Occurrence of Aspergillus allahabadii on sandstone at Bayon temple,Angkor Thom,Cambodia

Microbial biofilms on surface of sandstone is detrimental to the integrity of the substratum material and they are biodeteriogens responsible for the damage of sandstone over time. We observed that fungi formed extensive biofilms on areas previously colonized by autotrophic and heterotrophic microbial biofilms causing darkening of the stone surface. Appearance of fungi on these biofilms has resulted in removal of the preformed biofilm through extensive examination of sandstone surfaces in Angkor Thom, Cambodia. One fungus, isolated from the surface with capability of removing biofilms, was purified and identified as Aspergillus allahabadii during our survey and sampling of microbial biofilms at Bayon temple, Angkor Thom, Cambodia in 2008. Ribosomal RNA (ITS and 5.8S) and β-tubulin gene sequences were phylogenetically analyzed to confirm the taxonomy of this strain. In addition, its protein profile and enzyme assays were also carried out and β-galactosidase was the highest among 7 enzymes tested. Our results suggest that fungi may have an important role in removing microbial biofilms on surfaces of stone and potential mechanisms and applications are discussed.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

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.     

Effect of photosynthesis on pH variation in cyanobacterial biofilms from Roman catacombs

Cyanobacterial biofilms present on stone surfaces inRoman hypogea were studied with the aim of assessingtheir deteriogenic activity on the colonisedsubstrata. In order to achieve this, non-destructivemethods were developed and applied to measure pHvariation induced via photosynthesis and respirationin representative cyanobacteria from Roman catacombs.Amperometric and potentiometric microsensors were alsoused on Scytonema biofilms in culture in orderto measure photosynthesis and assess pH decreases andincreases during dark–light periods. Measurementsof pH showed that, starting with values slightly belowneutral, the pH in Scytonema biofilms increasedby 0.24–0.77 units in the transition from dark to1000 mol photon m^-2 s^-1 irradiance.Comparison of photosynthesis and pH curves recordedsimultaneously on the same artificial biofilm showeda maximum increase in pH value at irradiances higherthan those saturating photosynthesis. Alkalinisationof the substrate during illumination occurred to asufficient extent to induce precipitation of mineralcompounds, especially on calcareous substrates such asthose present in Roman hypogea.     

Nitrogen fixation andnif gene organization in branched heterocystous cyanobacteria: Variation in the presence ofxisA

Five branched heterocystous cyanobacteria (Scytonematopsis sp.,Scytonema sp.,Tolypothrix ceylonica, Mastigocladus sp. andFischerella sp.) were examined for their pattern of induction of nitrogenase activity andnif gene organization. All the forms showed the onset of nitrogenase activity after 12 h which could be correlated with the appearance of proheterocysts. The highest activity was exhibited byT. ceylonica. Hybridization studies revealed the presence of thenifD gene but the absence of thexisA gene inMastigocladus sp. andScytonematopsis sp. Interestingly,Tolypothrix sp. andScytonema sp. DNA samples hybridized withxisA. Hence no uniformity seems to exist regarding the presence ofxisA and the relatednif gene organization in branched heterocystous cyanobacteria. This investigation throws light on the primitive character and phylogenetic relatedness of branched forms to the coccoid/colonial forms. It also provides evidence for the proposition that stigonematacean cyanobacteria may not represent the most advanced cyanobacterial forms; rather they may link the coccoid and filamentous forms.     

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     

Microbial Community Analysis of Fresh and Old Microbial Biofilms on Bayon Temple Sandstone of Angkor Thom, Cambodia

The temples of Angkor monuments including Angkor Thom and Bayon in Cambodia and surrounding countries were exclusively constructed using sandstone. They are severely threatened by biodeterioration caused by active growth of different microorganisms on the sandstone surfaces, but knowledge on the microbial community and composition of the biofilms on the sandstone is not available from this region. This study investigated the microbial community diversity by examining the fresh and old biofilms of the biodeteriorated bas-relief wall surfaces of the Bayon Temple by analysis of 16S and 18S rRNA gene sequences. The results showed that the retrieved sequences were clustered in 11 bacterial, 11 eukaryotic and two archaeal divisions with disparate communities (Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Proteobacteria; Alveolata, Fungi, Metazoa, Viridiplantae; Crenarchaeote, and Euyarchaeota). A comparison of the microbial communities between the fresh and old biofilms revealed that the bacterial community of old biofilm was very similar to the newly formed fresh biofilm in terms of bacterial composition, but the eukaryotic communities were distinctly different between these two. This information has important implications for understanding the formation process and development of the microbial diversity on the sandstone surfaces, and furthermore to the relationship between the extent of biodeterioration and succession of microbial communities on sandstone in tropic region.     

Characteristics and role of the exocellular polysaccharides produced by five cyanobacteria isolated from phototrophic biofilms growing on stone monuments

Three coccoid and two filamentous cyanobacterial strains were isolated from phototrophic biofilms exposed to intense solar radiation on lithic surfaces of the Parasurameswar Temple and Khandagiri caves, located in Orissa State, India. Based on to their morphological features, the three coccoid strains were assigned to the genera Gloeocapsosis and Gloeocapsa, while the two filamentous strains were assigned to the genera Leptolyngbya and Plectonema. Eleven to 12 neutral and acidic sugars were detected in the slime secreted by the five strains. The secretions showed a high affinity for bivalent metal cations, suggesting their ability to actively contribute to weakening the mineral substrata. The secretion of protective pigments in the polysaccharide layers, namely mycosporine amino acid-like substances (MAAs) and scytonemins, under exposure to UV radiation showed how the acclimation response contributes to the persistence of cyanobacteria on exposed lithoid surfaces in tropical areas.     

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.     

Associative Cyanobacteria Isolated from the Roots of Epiphytic Orchids

Associative cyanobacteria were isolated from the rhizoplane and velamen of the aerial roots of the epiphytic orchids Acampe papillosa, Phalaenopsis amabilis, and Dendrobium moschatum and from the substrate roots of A. papillosa and D. moschatum. Cyanobacteria were isolated on complete and nitrogen-free variants of BG-11 medium. On all media and in all samples, cyanobacteria of the genus Nostoc predominated. Nostoc, Anabaena, and Calothrixwere isolated from the surface of the A. papillosa aerial roots, whereas the isolates from the substrate roots were Nostoc, Oscillatoria,and representatives of the LPP group (Lyngbia, Phormidium, and Plectonema, incapable of nitrogen fixation). On the D. moschatum substrate roots, Nostoc and LPP group representatives were also found, as well as Fischerella. On the aerial roots of P. amabilis and D. phalaenopsis grown in a greenhouse simulating the climate of moist tropical forest, cyanobacteria were represented by Nostoc, LPP group, and Scytonema in D. phalaenopsis and by Nostoc, Scytonema, Calothrix, Spirulina, Oscillatoria, and the LPP group in P. amabilis. For D. moschatum, the spectra of cyanobacteria populating the substrate root rhizoplane and the substrate (pine bark) were compared. In the parenchyma of the aerial roots of P. amabilis, fungal hyphae and/or their half-degraded remains were detected, which testifies to the presence of mycorrhizal fungi in this plant. This phenomenon is attributed to the presence of a sheath formed by cyanobacteria and serving as a substrate for fungi.     

The biology of carbonate precipitation by cyanobacteria

Summary In the freshwater areas of the Everglades, Florida, U.S.A., carbonate is precipitated in dense cyanobacterial mats. Precipitation is linked with photosynthesis in the mats in a quantitative relationship. On ground of field observations and experiments a model for precipitation in the filamentous cyanobacteriaScytonema is proposed, which links precipitation to bicarbonate use in photosynthesis and subsequent release of OH⁻ ions. Besides supersaturation of the water with respect to carbonate and photosynthetic bicarbonate use, precipitation requires a suitable sheath structure and composition. The characteristics of the sheath seem to be responsible for a distinct crystal morphology in the two generaScytonema andSchizothrix, as well as for the restriction of calcification to the outer sheath inScytonema. In the immediate vicinity of the trichom precipitation seems to be inhibited. Comparison of this form of calcifying cyanobacteria with calcification in calcareous algae shows many similarities and rises the question of the biological significance of calcification or precipitation. The precipitated carbonate shows equilibrium precipitation in its δ oxygen values, while it is enriched in¹³C relative to the ambient water. This agrees with a model of precipitation in which the carbonate derives from the water immediately surrounding the filament. There the water is depleted in¹²C which is preferably taken up for photosynthesis. No respiratory carbon is involved in precipitation. From measurements of the amount of precipitation in the field and in experiments the annual sedimentation rate is estimated to be 0.024 to 0.24 mm. These values fall within the range of laminae thicknesses in fossil algal laminites.     

Diversity and characteristics of terrestrial cyanobacteria near gimhae city,Korea

Cyanobacteria samples were collected from smears and mats growing on the surfaces of buildings, roadside walls, rocks, and monuments near Gimhae City, Korea. These populations were not as thick and dense, nor as diverse, as those found in temperate and tropical regions. Crusts at Gimhae City were dominated by colonial forms of species fromChroococcus, Aphanothece,Aphanocapsa, Gloeofthece,Gloeocapsa, andChroococidiopsis, as well as by filamentous forms from species ofScytonema andTolypothrix. The colonial forms appeared primarily as smears, especially for species ofGloeocapsa andChroococcidiopsis, whereas filamentous forms were the major constituents of mat populations. Invariably, the cells and trichomes of these colonial and filamentous forms were enclosed with a thick, firm, multi-colored laminar sheath, as seen by transmission electron microscopy. Such an association between cell walls and trichomes was stronger in the colonial forms, particularly forGloeocapsa, compared with the filamentous forms, in which the sheath did not detach from the wall even under arid conditions. The organization of cytoplasmic structures, particularly the thylakoid membranes, was least affected by acute dryness.     

Characterization of Microbial Mats from a Desert Wadi Ecosystem in the Sultanate of Oman

Desert wadis are widespread in the Arabian Peninsula and play a vital role in the ecology of the region; nevertheless, these ecosystems are among the least studied. Various types of microbial mats are predominant in wadis, but information on their bacterial diversity and spatial distribution is very scarce. We investigated bacterial diversity, pigments and lipid composition of ten mats located at the down-, mid- and upstream of a desert wadi in Oman. Direct microscopy revealed the existence of different unicellular and filamentous cyanobacteria, with the dominance of the heterocystous genera Calothrix and Scytonema. The majority of MiSeq 16S rRNA sequences (44-76%) were affiliated to Cyanobacteria and Proteobacteria. While Alphaproteobacteria was the most dominant proteobacterial class (10 to 48% of total sequences), Gamma- and Deltaproteobacteria were subdominant. Cluster analysis showed that the mats’ bacterial communities at the different locations along the wadi were different and shared less than 60% of their operational taxonomic units (OTUs). Chlorophyll a and scytonemin were the most predominant pigments in all mats. Different saturated, branched and mono- and poly-unsaturated fatty acids were detected in all mats, with C₁₆ and C₁₈ compounds as most dominant. The detected pigments and fatty acids indicate a major role of cyanobacteria in the wadi mats and the adaptation of microorganisms therein to the harsh wadi environment. Detection of diadinoxanthin and fucoxanthin confirmed the presence of diatoms. We conclude that microbial mats are important elements in wadi ecosystems and exist in a great variety of structure and community composition.     

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.     

Establishing oil-degrading biofilms on gravel particles and glass plates

A method is described for “artificially” establishing biofilms rich in hydrocarbon degrading bacteria on gravel particles and glass plates. The microbial consortia in the biofilms included in additions, filamentous cyanobacteria, picoplankton and diatoms. Phototrophic microorganisms were pioneer colonizers. Hydrocarbon utilizing bacteria, namely Acinetobacter calcoaceticus and nocardioforms were in part attached to filaments of cyanobacteria. In batch cultures, it was shown that those artificial biofilms had an attenuation effect on crude-oil in contaminated sea water samples. The potential use of these biofilms for preparing trickling filters (gravel particles), and in bioreactors (glass plates) for attenuating hydrocarbons in oily liquid wastes before their disposal in the open environment is suggested and discussed.     

Epilithic biofilms provide large amounts of nitrogen to tropical mountain landscapes

We show that epilithic biofilms are a relevant nitrogen (N) source in a rocky mountain range in Brazil. During different seasons, we quantified nitrate, ammonium, dissolved organic N (DON) and total dissolved N (TDN) leached by a simulated short rain event. We quantified the epilithic autotrophic biomass by taxonomic groups and its correlation with leached N. We hypothesized that leached N would be correlated to heterocystous cyanobacteria biomass since they are more efficient N₂ fixers. We estimated a landscape N supply of 8.5 kg.ha⁻¹.year⁻¹ considering the mean precipitation in the region. TDN in leachate was mainly composed of DON (83.8% ± 22%), followed by nitrate (12.1% ± 3%) and ammonium (5% ± 5%). The autotrophic epilithic community was mainly composed of non-heterocystous (Gloeocapsopsis) and heterocystous cyanobacteria (Scytonema and Stigonema), except for a site more commonly affected by fire events that showed a dominance of Chlorophyta. Biogeochemical upscaling was facilitated by the fact that N leaching was not different among sites or related to autotrophic epilithic biomass or assemblage composition. In conclusion, the capacity of epilithic biofilms to provide N to surrounding systems is an ecosystem service that underscores the necessity to conserve them and their habitats.     

Unravelling the microbial role in ooid formation – results of an in situ experiment in modern freshwater Lake Geneva in Switzerland

The microbial role in the formation of the cortex of low‐Mg calcite freshwater ooids in western part of Lake Geneva in Switzerland has been suggested previously, but not demonstrated conclusively. Early work mostly concentrated in hypersaline milieus, and hence little is known about their genesis in freshwater environments. We designed an in situ experiment to mimic the natural process of low‐Mg calcite precipitation. A special device was placed in the ooid‐rich bank of the lake. It contained frosted glass (SiO₂) slides, while quartz (SiO₂) is the most abundant mineral composition of ooid nuclei that acted as artificial substrates to favour microbial colonization. Microscopic inspection of the slides revealed a clear seasonal pattern of carbonate precipitates, which were always closely associated with biofilms that developed on the surface of the frosted slides containing extracellular polymeric substance, coccoid and filamentous cyanobacteria, diatoms and heterotrophic bacteria. Carbonate precipitation peaks during early spring and late summer, and low‐Mg calcite crystals mostly occur in close association with filamentous and coccoid cyanobacteria (e.g. Tolypothrix, Oscillatoria and Synechococcus, Anacystis, respectively). Further scanning electron microscope inspection of the samples revealed low‐Mg calcite with crystal forms varying from anhedral to euhedral rhombohedra, depending on the seasons. Liquid cultures corroborate the in situ observations and demonstrate that under the same physicochemical conditions the absence of biofilms prevents the precipitation of low‐Mg calcite crystals. These results illustrate that biofilms play a substantial role in low‐Mg calcite ooid cortex formation. It further demonstrates the involvement of microbes in the early stages of ooid development. Combined with ongoing microbial cultures under laboratory‐controlled conditions, the outcome of our investigation favoured the hypothesis of external microbial precipitation of low‐Mg calcite as the main mechanism involved in the early stage of ooid formation in freshwater Lake Geneva.     

Extracellular phycoerythrin-like protein released by freshwater cyanobacteria <Emphasis Type="Italic">Oscillatoria</Emphasis> and <Emphasis Type="Italic">Scytonema</Emphasis> sp.

During growth of the freshwater cyanobacteria, Oscillatoria sp. BTCC/A0004, and Scytonema sp. TISTR 8208, a pink pigment is released into the growth medium. The pigment from each source had a molecular weight of approximately 250 kDa and had adsorption maxima at 560 and 620 nm. These results suggest that pink pigment is a phycoerythrin-like protein. It inhibited the growth of green algae, Chlorella fusca and Chlamydomonas reinhardtii, but not other cyanobacteria or true bacteria. The concentration at which growth inhibition 50% occurred was 0.5, 6 and more than 10 mg ml⁻¹, respectively.     

Real-time PCR as an effective technique to assess the impact of phoresy by Paenibacillus sp. bacteria on Steinernema diaprepesi nematodes in nature

Quantitative real‐time PCR (qPCR) is a powerful tool to study species of cryptic organisms in complex food webs. This technique was recently developed to detect and quantify several species of entomopathogenic nematodes (EPNs), which are widely used for biological control of insects, and some natural enemies of EPNs such as nematophagous fungi and the phoretic bacteria Paenibacillus sp. and Paenibacillus nematophilus. A drawback to the use of primers and TaqMan probes designed for Paenibacillus sp. is that the qPCR also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two closely related species that are not phoretically associated with EPNs. Here, we report that the detection of Paenibacillus sp. DNA in nematode samples was two orders of magnitude greater (P < 0.001) when the bacterium was added to soil together with its EPN species‐specific host Steinernema diaprepesi than when it was added concomitantly with other EPNs or with species of bacterial‐feeding nematodes. Just 6% of samples detected trace amounts of P. thiaminolyticus and P. popilliae exposed to the same experimental conditions. Thus, although the molecular assay detects Paenibacillus spp. DNA in nonphoretic associations, the levels are essentially background compared to the detection of Paenibacillus sp. in association with its nematode host.     

Can tropical freshwater zooplankton graze efficiently on cyanobacteria?

Zooplankton may at times graze cyanobacteria. However, their top-down effects are considered to be low, particularly in tropical regions dominated by small-size grazers that may be unable to consume efficiently filamentous or colonial species. Recently, cyanobacteria blooms were reported in the Senegal River hydrosystem. We conducted feeding experiments to assess the ability of copepods (Pseudodiaptomus hessei and Mesocyclops ogunnus), cladocerans (Moina micrura and Ceriodaphnia cornuta), and rotifers (Brachionus angularis, B. falcatus, and Keratella sp.) to control different cyanobacteria (Cylindrospermopsis raciborskii, Anabaena solitaria, A. flos-aquae, and Microcystis aeruginosa). None of the zooplankton species ingested M. aeruginosa. Mesocyclops ogunnus did not consume any of the cyanobacteria. Both cladocerans consumed the smallest filaments of cyanobacteria, whereas all the rotifers and P. hessei consumed a broader food-size spectrum. The functional feeding responses suggest that the concentration and size of the filaments are not the sole criteria for food consumption. The high zooplankton community grazing rates, estimated by applying the clearance rates measured in the laboratory to the in situ zooplankton abundance, indicate that grazing by zooplankton potentially constitutes an important controlling factor for the filamentous cyanobacteria in the tropics.