Laura Bruno

Cytomorphological and Genetic Characterization of Troglobitic Leptolyngbya Strains Isolated from Roman Hypogea

ABSTRACT Six Leptolyngbya strains, isolated from the archaeological surfaces of hypogean sites, were phenotypically and genetically characterized by light and electron microscopy and 16S rRNA gene and 16S-23S internally transcribed spacer (ITS) sequencing. Three phycoerythrin-rich (red) and three phycocyanin-rich (green) isolates were assigned to different operational taxonomic units (OTUs). Among the green isolates, one strain showed an OTU intraspecific variation due to differences in the ITS sequences and genomic polymorphism. Within the ITS sequence, variable regions, conserved domains and tRNAIle and tRNAAla genes showed high sequence identity among the phylotypes. Together, these data indicated a relatedness of the six strains to other Leptolyngbya from subaerophytic and geothermal environments and allowed the definition of novel Leptolyngbya OTUs.

Microbial diversity in paleolithic caves: a study case on the phototrophic biofilms of the Cave of Bats (Zuheros, Spain)

The biological colonization of rocks in the Cave of Bats (Cueva de Los Murciélagos, Zuheros, Spain) was studied in order to reveal the diversity of microorganisms involved in the biofilm formation. The culturable, metabolically active fraction of biodeteriogens present on surfaces was investigated focusing on morphological, ultrastructural, and genetic features, and their presence related to the peculiar environmental conditions of the underground site. PCR-ITS analysis and 16S rDNA sequences were used to clusterize and characterize the isolated strains. The presence of bacterial taxa associated to the photosynthetic microflora and fungi within the biofilm contributed to clarify the relationships inside the microbial community and to explain the alteration observed at the different sites. These results will contribute to the application of more successful strategies for the preventive conservation of subterranean archaeological sites.

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.

Characterization of biofilm-forming cyanobacteria for biomass and lipid production

This work reports on one of the first attempts to use biofilm‐forming cyanobacteria for biomass and lipid production.

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 μmol 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.

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.

New strategies for the monitoring and control of cyanobacterial films on valuable lithic faces

Abstract A survey of studies on the structure and functioning of sub-aerial epilithic phototrophic biofilms, which develop inside hypogea sites, was conducted in the framework of projects whose aim is to investigate cyanobacteria and associated microorganisms causing deterioration of stone artefacts. Using different methodological approaches, it was possible to identify and characterize cyanobacterial taxa, and to deduce their relationships with other microorganisms and the mineral substrata. Part of studies also aimed at identifying biotransformation and biomineralization mechanisms affecting stone, and the biofilm response to various light conditions. The calcium distribution and the structure of the cyanobacterial sheath gave an indication on the most deteriogenic species. Monochromatic light experiments allowed us to develop non-destructive methodological approaches for the monitoring and control/prevention of cyanobacterial biofilm growth on stone.

Polyphasic characterization of a thermo-tolerant filamentous cyanobacterium isolated from the Euganean thermal muds (Padua, Italy)

In this paper we report a morphological, ultrastructural, biochemical and molecular (16S rRNA, 16S–23S ITS, rbcL and rpoC1 gene sequencing) survey on a very thin, non-heterocystous, filamentous cyanobacterium, isolated from mats covering several mud maturation tanks of the Euganean Thermal District, at temperatures ranging from 26 to 59°C. Denaturing gradient gel electrophoresis results, obtained using cyanobacterial primers targeting the 16S rRNA gene, confirmed that this cyanobacterium is one of the commonest taxa growing in the mud tanks. Comparison with Geitlerinema sp. PCC 8501 (=Phormidium laminosum Gomont ex Gomont strain OH-1-p Cl 1), a thin thermobiotic species isolated from hot springs of Oregon and morphologically similar to our isolate, led us to hypothesize that the Euganean and PCC 8501 strains are either very similar sister species or ecotypes of the same species in a yet to be defined clade, clearly distinct within the paraphyletic Leptolyngbya group.

Characterization of exopolysaccharides produced by seven biofilm-forming cyanobacterial strains for biotechnological applications

The molecular identification of seven biofilm-forming cyanobacteria and the characterization of their exopolysaccharides were made and considered in terms of potential biotechnological applications. The studied strains were isolated from phototrophic biofilms taken from various Italian sites including a wastewater treatment plant, an eroded soil, and a brackish lagoon. The polysaccharides were characterized by use of ion exchange chromatography, circular dichroism, and cytochemical stains. All strains produced exopolysaccharides with differing ratios of hydrophobic and hydrophilic moieties depending on the species, the polysaccharide fraction (i.e., whether capsular or released), and the ambient conditions. It was shown that the anionic nature of the exopolysaccharides was due to the presence of carboxylic and sulfated groups and is likely the main characteristic with industrial applicability. Potential biotechnological applications are discussed.

Photoinhibition of Cyanobacteria and its Application in Cultural Heritage Conservation

Light has bilateral effects on phototrophic organisms. As cyanobacteria in Roman hypogea are long acclimatized to dim environment, moderate intensity of illumination can be used to alleviate biodeterioration problems on the stone substrata. Moderate intensity of light inactivates cyanobacteria by causing photoinhibition, photobleaching and photodamage to the cells. The effectiveness of light depends not only on its intensity but also on the composition and pigmentation of the component cyanobacteria in the biofilms. Red light is the most effective for the species rich in phycocyanin and allophycocyanin, such as Leptolyngbya sp. and Scytonema julianum, whereas green light is effective to inhibit the species rich in phycoerythrin, like Oculatella subterranea. White light is effective to control the grayish and the black cyanobacteria, such as Symphyonemopsis sp. and Eucapsis sp. abundant in all of these pigments. Blue light is the least effective. 150 μmol photons m−2 s−1 of blue light cannot cause biofilm damage while the same intensity of red, green or white irradiation for 14 days can severely damage the cyanobacterial cells in the biofilms due to ROS formation. Electron spin resonance spectroscopy detected the formation of radicals in different cyanobacterial cellular extracts exposed to 80 μmol photons m−2 s−1 of light.