Phototrophic Biofilms on Ancient Mayan Buildings in Yucatan, Mexico

Buildings at the important archaeological sites of Uxmal and Kabah, Mexico, are being degraded by microbial biofilms. Phospholipid fatty acid (PLFA) and chlorophyll a analyses indicated that phototrophs were the major epilithic microorganisms and were more prevalent on interior walls than exterior walls. Culture and microscopical techniques showed that Xenococcus formed the major biomass on interior surfaces, but the stone-degrading genera Gloeocapsa and Synechocystis were also present in high numbers. Relatively few filamentous algae and cyanobacteria were detected. The fatty acid analysis also showed that complex biofilms colonize these buildings. Circular depressions observed by scanning electron microscopy (SEM) on stone and stucco surfaces beneath the biofilm corresponded in shape and size to coccoid cyanobacteria. SEM images also demonstrated the presence of calcareous deposits on some coccoid cells in the biofilm. Phototrophic biofilms may contribute to biodegradation by (1) providing nutrients that support growth of acid-producing fungi and bacteria and (2) active “boring” behavior, the solubilized calcium being reprecipitated as calcium carbonate. Received: 15 March 1999 / Accepted: 24 June 1999     

Biodeterioration of Mayan archaeological sites in the Yucatan Peninsula, Mexico

Many of the monuments of the Mayan civilization are suffering deterioration caused by environmental factors (high temperatures and relative humidities), increasing contamination from natural and anthropogenic sources, and by the action of micro- and macro-biological communities. Archaeological sites and historical monuments in the Mayan area were constructed with different limestones which offer different resistances to degradation by the various types of contamination. Two different sampling sites were chosen at the archaeological site of Uxmal in the Yucatan Peninsula, Mexico. Heterotrophic bacteria, cyanobacteria and different fungi were isolated and classified taxonomically. The other archaeological site chosen for this study was the fortress of Tulum, located at the side of the Caribbean Sea and exposed to chloride of marine spray and sand erosion. In this case, heterotrophic aerobic and anaerobic bacteria, cyanobacteria and fungi were isolated from the four sampling areas selected. In both archaeological sites crust deposits were observed by using light microscopy, SEM and ESEM. Surface analyses were made by means of EDAX and electron microprobe. Possible mechanisms of stone decay, based on the type of microorganisms isolated, the physico-chemical characteristics of the constructional materials and environmental factors are discussed.     

Microbial Biofilms on the Sandstone Monuments of the Angkor Wat Complex,Cambodia

Discoloring biofilms from Cambodian temples Angkor Wat, Preah Khan, and the Bayon and West Prasat in Angkor Thom contained a microbial community dominated by coccoid cyanobacteria. Molecular analysis identified Chroococcidiopsis as major colonizer, but low similarity values (<95%) suggested a similar genus or species not present in the databases. In only two of the six sites sampled were filamentous cyanobacteria, Microcoleus, Leptolyngbya, and Scytonema, found; the first two detected by sequencing of 16S rRNA gene library clones from samples of a moist green biofilm on internal walls in Preah Khan, where Lyngbya (possibly synonymous with Microcoleus) was seen by direct microscopy as major colonizer. Scytonema was detected also by microscopy on an internal wall in the Bayon. This suggests that filamentous cyanobacteria are more prevalent in internal (high moisture) areas. Heterotrophic bacteria were found in all samples. DNA sequencing of bands from DGGE gels identified Proteobacteria (Stenotrophomonas maltophilia and Methylobacterium radiotolerans) and Firmicutes (Bacillus sp., Bacillus niacini, Bacillus sporothermodurans, Lysinibacillus fusiformis, Paenibacillus sp., Paenibacillus panacisoli, and Paenibacillus zanthoxyli). Some of these bacteria produce organic acids, potentially degrading stone. Actinobacteria, mainly streptomycetes, were present in most samples; algae and fungi were rare. A dark-pigmented filamentous fungus was detected in internal and external Preah Khan samples, while the alga Trentepohlia was found only in samples taken from external, pink-stained stone at Preah Khan. Results show that these microbial biofilms are mature communities whose major constituents are resistant to dehydration and high levels of irradiation and can be involved in deterioration of sandstone. Such analyses are important prerequisites to the application of control strategies.     

Algal and Cyanobacterial Biofilms on Calcareous Historic Buildings

Major microorganisms in biofilms on external surfaces of historic buildings are algae, cyanobacteria, bacteria, and fungi. Their growth causes discoloration and degradation. We compared the phototrophs on cement-based renderings and limestone substrates at 14 historic locations (47 sites sampled) in Europe and Latin America. Most biofilms contained both cyanobacteria and algae. Single-celled and colonial cyanobacteria frequently constituted the major phototroph biomass on limestone monuments (32 sites sampled). Greater numbers of phototrophs, and especially of algae and of filamentous morphotypes, were found on cement-based renderings (15 sites), probably owing to the porosity and small pore size of the latter substrates, allowing greater entry and retention of water. All phototrophic groups were more frequent on Latin American than on European buildings (20 and 27 sites, respectively), with cyanobacteria and filamentous phototrophs showing the greatest differences. The results confirm the influence of both climate and substrate on phototroph colonization of historic buildings. Received: 7 March 2002 / Accepted: 8 April 2002     

A comparative study of the major microbial biomass of biofilms on exteriors of buildings in Europe and Latin America

Microorganisms in biofilms on building surfaces include algae, bacteria and fungi and cause discolouration and degradation, but definitive information about preferences of microbial groups for given building substrates and how this is affected by environmental conditions is lacking. Major biomass in 230 biofilms from buildings in seven Latin American and six European countries was analysed. Substrates included composites (cement, mortar, concrete, brick), painted surfaces and dimensional stone. Cyanobacteria, mostly coccoid types, were most frequently present as major biomass in LA, followed by fungi, whereas in Europe algae were most common, followed by cyanobacteria. Algae were more frequent than other groups on all substrates in Europe. Fungi, particularly uncommon as major biomass on stone, were more frequent on paint than on all other substrates (40% cf. 12%). Actinomycetes (frequently streptomycetes) were detected occasionally as major biomass, mainly on stone; climatic differences may explain the relative prevalence of this group in Europe.     

Exopolysaccharides of Two Cyanobacterial Strains from Roman Hypogea

The cytomorphology and the exopolysaccharides of two heterocystous cyanobacteria, Scytonema ocellatum CP8-2 and Fischerella maior NAV 10 bis, isolated from biofilms collected in two Roman hypogea, were investigated from a cytochemical, ultrastructural and biochemical point of view. Cytochemical stains and observation using light and transmission electron microscopy showed a bi-layered sheath in both cyanobacteria, with a different composition in sulphated and carboxylic groups. RP-HPLC of the extracted polysaccharides forming the mucous matrix showed the neutral sugars glucose and arabinose and the charged galacturonic acid as the main monosaccharides. Circular dichroism spectra recorded at varying pH and temperature showed that these polymers followed a random coil model and did not present a structured organised conformation at ambient temperature. The outcomes of this study increase the understanding of colonization and biodeterioration of important archaeological and cultural sites by cyanobacterial biofilms and the role of the exopolymeric substances involved.     

Lichen-like colonies of pure Trentepohlia on limestone monuments

Biofilms were collected on discoloured limestone samples and on adhesive tape from historic buildings at the Mayan site of Edzna, in Campeche, Mexico. Grey, brown, and black areas were colonised predominantly by coccoid and colonial cyanobacteria, also detected as endoliths. The major biomass on the pink stone surface was Trentepohlia. At low power, surface micropits containing crustose-lichen-like black colonies were seen within these pink areas. These were apparently pure colonies of T. umbrina; no filamentous fungi were detected here. The black colour was produced by dense, amorphous pigment deposits in the colony centres. Insolation levels were high (400 μmphotons m⁻² s⁻¹) and the moisture content of the stone was 0.7%. The high carotenoid:chlorophyll-α ratio of Trentepohlia biofilms (3.9:1) is an adaptation protecting against environmental stress. Trentepohlia caused severe local erosion (pitting) and, when present as a more uniform biofilm, the well-known pink surface discoloration.     

Cyanobacteria-containing biofilms from a Mayan monument in Palenque,Mexico

Surfaces of buildings at the archaeological site of Palenque, Mexico, are colonized by cyanobacteria that form biofilms, which in turn cause aesthetic and structural damage. The structural characterization and species composition of biofilms from the walls of one of these buildings, El Palacio, are reported. The distribution of photosynthetic microorganisms in the biofilms, their relationship with the colonized substratum, and the three-dimensional structure of the biofilms were studied by image analysis. The differences between local seasonal microenvironments at the Palenque site, the bioreceptivity of stone and the relationship between biofilms and their substrata are described. The implications for the development and permanence of species capable of withstanding temporal heterogeneity in and on El Palacio, mainly due to alternating wet and dry seasons, are discussed. Knowledge on how different biofilms contribute to biodegradation or bioprotection of the substratum can be used to develop maintenance and conservation protocols for cultural heritage.     

Influence of ripening container on the lactic acid bacteria population in Tulum cheese

The objective of the present study was to investigate the influence of container material (plastic or goat-skin bag) on the growth of lactic acid bacteria in Tulum cheese during 9 months of ripening. The lactic acid bacteria in Tulum cheeses were periodically counted on MRS and M17 agars throughout ripening. Results showed that the highest counts of lactic acid bacteria on MRS or M17 were observed at the beginning of ripening and their counts decreased during later stages of ripening. The cheese samples ripened in plastic bags exhibited higher numbers of LAB on MRS and M-17 agars than those ripened in goat-skin bags. A total of 112 strains of lactic acid bacteria were isolated from Tulum cheeses ripened in plastic or goat-skin bags during ripening. The lactic acid bacteria present in the cheese were classified by Microbial Identification System (MIS) based on a comparison of the fatty acid methyl ester profiles. Different species including Enteroccocus, Lactobacillus, Streptococcus, Lactococcus and Pediococcus genera were found in unripened cheese. As ripening proceeded, the species Streptococcus and Lactococcus disappeared and the percentages of the species Enterococcus was unchanged in both containers. There were slight differences between the cheeses ripened in plastic or goat-skin bags in terms of the profiles of lactic acid bacteria isolated. Some species including L. brevis, L. mesenteroides subsp. dextranicum, P. damnosus and E. mundtii were isolated only in the cheeses ripened in plastic bags; however, L. coryniformis and L. malafermentans were isolated only in the cheeses ripened in goat-skin bags at 6 or 9 months of ripening. Also the numbers of E. faecalis isolates were higher in the cheeses ripened in plastic containers than cheeses ripened goat-skin bags at the 6 or 9 months of ripening. The results showed that Lactobacillus and Enterococcus were the predominant species in matured Tulum cheeses in both ripening containers. It seemed possible to produce Tulum cheese with similar characteristics from both the containers used.     

Genetic Characterization of Epilithic Cyanobacteria and Their Associated Bacteria

Epilithic phototrophic biofilms develop inside Roman Necropolis and Catacombs on rock surfaces exposed to artificial light sources and are composed by a microbial consortium dominated by cyanobacteria. In this work, six non-axenic cultures of Leptolyngbya sp. strains isolated from biofilms from different Roman hypogea and maintained in cultures from 11 to 20 years were analysed along with their associated bacteria isolated in culture. The employment of PCR-fingerprinting techniques, using HIP1 and ERIC derived primers, allowed the clustering in three groups of the six Leptolyngbya strains and the typing of their isolated bacteria. The bacterial fingerprinting patterns were in agreement with the 16S rRNA gene sequencing and showed the presence in Leptolyngbya isolates of Pseudomonas, Stenotrophomonas, Agrobacterium and Bacillus representatives that were detected also in biofilms sampled from catacombs.     

Nitrogen fixation in lichens is important for improved rock weathering

It is known that cyanobacteria in cyanolichens fix nitrogen for their nutrition. However, specific uses of the fixed nitrogen have not been examined. The present study shows experimentally that a mutualistic interaction between a heterotrophic N₂ fixer and lichen fungi in the presence of a carbon source can contribute to enhanced release of organic acids, leading to improved solubilization of the mineral substrate. Three lichen fungi were isolated fromXanthoparmelia mexicana, a foliose lichen, and they were cultured separately or with a heterotrophic N₂ fixer in nutrient broth media in the presence of a mineral substrate. Cells of the N₂-fixing bacteria attached to the mycelial mats of all fungi, forming biofilms. All biofilms showed higher solubilizations of the substrate than cultures of their fungi alone. This finding has bearing on the significance of the origin and existence of N₂-fixing activity in the evolution of lichen symbiosis. Further, our results may explain why there are N₂-fixing photobionts even in the presence of non-fixing photobionts (green algae) in some remarkable lichens such asPlacopsis gelida. Our study sheds doubt on the idea that the establishment of terrestrial eukaryotes was possible only through the association between a fungus and a phototroph.     

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

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

Nutritional quality of biofilms with respect to light regime in Lake Saint-Pierre (Québec, Canada)

1. In situ experiments were conducted using specialised incubation devices to grow biofilms under varying light regimes and grazing intensities (by excluding fish and large‐sized zooplankton, >2 mm) both within and between two sites in Lake Saint‐Pierre. 2. Biofilms growing under greater in situ UVR and light exposures found in the south water mass were characterised by a greater biomass and nutrient content, but their total fatty acid (FA) contents and ratios of elemental nutrients were not significantly different from the north. There was a relatively greater abundance of chlorophytes and cyanobacteria in the south water mass, along with a greater proportion of low nutritional quality saturated fatty acids (SAFA). Conversely, biofilms growing in the north had a greater relative abundance of diatoms, as well as greater eicosapentaneoic acid (20:5ω3) and docosahexaneoic acid (22:6ω3) concentrations (two FAs implicated in the physiological competency of grazers). 3. The prevailing community structures created differences in terms of nutritional status of the biofilms for benthic grazers and their predators at the two sites. The biofilms from the southern site were characterised by greater food quantity at the expense of quality, while biofilms from the northern site contained less food of a better quality. Despite this, the nutritional regime in the south supported a greater productivity at higher trophic levels. The secondary treatments (light and grazing by fish and macro‐invertebrates) had lesser effects on food quality.     

Cyanobacteria cause black staining of the National Museum of the American Indian Building, Washington, DC, USA

Microbial deterioration of stone is a widely recognised problem affecting monuments and buildings all over the world. In this paper, dark-coloured staining, putatively attributed to microorganisms, on areas of the National Museum of the American Indian Building, Washington, DC, USA, were studied. Observations by optical and electron microscopy of surfaces and cross sections of limestone indicated that biofilms, which penetrated up to a maximum depth of about 1?mm, were mainly composed of cyanobacteria, with the predominance of Gloeocapsa and Lyngbya. Denaturing gradient gel electrophoresis analysis revealed that the microbial community also included eukaryotic algae (Trebouxiophyceae) and fungi (Ascomycota), along with a consortium of bacteria. Energy-dispersive X-ray spectroscopy analysis showed the same elemental composition in stained and unstained areas of the samples, indicating that the discolouration was not due to abiotic chemical changes within the stone. The dark pigmentation of the stone was correlated with the high content of scytonemin, which was found in all samples.     

Relationship between color and pigment production in two stone biofilm-forming cyanobacteria (Nostoc sp. PCC 9104 and Nostoc sp. PCC 9025)

Previous studies have provided evidence that color measurements enable on site quantification of superficial biofilms, thereby avoiding the need for sampling. In the present study, the efficiency of color measurements to evaluate to what extent pigment production is affected by environmental parameters such as light intensity, combined nitrogen and nutrient availability, was tested with two cyanobacteria, Nostoc sp. strains PCC 9104 and PCC 9025, which form biofilms on stone. Both strains were acclimated, in aerated batch cultures for 2 weeks, to three different culture media: BG-11, BG-11₀, and BG-11₀/10 at either high or low light intensity. The content of chlorophyll a, carotenoids, and phycocyanins was measured throughout the experiment, together with variations in the color of the cyanobacteria, which were represented in the CIELAB color space. The results confirmed that the CIELAB color parameters are correlated with pigment content in such a way that variations in the latter are reflected as variations in color.     

Spatial Dominance and Inorganic Carbon Assimilation by Conspicuous Autotrophic Biofilms in a Physical and Chemical Gradient of a Cold Sulfurous Spring: The Role of Differential Ecological Strategies

The community composition and ecophysiological features of microbial autotrophic biofilms were studied in Fuente Podrida, a cold sulfur spring located in East Spain. We demonstrated how different ecophysiological strategies, such as resistance and/or utilization of sulfide and oxygen, light adaptation, or resistance to high water flow, allow each of the microorganisms described to efficiently colonize several areas within the environmental gradient. In the zone of the spring constantly influenced by sulfide-rich waters, biofilms were formed by purple bacteria, cyanobacteria, and filamentous colorless sulfur bacteria. Purple bacteria showed higher photosynthetic efficiency per pigment unit than cyanobacteria, although they were dominant only in anoxic areas. Two filamentous cyanobacteria, strain UVFP1 and strain UVFP2, were also abundant in the sulfide-rich area. Whereas the cyanobacterial strain UVFP2 shows a strategy based on the resistance to sulfide of oxygenic photosynthesis, strain UVFP1, additionally, has the capacity for sulfide-driven anoxygenic photosynthesis. Molecular phylogenetic analyses cluster the benthic strain UVFP1 with genus Planktothrix, but with no particular species, whereas UVFP2 does not closely cluster with any known cyanobacterial species. The colorless sulfur bacterium Thiothrix sp. extended throughout the zone in which both sulfide and oxygen were present, exhibiting its capacity for chemolithoautotrophic dark carbon fixation. Downstream from the source, where springwater mixes with well-oxygenated stream water and sulfide disappears, autotrophic biofilms were dominated by diatoms showing higher photosynthetic rates than cyanobacteria and, by a lesser extent, by a sulfide-sensitive cyanobacterium (strain UVFP3) well adapted to low light availability, although in the areas of higher water velocity far from the river shore, the dominance shifted to crust-forming cyanobacteria. Both types of microorganisms were highly sensitive to sulfide impeding them from occupying sulfide-rich areas of the spring. Sulfide, oxygen, light availability, and water velocity appear as the main factors structuring the autotrophic community of Fuente Podrida spring. An erratum to this article is availbale at .     

Wastewater pollution differently affects the antibiotic resistance gene pool and biofilm bacterial communities across streambed compartments

Wastewater discharges introduce antibiotic residues and antibiotic‐resistant bacteria (ARB) into surface waters. Both inputs directly affect the streambed resistome, either by exerting a selective pressure that favour the proliferation of resistant phenotypes or by enriching the resident communities with wastewater‐associated ARB. Here, we investigated the impact of raw and treated urban wastewater discharges on epilithic (growing on rocks) and epipsammic (growing on sandy substrata) streambed biofilms. The effects were assessed by comparing control and impact sites (i) on the composition of bacterial communities; (ii) on the abundance of twelve antibiotic resistance genes (ARGs) encoding resistance to β‐lactams, fluoroquinolones, sulphonamides, tetracyclines, macrolides and vancomycin, as well as the class 1 integron‐integrase gene (intI1); (iii) on the occurrence of wastewater‐associated bacteria, including putative pathogens, and their potential linkage to target ARGs. We measured more pronounced effects of raw sewage than treated wastewater at the three studied levels. This effect was especially noticeable in epilithic biofilms, which showed a higher contribution of wastewater‐associated bacteria and ARB than in epipsammic biofilms. Comparison of correlation coefficients obtained between the relative abundance of both target ARGs and operational taxonomic units classified as either potential pathogens or nonpathogens yielded significant higher correlations between the former category and genes intI1, sul1, sul2 and ermB. Altogether, these results indicate that wastewater‐associated micro‐organisms, including potential pathogens, contribute to maintain the streambed resistome and that epilithic biofilms appear as sensitive biosensors of the effect of wastewater pollution in surface waters.     

Algal biofilms on tree bark to monitor airborne pollutants

Algae are used in biomonitoring systems to detect water or soil pollution. So it is conceivable to establish a biomonitoring system for the detection of airborne pollutants (ozone and particulate matter (PM-10)) in urban habitats by algae. Autotrophic biofilms are widely present, cover nearly every exposed surface, especially tree bark and consist of a large variety of species of algae, cyanobacteria and fungi. To explore the diversity of green algae at different air pollution monitoring sites we choose trees with different structures of bark at three locations in and near Leipzig. We compared the measured levels of air pollution with the algal species and communities present. The sites differed in the quality and amount of airborne pollutants, among which we concentrated on ozone and particulate matter (PM-10). The collection sites were Leipzig-Centre, Leipzig-West and a forest area east of Leipzig (Collmberg). Autotrophic biofilms were collected, algae cultures established and taxonomic and morphological studies were carried out with light microscopy. Green algae were present on tree bark at all sites and forty-eight different algal species and cyanobacteria were isolated. Preliminary results suggested a correlation between pollutants and occurrence of some specific algal species and the specific algal assemblages at a given site. It is concluded that this could provide the basis for a biomonitoring system involving aero-terrestrial algae for the detection of airborne pollutants. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Development of cyanobacterium-based biofilms and their in vitro evaluation for agriculturally useful traits

The ability of cyanobacteria to be useful as matrices for agriculturally important bacteria was evaluated. Biofilms were generated with the selected strain Anabaena torulosa after co-culturing with Azotobacter chroococcum, Pseudomonas striata, Serratia marcescens, and Mesorhizobium ciceri. The biochemical attributes were compared with individual bacterial and cyanobacterial cultures. The biofilms were characterized in terms of proteins, chlorophyll, IAA production, acetylene-reducing activity, phosphate solubilization, and antagonism towards selected phytopathogenic fungi. An enhancement in the population counts was recorded in A. torulosa–S. marcescens and A. torulosa–P. striata biofilms. The A. torulosa–A. chroococcum and A. torulosa–M. ciceri biofilms were also able to utilize new saccharides as compared to the individual cultures. Such novel biofilms with agriculturally useful traits can provide additional advantages including the broader spectrum of activity and the presence or formation of biologically active compounds; they also suggest the way to effective inoculants for sustainable and environment friendly agriculture.     

STIMULATING EFFECT OF ANABAENA SP. (CYANOBACTERIA) EXUDATE ON PRYMNESIUM PARVUM (HAPTOPHYTA)1

Prymnesium parvum blooms have become more frequent in the south‐central United States, leading to significant ecological and economic impacts. Allelopathic effects from cyanobacteria were suggested as a mechanism that might limit the development of P. parvum blooms. This research focused on the effects of cultured cyanobacteria, Anabaena sp., on P. parvum. Over a 6‐d period, daily additions of filtrate from the senescent Anabaena culture were made to P. parvum cultures growing in log phase. All treatments, including several types of controls, showed reductions in P. parvum biomass over the course of the experiment, but the treatments receiving Anabaena filtrate were reduced to a lesser degree, suggesting that filtrate from the senescent cyanobacteria culture was beneficial to P. parvum in some way. This unexpected outcome may have resulted from stimulation of heterotrophic bacteria by the addition of Anabaena filtrate, which likely contained exudates rich in dissolved organic carbon compounds. P. parvum was then able to supplement its nutritional requirements for growth by feeding on the elevated bacteria population. These findings coupled to previous observations suggest that interactions between cyanobacteria and P. parvum in natural environments are complex, where both allelopathic and growth‐stimulating interactions are possible.