Virginia Souza-Egipsy

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.

Low-Temperature Growth of Shewanella oneidensis MR-1

ABSTRACT Shewanella oneidensis MR-1 is a mesophilic bacterium with a maximum growth temperature of ≈35°C but the ability to grow over a wide range of temperatures, including temperatures near zero. At room temperature (≈22°C) MR-1 grows with a doubling time of about 40 min, but when moved from 22°C to 3°C, MR-1 cells display a very long lag phase of more than 100 h followed by very slow growth, with a doubling time of ≈67 h. In comparison to cells grown at 22°C, the cold-grown cells formed long, motile filaments, showed many spheroplast-like structures, produced an array of proteins not seen at higher temperature, and synthesized a different pattern of cellular lipids. Frequent pilus-like structures were observed during the transition from 3 to 22°C.

Geomicrobiology of La Zarza-Perrunal Acid Mine Effluent (Iberian Pyritic Belt, Spain)

ABSTRACT Effluent from La Zarza-Perrunal, a mine on the Iberian Pyrite Belt, was chosen to be geomicrobiologically characterized along a 1,200-m stream length. The pH at the origin was 3.1, which decreased to 1.9 at the final downstream sampling site. The total iron concentration showed variations along the effluent, resulting from (i) significant hydrolysis and precipitation of Fe(III) (especially along the first reach of the stream) and (ii) concentration induced by evaporation (mostly in the last reach). A dramatic increase in iron oxidation was observed along the course of the effluent [from Fe(III)/Fetotal = 0.11 in the origin to Fe(III)/Fetotal = 0.99 at the last sampling station]. A change in the O2 content along the effluent, from nearly anoxic at the origin to saturation with oxygen at the last sampling site, was also observed. Prokaryotic and eukaryotic diversity throughout the effluent was determined by microscopy and 16S rRNA gene cloning and sequencing. Sulfate-reducing bacteria (Desulfosporosinus and Syntrophobacter) were detected only near the origin. Some iron-reducing bacteria (Acidiphilium, Acidobacterium, and Acidosphaera) were found throughout the river. Iron-oxidizing microorganisms (Leptospirillum spp., Acidithiobacillus ferrooxidans, and Thermoplasmata) were increasingly detected downstream. Changes in eukaryotic diversity were also remarkable. Algae, especially Chlorella, were present at the origin, forming continuous, green, macroscopic biofilms, subsequently replaced further downstream by sporadic Zygnematales filaments. Taking into consideration the characteristics of this acidic extreme environment and the physiological properties and spatial distribution of the identified microorganisms, a geomicrobiological model of this ecosystem is advanced.

Extracellular matrix assembly in extreme acidic eukaryotic biofilms and their possible implications in heavy metal adsorption

To evaluate the importance of the extracellular matrix in relation to heavy metal binding capacity in extreme acidic environments, the extracellular polymeric substances (EPS) composition of 12 biofilms isolated from Río Tinto (SW, Spain) was analyzed. Each biofilm was composed mainly by one or two species of eukaryotes, although other microorganisms were present. EPS ranged from 130 to 439 mg g(-1) biofilm dry weight, representing between 15% and the 40% of the total biofilm dry weight (DW). Statistically significant differences (p<0.05) were found in the amount of total EPS extracted from biofilms dominated by the same organism at different sampling points. The amount of EPS varied among different biofilms collected from the same sampling location. Colloidal EPS ranged from 42 to 313 mg g(-1) dry weight; 10% to 30% of the total biofilm dry weight. Capsular EPS ranged from 50 to 318 mg g(-1) dry weight; 5% to 30% of the total biofilm dry weight. Seven of the 12 biofilms showed higher amounts of capsular than colloidal EPS (p<0.05). Total amount of EPS decreased when total cell numbers and pH increased. There was a positive correlation between EPS concentration and heavy metal concentration in the water. Observations by low temperature scanning electron microscopy (LTSEM) revealed the mineral adsorption in the matrix of EPS and onto the cell walls. EPS in all biofilms were primarily composed of carbohydrates, heavy metals and humic acid, plus small quantities of proteins and DNA. After carbohydrates, heavy metals were the second main constituents of the extracellular matrix. Their total concentrations ranged from 3 to 32 mg g(-1) biofilm dry weight, reaching up to 16% of the total composition. In general, the heavy metal composition of the EPS extracted from the biofilms closely resembled the metal composition of the water from which the biofilms were collected.

Water distribution within terricolous lichens revealed by scanning electron microscopy and its relevance in soil crust ecology

The effects of the structural features of the thallus and the lichen-substratum interface on soil water infiltration were investigated using low temperature scanning electron microscopy and scanning electron microscopy with backscattered electron imaging. Two morphologically different lichen species were examined, the crustose Diploschistes diacapsis and the squamulose Squamarina lentigera. Water distribution within lichen-forming soil crusts explains the results of earlier infiltration studies. Several anatomical features of the upper cortex gave rise to similar impervious characteristics of the thallus. The fungal material comprising the lichen thallus showed significant hydrophobic features and only when in contact with calcium oxalate crystals or minerals in the soil was this material able to retain water. The lichen-substratum interface appears to play an important role in the hydric strategy of these lichens which in turn affects the penetration of water into the soil.

Micrometer-scale porosity as a biosignature in carbonate crusts

We formed calcite crusts in the presence and absence of the heterotrophic bacterium Desulfovibrio desulfuricans strain G20 to investigate microbial morphological signatures in fast-accreting carbonate precipitates. Submicrometer- to micrometer-sized pores (micropores) were present and ubiquitous in the G20 crusts but absent in abiotically precipitated crusts. Bacterial micropores resemble inclusions under transmitted light, but have distinct size, biological shapes and patterns (swirling or dendritic) and are distributed differently from common fluid inclusions. We observed similar porosity in both modern and ancient carbonate crusts of putative biotic origin. Our experiments support the microbial origin of micropores and help define specific criteria whereby to recognize these features as biosignatures in the rock record.

Adaptation strategies of endolithic chlorophototrophs to survive the hyperarid and extreme solar radiation environment of the Atacama Desert

The Atacama Desert, northern Chile, is one of the driest deserts on Earth and, as such, a natural laboratory to explore the limits of life and the strategies evolved by microorganisms to adapt to extreme environments. Here we report the exceptional adaptation strategies of chlorophototrophic and eukaryotic algae, and chlorophototrophic and prokaryotic cyanobacteria to the hyperarid and extremely high solar radiation conditions occurring in this desert. Our approach combined several microscopy techniques, spectroscopic analytical methods, and molecular analyses. We found that the major adaptation strategy was to avoid the extreme environmental conditions by colonizing cryptoendolithic, as well as, hypoendolithic habitats within gypsum deposits. The cryptoendolithic colonization occurred a few millimeters beneath the gypsum surface and showed a succession of organized horizons of algae and cyanobacteria, which has never been reported for endolithic microbial communities. The presence of cyanobacteria beneath the algal layer, in close contact with sepiolite inclusions, and their hypoendolithic colonization suggest that occasional liquid water might persist within these sub-microhabitats. We also identified the presence of abundant carotenoids in the upper cryptoendolithic algal habitat and scytonemin in the cyanobacteria hypoendolithic habitat. This study illustrates that successful lithobiontic microbial colonization at the limit for microbial life is the result of a combination of adaptive strategies to avoid excess solar irradiance and extreme evapotranspiration rates, taking advantage of the complex structural and mineralogical characteristics of gypsum deposits—conceptually called “rocks habitable architecture.” Additionally, self-protection by synthesis and accumulation of secondary metabolites likely produces a shielding effect that prevents photoinhibition and lethal photooxidative damage to the chlorophototrophs, representing another level of adaptation.

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.

Structure and temporal dynamics of the bacterial communities associated to microhabitats of the coral Oculina patagonica

Corals are known to contain a diverse microbiota that plays a paramount role in the physiology and health of holobiont. However, few studies have addressed the variability of bacterial communities within the coral host. In this study, bacterial community composition from the mucus, tissue and skeleton of the scleractinian coral Oculina patagonica were investigated seasonally at two locations in the Western Mediterranean Sea, to further understand how environmental conditions and the coral microbiome structure are related. We used denaturing gradient gel electrophoresis in combination with next-generation sequencing and electron microscopy to characterize the bacterial community. The bacterial communities were significantly different among coral compartments, and coral tissue displayed the greatest changes related to environmental conditions and coral health status. Species belonging to the Rhodobacteraceae and Vibrionaceae families form part of O. patagonica tissues core microbiome and may play significant roles in the nitrogen cycle. Furthermore, sequences related to the coral pathogens, Vibrio mediterranei and Vibrio coralliilyticus, were detected not only in bleached corals but also in healthy ones, even during cold months. This fact opens a new view onto unveiling the role of pathogens in the development of coral diseases in the future.

Calcium Phosphate Preservation of Faecal Bacterial Negative Moulds in Hyaena Coprolites

The vertebrate fossil locality of La Roma 2, Spain (Upper Miocene, Late Vallesian, MN10) is characterised by a high abundance of mammalian coprolites, which provide direct clues to the diets and habitats of the organisms that produced them. X-ray diffraction analysis showed a sample of hyaena (cf. Lycyaena chaeretis) coprolites to be mostly composed of calcium phosphate. Ultrastructural SEM and TEM studies revealed three successive phases of preservation, including an initial phase of mineralisation that produced microspherulites within a very fine-grained cement. This indicates that most of the calcium phosphate present in the coprolites precipitated rapidly, which in turn facilitated the formation of negative moulds of faecal bacteria within the coprolite matrix.