A. de los Ríos

In situ evaluation of the biodeteriorating action of microorganisms and the effects of biocides on carbonate rock of the Jeronimos Monastery (Lisbon)

The biodeterioration effects of microorganisms colonizing the cloister terrace wall of the Jeronimos Monastery (Lisbon) were evaluated using several microscopy techniques that allow the in situ examination of lithobiontic communities. The techniques applied were: scanning electron microscopy with back-scattered electron imaging (SEM-BSE), low temperature scanning electron microscopy (LTSEM), transmission electron microscopy (TEM) and an X-ray energy dispersive spectroscopy (EDS) microanalytical system. The stone was seen to be colonized by different lichens and microorganisms and lichen thalli of Thyrea, Aspicilia, Verrucaria and Caloplaca were identified. Cyanobacteria were frequently observed close-by, as single cells or colonies and heterotrophic bacteria were also found among these. The lithobiontic community showed biogeophysical and biogeochemical effects on the substrate. Cyanobacteria produced bowl- or pear-shaped cavities. Using SEM-BSE and TEM we were able to observe a mineral network structure adjacent to the cyanobacterial wall that might be related to calcium biomobilization processes. Neoformation of biogenic carbonate was detected in thalli of the lichen Thyrea. This information was complemented by observing details of the response of these biological components to the biocidal agents, ALGOPHASE®, METATIN® and PREVENTOL R80®. After treatment, Thyrea remained on the stone, although ultrastructural alterations were observed in the photobiont. When the effects of the biocides on the ultrastructure of the cyanobacteria were analyzed, ALGOPHASE® proved to be the least efficient, while PREVENTOL R80® led to the complete disorganization of the prokaryotic cyanobacterial cell. These results point to the importance of evaluating biodeterioration processes and possible treatment measures without extracting the microorganisms from their microhabitat.

Viability of endolithic micro-organisms in rocks from the McMurdo Dry Valleys of Antarctica established by confocal and fluorescence microscopy

The rocks of the McMurdo Dry Valleys desert in Antarctica harbour endolithic communities of micro‐organisms such as lichens, fungi, cyanobacteria and bacteria. Establishing the physiological status and viability of these microbial colonies in their natural microhabitat has far‐reaching implications for understanding the microbial ecology of the harsh environment of this polar desert. Here we describe the use of confocal microscopy and a specific fluorescent probe (FUN‐1) to evaluate the metabolic activity of fungal cells. Application of confocal microscopy also served to identify living and dead bacteria or cyanobacteria using the fluorescent assay reagents Live/Dead SYTO 9 and propidium iodide or SYTOX Green, respectively. In addition, through the use of epifluorescence microscopy, live/dead bacteria and cyanobacteria could be detected by estimating fluorescence from their cell components provoked by simultaneously staining with nucleic acids stains such as DAPI and SYTOX Green.

An exceptional group-I intron-like insertion in the SSU rDNA of lichen mycobionts

Abstract An exceptional group-I intron-like insertion at position 940 of the nuclear small subunit rDNA is found in lichen mycobionts of the families Parmeliaceae and Lecanoraceae. This shared insertion site is exceptional as it follows a G. Although several features of the self-splicing Tetrahymena intron are missing, the conserved structure of the presumed core region indicates that the new intron-like insertion, which is missing in mature transcripts, is not part of a silenced ribosomal repeat. It is unlikely that the new insertion is horizontally transferred from the adjacent position 943. A phylogenetic analysis indicates congruence with lichen phylogeny and suggests that the insertion has been vertically inherited.

Comparative ecophysiology of three Placopsis species, pioneer lichens in recently exposed Chilean glacial forelands

Lichen species belonging to the genus Placopsis are early colonisers on snow free moraines of exposed land surfaces in the subantarctic region of Tierra de Fuego, South Chile. The physiological performance of three co-occurring species, P. pycnotheca, (terricolous), and P. perrugosa, and P. stenophylla (both saxicolous) was studied. All, possess green algal photobionts but have cyanobacteria in cephalodia. It was found that there was (i) a strong positive correlation between the acetylene reduction rate (AR) and the maximum photosynthetic rate (Amax), between the N content and the AR rate, and between the N and P contents, and (ii) the relationship between the CO2-exchange rates and the responses obtained in the laboratory reflected the ecology of these three lichens in the field. The results provide new information about the dynamics of some of the fastest growing crustose lichens. We hypothesize that the performance of these three species may have developed as a response to growing in an unstable environment that resulted from frequent glacial fluctuations.

MICROBIAL BIOFILMS ON CARBONATE ROCKS FROM A QUARRY AND MONUMENTS IN NOVELDA (ALICANTE, SPAIN)

Dolomite complex rock of the Upper Jurassic and biocalcarenite (Bateig stone) used for monuments in Novelda (Alicante, Spain) support a considerable biocomplexity of biofilms. The climate of the Novelda region is described as Iberolevantinemesomediterranean. The La Mola quarry, from which samples were obtained for the present study, was opened at the end of the 19th century and remained in use until the end of the 1970s. The Novelda monuments studied were constructed at the end of the 19th century. In the quarry boundary zone there is an abundance of lichens, such as Collema,Catapyrenium and Lecidea, mosses and chroococcoid cyanobacteria (Gloeocapsa sp. and Chroococcus sp.). Fragments of quarry rock and monument stone were investigated by SEM-BSE and/or EDS analysis according to a new methodology. In the Mola quarry, the combined action of epilithic and endolithic microorganisms (mostly cyanobacteria) degrade the rock, becoming truly euendolithic since they bore into the rock. Alteration of the biocalcarenite by lichen thalli and microorganisms can be modelled: the mycobiont of the algal zone and upper cortex harbor biomineral fragments of the substratum including calcium oxalate. A second layer lying close to the lithic substratum is composed on the mycobiont intermixed with the bioclasts and minerals detached from it, resulting in significant mechanical alteration. Within the rock, microorganisms are present between the hollows and cavities: here the bioclasts and minerals shelter the microorganisms and important element biomobilization phenomena take place.

Long-term responses of the green-algal lichen Parmelia caperata to natural CO2 enrichment

Abstract Acclimation to elevated CO2 was investigated in Parmelia caperata originating from the vicinity of a natural CO2 spring, where the average daytime CO2 concentration was 729 ± 39 μmol mol−1 dry air. Thalli showed no evidence of a down-regulation in photosynthetic capacity following long-term exposure to CO2 enrichment in the field; carboxylation efficiency, total Ribulose bisphosphate carboxylase/oxygenase (Rubisco) content, apparent quantum yield of CO2 assimilation, and the light-saturated rate of CO2 assimilation (measured under ambient and saturating CO2 concentrations) were similar in thalli from the naturally CO2 enriched site and an adjacent control site where the average long-term CO2 concentration was about 355 μmol mol−1. Thalli from both CO2 environments exhibited low CO2 compensation points and early saturation of CO2 uptake kinetics in response to increasing external CO2 concentrations, suggesting the presence of an active carbon-concentrating mechanism. Consistent with the lack of significant effects on photosynthetic metabolism, no changes were found in the nitrogen content of thalli following prolonged exposure to elevated CO2. Detailed intrathalline analysis revealed a decreased investment of nitrogen in Rubisco in the pyrenoid of algae located in the elongation zone of thalli originating from elevated CO2, an effect associated with a reduction in the percentage of the cell volume occupied by lipid bodies and starch grains. Although these differences did not affect the photosynthetic capacity of thalli, there was evidence of enhanced limitations to CO2 assimilation in lichens originating from the CO2-enriched site. The light-saturated rate of CO2 assimilation measured at the average growth CO2 concentration was found to be significantly lower in thalli originating from a CO2-enriched atmosphere compared with that of thalli originating and measured at ambient CO2. At lower photosynthetic photon flux densities, the light compensation point of net CO2 assimilation was significantly higher in thalli originating from elevated CO2, and this effect was associated with higher usnic acid content.

Towards a more realistic picture of in situ biocide actions: combining physiological and microscopy techniques.

In this study, we combined chlorophyll a fluorescence (ChlaF) measurements, using pulse-amplitude-modulate (PAM) equipment, with scanning electron microscopy in backscattered electron mode (SEM-BSE) and transmission electron microscopy (TEM) images to evaluate the actions of Koretrel at lower concentrations on Verrucaria nigrescens colonising a dolostone. ChlaF measurements are good indicators of the damaging effects of biocides. However, these indicators only provide an incomplete view of the mechanism of biocides used to control biodeterioration agents. The death of the V. nigrescens photobiont at two biocide concentrations was revealed by PAM, SEM-BSE and TEM. Once Koretrel was applied, the Fv/Fm ratios markedly fell in the first few hours after the 1.5% treatment, and ratios for the 3% dilution remained close to zero throughout the study. The algal zone shows the plasmolysed appearance of the photobiont cells, and important aspects related to the action of the biocide on free and lichenised fungi were also detected using SEM-BSE. Many of the mycobiont cells had only their cell walls preserved; although, some fungal hyphae in lichen thalli and some microorganisms in endolithic clusters maintained lipid storage in their cytoplasm. These results indicated that the combination of physiological and microscopy techniques improves the assessment of biocide action in situ and this will help to optimize protocols in order to reduce the emission of these compounds to the environment.

Diversity of Endosymbiotic Nostoc in Gunnera magellanica (L) from Tierra del Fuego, Chile

Global warming is causing ice retreat in glaciers worldwide, most visibly over the last few decades in some areas of the planet. One of the most affected areas is the region of Tierra del Fuego (southern South America). Vascular plant recolonisation of recently deglaciated areas in this region is initiated by Gunnera magellanica, which forms symbiotic associations with the cyanobacterial genus Nostoc, a trait that likely confers advantages in this colonisation process. This symbiotic association in the genus Gunnera is notable as it represents the only known symbiotic relationship between angiosperms and cyanobacteria. The aim of this work was to study the genetic diversity of the Nostoc symbionts in Gunnera at three different, nested scale levels: specimen, population and region. Three different genomic regions were examined in the study: a fragment of the small subunit ribosomal RNA gene (16S), the RuBisCO large subunit gene coupled with its promoter sequence and a chaperon-like protein (rbcLX) and the ribosomal internal transcribed spacer (ITS) region. The identity of Nostoc as the symbiont was confirmed in all the infected rhizome tissue analysed. Strains isolated in the present study were closely related to strains known to form symbioses with other organisms, such as lichen-forming fungi or bryophytes. We found 12 unique haplotypes in the 16S rRNA (small subunit) region analysis, 19 unique haplotypes in the ITS region analysis and 57 in the RuBisCO proteins region (rbcLX). No genetic variability was found among Nostoc symbionts within a single host plant while Nostoc populations among different host plants within a given sampling site revealed major differences. Noteworthy, interpopulation variation was also shown between recently deglaciated soils and more ancient ones, between eastern and western sites and between northern and southern slopes of Cordillera Darwin. The cell structure of the symbiotic relationship was observed with low-temperature scanning electron microscopy, showing changes in morphology of both cyanobiont cells (differentiate more heterocysts) and plant cells (increased size). Developmental stages of the symbiosis, including cell walls and membranes and EPS matrix states, were also observed.

Effect of copper and lead on two consortia of phototrophic microorganisms and their capacity to sequester metals.

The roles of consortia of phototrophic microorganisms have been investigated in this paper to determine their potential role to tolerate or resist metals and to capture them from polluted cultures. With this purpose, two consortia of microorganisms: on one hand, Geitlerinema sp. DE2011 (Ge) and Scenedesmus sp. DE2009 (Sc) (both identified in this paper by molecular biology methods) isolated from Ebro Delta microbial mats, and on the other, Spirulina sp. PCC 6313 (Sp) and Chroococcus sp. PCC 9106 (Ch), from Pasteur culture collection were polluted with copper and lead. In order to analyze the ability of these consortia to tolerate and capture metals, copper and lead were selected, because both have been detected in Ebro Delta microbial mats. The tolerance-resistance to copper and lead for both consortia was determined in vivo and at cellular level by Confocal Laser Scanning Microscopy (CLSM-λscan function). The results obtained demonstrate that both consortia are highly tolerant-resistant to lead and that the limits between the copper concentration having cytotoxic effect and that having an essential effect are very close in these microorganisms. The capacity of both consortia to capture extra- and intracellular copper and lead was determined by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) respectively, coupled to an Energy Dispersive X-ray detector (EDX). The results showed that all the microorganisms assayed were able to capture copper extracellularly in the extrapolymeric substances, and lead extra- and intracellularly in polyphosphate inclusions. Moreover, the studied micro-organisms did not exert any inhibitory effect on each others metal binding capacity. From the results obtained in this paper, it can be concluded that consortia of phototrophic microorganisms could play a very important role in biorepairing sediments polluted by metals, as a result of their ability to tolerate or resist high concentrations of metals and to bioaccumulate them, extra- and intracellulary.

Erratum to: Diversity of Endosymbiotic Nostoc in Gunnera magellanica from Tierra del Fuego, Chile

Industrial units, manufacturing dyes, chemicals,solvents, and xenobiotic compounds, produce liquid and solid wastes, which upon conventional treatment are released in the nearby environment and thus are the major cause of pollution. Soil collected from contaminated Kharicut Canalbank (N 22°57.878′; E 072°38.478′), Ahmeda bad, Gujarat,India was used for metagenomic DNA preparation to study the capabilities of intrinsic microbial community in dealing with xenobiotics. Sequencing of metagenomic DNA on the Genome Sequencer FLX System using titanium chemistry resulted in 409,782 reads accounting for 133,529,997 bases of sequence information. Taxonomic analyses and gene annotations were carried out using the bioinformatics platform Sequence Analysis and Management System for Metagenomic Datasets. Taxonomic profiling was carried out by three different complementary approaches: (a) 16S rDNA, (b) environmental gene tags, and (c) lowest common ancestor. The most abundant phylum and genus were found to be “Proteobacteria”and “Pseudomonas,” respectively. Metagenome reads were mapped on sequenced microbial genomes and the highest numbers of reads were allocated to Pseudomonas stutzeri A1501. Assignment of obtained metagenome reads to Gene Ontology terms, Clusters of Orthologous Groups of protein categories, protein family numbers, and Kyoto Encyclopedia of Genes and Genomes hits revealed genomic potential of indigenous microbial community. In total, 157,024 reads corresponded to 37,028 different KEGG hits, and amongst them, 11,574 reads corresponded to 131 different enzymes potentially involved in xenobiotic biodegradation. These enzymes were mapped on biodegradation pathways of xenobiotics to elucidate their roles in possible catalytic reactions. Consequently, information obtained from the present study will act as a baseline which, subsequently along with other“-omic” studies, will help in designing future bioremediation strategies in effluent treatment plants and environmental cleanup projects.