweathering, that lead to the formation of crusts (Chilver, 2004). Themicrobial (epilithic) contribution to the formation of patinas hasbeen previously highlighted for European (Warscheid and Braams,2000; Thornbush and Viles, 2004) and Latin American monuments(Ortega-Morales et al., 2004; Gaylarde et al., 2007). The array ofcolor displayed by biological patinas has often been correlated with

affects Trentepohlia-dominated biofilms on Mayan monuments of thep://dx.doi.org/10.1016/j.ibiod.2012.07.014

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Fig. 1. Structure VIII at Becán, showing the occurrence of Trentepohlia appearing aseither yellow filamentous forms (arrows) or adhered orange biofilms (upper part ofimage) of west facing building blocks. (For interpretation of the references to colour inthis figure legend, the reader is referred to the web version of this article.)

Fig. 2. Biplot of correspondence analysis (CA) of red and black patina colonizationpatterns in Rio Bec Mayan monuments. Note that red patinas were mainly associatedwith North facing walls, where black patina occurred predominantly on south-facingsurfaces.

B.O. Ortega-Morales et al. / International Biodeterioration & Biodegradation xxx (2012) 1e62

the occurrence of specific microorganisms (Kumar and Kumar,1999; Barberousse et al., 2006). Because of the lack of local sour-ces of atmospheric pollutants, the Rio Bec archaeological sites areperfect locations for monitoring the composition of undisturbedmicrobial patinas. Prevailing microclimatic conditions of lightregime, humidity and temperature have been considered the majorfactors determining the colonization of building surfaces bymicroorganisms (Ortega-Morales et al., 2004; Gaylarde et al.,2006). These factors are influenced by building orientation(Barberousse et al., 2006). To our knowledge, there has been nostudy dealing with the influence of building orientation onmicrobial biofilm development in the tropics. The aim of this studywas to identify the main microorganisms comprising selectedpatinas from these sites and determine the effect of orientation ontheir occurrence and distribution. Of structures situated in a similartype of environment, the Mayan buildings at Tikal, Guatemala, andat Palenque, Chiapas, México, are the only ones for which micro-biological information is available on surface biofilms (Garcia deMiguel et al., 1995; Laiz et al., 2004; Ramírez et al., 2010). Weemphasized phototrophic microorganisms (algae and cyanobac-teria) because they are often the major biomass and are consideredthe first colonizers of building surfaces (Gaylarde and Gaylarde,2005; Barberousse et al., 2006).

2. Materials and methods

2.1. Study sites and sampling strategy

The orientation and appearance of the walls of various buildingsat the Mayan sites of Hormiguero, Chicanná and Becán, Campeche,were inspected. Samples of the stone surface were taken froma number of buildings displaying the major types of biogenicpatinas (red and black) in the month of February, 2010. The Mayanarchaeological sites in México are protected and thus only limitedsampling of buildings is possible. Sampling was carried out usingadhesive tape (Shirakawa et al., 2002), sterile swabs moistened insterile saline, or scrapingwith a sterile plastic implement to removerelatively poorly adherent surface biofilms. The total areas sampledby the 3 different methods (swabs, scraping and adhesive tape)covered about 10 cm2. Loose stone material was also collected,where available. We aimed to have the same number of samplesper site, but this was not always possible because of legal limita-tions, as explained. Sites and structures included in the assessmentwere: Hormiguero, structures II and V; Chicanná, structures I, IVand XX; Becán, structures IV, VIII and X, in addition to steps(designated S) leading to the patio and the circular altar (designatedA) in this patio, located near Structure III.

2.2. Statistical analysis

A correspondence analysis (CA) was carried out to test theinfluence of building orientation on the occurrence of major typesof patinas (Zar, 1999). CA analysis sorts the objects in a chi-squarespace (Legendre and Legendre, 1998). The outcome of a 2 � 4 chi-square analysis was then reported and the resulting CA graph dis-played in Fig. 2. CA has been used in microbial ecology to studypatterns of distribution and composition of bacterial communitiesas a function of environmental factors (Ramette, 2007).

2.3. Microbiological analysis

Several techniques were used at each sampling site. Swabs wereimmediately placed into sterile saline and scrapings into sterileEppendorf tubes. In the laboratory, adhesive tape samples wereexamined microscopically, with rehydration in water, to determine

Please cite this article in press as: Ortega-Morales, B.O., et al., OrientationRio Bec style, International Biodeterioration & Biodegradation (2012), htt

the major biomass. They were incubated on solid MKM(Gaylarde and Gaylarde, 2005) under constant light conditions(80 mmol photons/m2 sec PAR). Plateswere examined after 24 h andthen at various intervals up to 4 weeks. When growth becameobvious, the tape was moved to another area of the plate and thegrowth scraped from the agar surface into liquid MKM mediumsupplemented with the antifungal agents cycloheximide andnystatin (both at 100 mg l�1), and shaken at 100 rpm underconstant illumination prior to further microscope examination.

Swab and scraping samples, after homogenization in sterilesaline, were also inoculated into liquid media, MKM, BG11 (Rippkaet al., 1979) and Bold’s Basal Medium, and treated as above. Allsampleswereexaminedmicroscopicallyat intervals and identifiedasfar as possible, using as keys Castenholz (2001) and Prescott (1964).


Table 1Phototrophs identified in limestone samples from Mayan buildings at Hormiguero,Chicanná and Becan, South Campeche, México.

Microorganism Hormiguero Chicanná Becan

Structure II/V I/IV/XX IV/S/A/VIII/X

Gloeocapsa þ/þ þ/�/þ þ/�/�/þ/�Chroococcidiopsis þ/þ þ/�/þ þ/�/�/�/�Leptolyngbya þ/þ �/�/� �/�/�/�/-Scytonema �/� �/�/þ þ/�/�/�/�Trentepohlia þ/þ þ/þ/þ þ/þ/þ/�/�Chlorella �/� �/�/þ þ/�/�/�/�

S ¼ steps leading to patio.A ¼ circular altar in patio near structure X.þ: present.�: absent.

Fig. 3. South-facing side of Structure II at Hormiguero exhibiting a heterogeneouscoverage of black patina (biofilms).

B.O. Ortega-Morales et al. / International Biodeterioration & Biodegradation xxx (2012) 1e6 3

2.4. Patina-derived pigment analysis

Selected samples of red patina were removed for pigmentsanalysis. Pigments were extracted with acetone. The extract waskept in the dark at �40 �C until analyzed. Chromatography wascarried out on a 150 � 4.6 mm Agilent C18 column (4 mm particlesize). The sample (5 ml) was injected with an automatic injector andmobile phases were pumped by a high pressure pump at a flow rateof 1 ml min�1. Peaks were detected at 450 nm by an UVeVISdetector (Agilent Technologies 1260 infinity). The column wasequilibrated prior to injecting each sample by flushing with ace-tonitrile:methanol (7:1, mobile phase A) for 7 min. The sample wasinjected into the column and mobile phase A was passed foranother 2 min. A mixture of acetonitrile:methanol:water:ethylacetate (7:0.96:0.04:2, mobile phase B) was then passed for 1 min.Finally, acetonitrile:methanol:water:ethyl acetate (7:0.96:0.04:8,mobile phase C) was passed until b-carotene was eluted. Peakswere identified by standard methods described previously (De lasRivas et al., 1989). The results are presented solely as qualitativedata, quantitative measurement being impossible due to thevarying amount of material that was available as samples.

2.5. Estimation of solar irradiance at Becán

Solar radiation is the primary source of energy for many bio-logical processes. Total solar irradiance, the sum of direct diffuseand deflected radiation, was calculated for the Becán, the mostimportant of the studied sites, using a Geographic InformationSystem, GIS e based approach (Rich et al., 1994, 1995; Fu and Rich,1999). A Digital Elevation Model for the main structures of thenuclear area of Becán was created, and from this three surfaceswere derived to estimate solar irradiance: a hemisphericalviewshed map, a solar map, and a sky map. The hemisphericalviewshed is a raster representation of the visible sky, whichincludes cleared and obstructed areas that take into accounthorizon bearings and maximum angle of sky obstruction, verysimilar to a “fish-eye” lens of a photographic camera lookingdirectly up at the zenith. A solar map estimates the direct solarirradiance originating from each of the sky directions defined in theprevious map. In essence, the solar map is a raster surface thatrepresents the path of the sun across the ecliptic. The solar trajec-tory is then calculated on the basis of latitude and seasonality. Aunique value was generated for each map sector that, along withzenith location and azimuth angle, calculates solar irradiance persector. The resulting values are then overlaid on the values of thehemispherical viewshed values in order to calculate direct solarirradiance. The sky map enables us to calculate diffuse solar irra-diance. This map constitutes a hemispheric viewshed of the skydivided in sectors that are defined by zenith and azimuth angles,and a value is assigned that will be applied to the estimation ofdiffuse solar irradiance. Lastly, to estimate direct and diffuse solarirradiance received from every sky direction, the hemisphericviewshed, solar, and sky maps are overlaid, and then the ratio ofvisible sky areas per sector is calculated by dividing the number ofunobstructed pixels by the total pixels per sector.

3. Results and discussion

Algae and cyanobacteria were the principal organisms seenunder direct light microscopy of samples. Other types of bacteriaand fungi (ascomycetes) were also noted, but occurred at lowerabundances. The main types identified in the patinas are shown inTable 1. The major biomass seen on rehydrated tape strips wasalways either the cyanobacterium Gloeocapsa or the alga Trente-pohlia. Filamentous cyanobacteria were seen at three sites.


Trentepohlia was always the main biomass in samples taken fromred-stained areas of the stone, appearing either as cushion-likeyellow filaments or red patches firmly attached to the substratumin a scattered manner (Fig. 1); the latter type of algal growth wasthe most predominant form. Red patinas at Mayan sites havepreviously been reported as inorganic coatings of hematite mixedwith calcite (Goodall et al., 2007) or biogenic (algal) films (Gaylardeet al., 2006). Our results confirm that the red discoloration on thesebuildings in Southern Campeche is caused by Trentepohlia coloni-zation. In contrast, cyanobacteria (Gloeocapsa and Chroococci-diopsis) were more prevalent in dark patinas. This dominance ofTrentepohlia and cyanobacteria in red and black patinas, respec-tively, was expected from previous studies and the literature (Weeand Lee, 1980; Gaylarde et al., 2006, 2007).

The first axis of the CA biplot (Fig. 2) showed a clear differenceon the spatial occurrence of red and black patinas onmonuments atthe studied archaeological sites. Red patinas were predominantlyassociated on surfaces facing North, and to a lesser extent to theEast. In contrast, black patinas displayed the highest frequency onSouth andWest oriented surfaces (Fig. 2). These findings show thatthese spatial patterns of microbial colonization were greatlyinfluenced by the orientation of the monuments (c2 0.05, 3¼ 17.25,p ¼ 0.0006).

Black coloration of patinas (Fig. 3) is predominantly due to thepresence of the biogenic pigment scytonemin, as previouslyreported on other limestone monuments (Ortega-Morales et al.,2005; Gaylarde et al., 2007). In contrast and consistent with


Table 2Orientation of red-stained areas at the 3 sites.

Site and structure N-facing E-facing S-facing W-facing

HormigueroS II þ þ e e

Chicanná XXS I þ þ e e

IV þ e e e

XX þ e þa

BecanIV þ þ e e

S þ þ e e

A þ þ þa e

VIII þa e e þa

S ¼ steps.A ¼ altar.þ: present.�: absent.

a Tree shade.

Fig. 5. Structures IV and VIII at Becán, showing that N-facing walls and some W-facingwalls are generally cooler, with S and E generally (but not always) having highertemperatures. Exceptions to this trend are seen in walls surrounded by vegetation orwhose slope/design creates microclimates that lead to increased wetting times.


a previous study (Mukherjee et al., 2010), carotenoids (beta caro-tene and luteine detected by HPLC e data not shown e ) areresponsible for the red coloration of Trentepohlia-colonized wallsfacing North or East, or on differently orientated sites if close to treecover (Table 2). Particularly impressive was the circular altar ina patio close to Structure III in Becán (Fig. 4). The pink/red colora-tion was seen only on the North and on East facing sides, as well aswhere there were overhanging trees. Structure VIII at the same siteshowed the influence of treesmore clearly. Its North facing sidewasabout 2 m from the vegetation (mainly trees) and was intenselycolonized with macroscopically apparent mosses and higher plantsin lower parts. In the upper regions, without mosses, red biofilms ofTrentepohlia were visible. Higher sections of buildings experiencemuch greater external temperatures and global solar radiation thanlower and middle sections (Fig. 5) and protection of protectivepigments by the cells becomes a necessity. Ismail (1996) andKrishan et al. (1995) stated that low-rise buildings can more easilybe shaded by surrounding vegetation, thus minimizing the impactof sunlight on the thermal regime of the building.

The West facing wall of Structure VIII close to the trees was alsocolored red, the degree of red discoloration showing a gradientfrom left to right and from lower to higher sections (Fig. 6). On the

Fig. 4. The altar in the patio near Structure III at Becán, showing red staining mainly tothe North. (For interpretation of the references to colour in this figure legend, thereader is referred to the web version of this article.)


other hand, the same wall, on the opposite side of the steps leadingto the top of the structure and further from the vegetation cover,showed only a black biofilm, which was composed almost entirelyof variously pigmented cells of Gloeocapsa. The red, purple and

Fig. 6. Structure VIII at Becán, showing vegetation and red-stained walls facing N (tothe left of the photograph) andW. Red stained areas are arrowed. (For interpretation ofthe references to colour in this figure legend, the reader is referred to the web versionof this article.)


B.O. Ortega-Morales et al. / International Biodeterioration & Biodegradation xxx (2012) 1e6 5

brown pigments within the cells and/or capsules confer protectionon these UV-exposed biofilms, but, without tree cover, this areaapparently did not retain sufficient humidity for the growth ofTrentepohlia. On a local scale, epilithic community composition ismainly related tomicroclimatic and substrate factors. The influenceof orientation on the spatial distribution of Trentepohlia films isexplained in terms of protection against excessive damaging solarradiation and longer times of wetness on limestone surfaces facingNorth and East. The GIS-based solar radiation analysis showed lessincident solar radiation (Fig. 6), particularly evident in lowersections of structures IV and VIII. Even in sections with low sunlightexposure (blue sections in Fig. 5), carotenoids (seen as red discol-oration) accumulated in Trentepohlia; this is a functional adaptationto copewith stress resulting from the combined action of damaginglevels of solar (UV) irradiation, thermal stress and desiccation(Adams et al., 1992; Zhang et al., 1997). Differently orientated walls,however, carried red patinas under lower levels of light. Carotenoidaccumulation is also increased in shade conditions to increase lightharvest.

The differential formation of patinas and their associatedmicrobial components on limestone walls at these sites may affectdeterioration processes in various ways. Some locations exhibiteda certain degree of association between microbially dominatedpatinas and surfaces showing less obvious (macroscopic) deterio-ration (Fig. 7). This could be related to the excretion of exopoly-meric substances (EPS) (Ortega-Morales et al., 2001), which havebeen shown to confer cohesion on stone particles (Yallop et al.,2000) and may minimize thermal stress through their hygro-scopic nature (Carter and Viles, 2005; Or et al., 2007). Walls devoidof patina and thus receiving higher levels of sunlight may be moreprone to hygrothermal deterioration.

Biofilmsmay also be destructive. Coccoid-shaped cyanobacterialcells like Gloeocapsa have been shown to be involved in degradationand boring into limestone (Hoffman, 1989; Ortega-Morales et al.,2001; Gaylarde and Englert, 2006). On our buildings the redpatinas were more difficult to remove than the black, probablybecause the underlying stone was less degraded. However, Tren-tepohlia may, in some cases, have deteriogenic activity; its occur-rence has been associated with micropits in limestone structures(Gaylarde et al., 2006).

One interesting observation was that the white crustose lichen(unidentified) very prevalent at all the sites was mainly associated

Fig. 7. Structure I at Chicanná, showing different biofilms on N-facing (red, facing toleft of picture) and W-facing (black, on right of photo) walls. Note that lower sectionsapparently devoid of patina are badly degraded, probably due to karstic dissolution.(For interpretation of the references to colour in this figure legend, the reader isreferred to the web version of this article.)


with the red Trentepohlia biofilms (see Fig. 6). A scratch testconfirmed that Trentepohlia was present in the lichen. There aremany crustose lichens with this alga as the phycobiont, 21 alone inthe British Isles (James and Coppins, 1979), but comparison of themacroscopic appearance of this fungus with photographs on theInternet (Pictures of Tropical Lichens), suggests that it could beAcanthothecis clavulifera, which contains Trentepohlia as the phy-cobiont. It has been suggested that the increase in prevalence ofTrentepohlia-containing lichens in Southern Europe is associatedwith global warming and the alga is considered to be the symbiontthat is mainly affected (Aptroot and van Herk, 2007). This suggeststhat we may expect an increase of red patinas on buildings inpreviously temperate environments in the future and it will beimportant to verify the degradative or protective effects of this algaon building stone.

4. Conclusions

Red and grey/black patinas on limestone buildings of the Rio Becperiod in Southern Campeche were composed principally of thealga Trentepohlia and coccoid cyanobacteria of the genus Gloeo-capsa, respectively.

Orientation of the buildings had a major influence on theappearance of the biofilms, red staining occurring principally onNorth and East facing walls, or on those close to vegetation.

Measurement of global irradiance at one of the sites suggestedthat this was the most important factor in determining microbialcolonization, high radiation reducing time of wetness andincreasing UV exposure, thus inhibiting algal growth while allow-ing resistant cyanobacteria to survive.

There was some suggestion that tightly-adherent microbialbiofilms could protect the limestone from non-biologicalweathering.

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orientation affects trentopohia dominated biofilms on maya monuments

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