Ultrastructural and Biogeochemical Features of Microbiotic Soil Crusts and their Implications in a Semi-Arid Habitat

This investigation was designed to explore the relationships between lichen symbionts (phycobiont and mycobiont) and the substrate on which they grow by examining the chemical and ultrastructural features of the lichen-soil interface. These lichens form an integral part of microbiotic soil crusts. Fragments of three different lichen biotypes growing over gypsum crystals and marls were fixed and embedded in resin. The lichen-substratum interface was then examined by scanning electron microscopy with backscattered electron imaging. In situ observation, microanalytical (EDS), and FT-Raman plus infrared spectroscopy of the lichen-substratum interface indicated that different ultrastructural features of the mycobiont were related to biogeochemical processes and Ca 2+ distribution in the soil crust. Phycobionts were observed to make direct contact with the substratum and to be surrounded by a nondifferentiated thallus structure. These observations suggest that they can grow outside the thallus in the early stages of lichen development in the semi-arid conditions of their habitat. The particular ultrastructural features of the lichen thallus and of the lichen-substratum interface appear to have marked effects on runoff phenomena and ponding generation of the surface.     

Fossilization of Acidophilic Microorganisms

This study examines fossil microorganisms found in iron-rich deposits in an extreme acidic environment, the Tinto River in SW Spain. Both electron microscopy (SEM and TEM) and non-destructive in situ microanalytical techniques (EDS, EMP and XPS) were used to determine the role of permineralization and encrustation in preserving microorganisms forming biofilms in the sediments. Unicellular algae were preserved by silica permineralization of their cell walls. Bacterial biofilms were preserved as molds by epicellular deposition of schwertmannite around them. In the case of fungi and filamentous algae, we observed permineralization of cell structures by schwertmannite in the sediments. The extracellular polymeric matrix around the cells was also preserved through permineralization of the fibrillar component. The process of permineralization and deposition of iron-rich precipitates present in the acidic waters of Rio Tinto served to preserve many microfossils in an oxidizing environment, in which organic compounds would not normally be expected to persist. Studies of microbial fossil formation mechanisms in modern extreme environments should focus on defining criteria to identify inorganic traces of microbial life in past environments on Earth or other planets.     

Reproduction and metabolism at - 10 degrees C of bacteria isolated from Siberian permafrost

We report the isolation and properties of several species of bacteria from Siberian permafrost. Half of the isolates were spore-forming bacteria unable to grow or metabolize at subzero temperatures. Other Gram-positive isolates metabolized, but never exhibited any growth at - 10 degrees C. One Gram-negative isolate metabolized and grew at - 10 degrees C, with a measured doubling time of 39 days. Metabolic studies of several isolates suggested that as temperature decreased below + 4 degrees C, the partitioning of energy changes with much more energy being used for cell maintenance as the temperature decreases. In addition, cells grown at - 10 degrees C exhibited major morphological changes at the ultrastructural level.     

Photosynthetic performance of phototrophic biofilms in extreme acidic environments

Photosynthesis versus irradiance curves and their associated photosynthetic parameters from different phototrophic biofilms isolated from an extreme acidic environment (Río Tinto, SW, Spain) were studied in order to relate them to their species composition and the physicochemical characteristics of their respective sampling locations. The results indicated that the biofilms are low light acclimated showing a photoinhibition model; only floating communities of filamentous algae showed a light saturation model. Thus, all the biofilms analysed showed photoinhibition over 60 μmol photon m(-2) s(-1) except in the case of Zygnemopsis sp. sample, which showed a light-saturated photosynthesis model under irradiations higher that 200 μmol photon m(-2) s(-1). The highest values of compensation light intensity (I(c)) were showed also by Zygnemosis sp. biofilm (c. 40 μmol photon m(-2) s(-1)), followed by Euglena mutabilis and Chlorella sp. samples (c. 20 μmol photon m(-2) s(-1)). The diatom sample showed the lowest I(c) values (c. 5 μmol photon m(-2) s(-1)). As far as we know this is the first attempt to determine the photosynthetic activity of low pH and heavy metal tolerant phototrophic biofilms, which may give light in the understanding of the ecological importance of these biofilms for the maintenance of the primary production of these extreme and unique ecosystems.     

Low-temperature growth of Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a mesophilic bacterium with a maximum growth temperature of approximately 35 degrees C but the ability to grow over a wide range of temperatures, including temperatures near zero. At room temperature ( approximately 22 degrees C) MR-1 grows with a doubling time of about 40 min, but when moved from 22 degrees C to 3 degrees 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 approximately 67 h. In comparison to cells grown at 22 degrees 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 degrees C.     

Extraction of extracellular polymeric substances from extreme acidic microbial biofilms

The efficiency of five extraction methods for extracellular polymeric substances (EPS) was compared on three benthic eukaryotic biofilms isolated from an extreme acidic river, Río Tinto (SW, Spain). Three chemical methods (MilliQ water, NaCl, and ethylenediamine tetraacetic acid [EDTA]) and two physical methods (Dowex 50.8 and Crown Ether cation exchange resins) were tested. The quality and quantity of the EPS extracted from acidic biofilms varied according to which EPS extraction protocol was used. Higher amounts were obtained when NaCl and Crown Ether resins were used as extractant agents, followed by EDTA, Dowex, and MilliQ. EPS amounts varied from approximately 155 to 478 mg g⁻¹ of dry weight depending on the extraction method and biofilm analyzed. EPS were primarily composed of carbohydrate, heavy metals, and humic acid, plus small quantities of proteins and DNA. Neutral hexose concentrations corresponded to more than 90% of the total EPS dry weight. The proportions of each metals in the EPS extracted with EDTA are similar to the proportions present in the water from each locality where the biofilms were collected except for Al, Cu, Zn, and Pb. In this study, the extracellular matrix heavy metal sorption efficiencies of five methods for extracting EPS from eukaryotic acidic biofilms were compared.     

Low-Temperature Growth of Shewanella oneidensis MR-1

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)

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)/Fe(total) = 0.11 in the origin to Fe(III)/Fe(total) = 0.99 at the last sampling station]. A change in the O(2) 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.     

Development and Structure of Eukaryotic Biofilms in an Extreme Acidic Environment,Río Tinto (SW,Spain)

An in situ colonization assay was performed to study the early stages of biofilm formation in Río Tinto (SW, Spain), an extremely acidic environment (pH ca. 2). Eukaryotic assemblages were monitored at monthly intervals for 1 year. Diversity, colonization rates, and seasonal variations were analyzed. Structural features of naturally grown biofilms were explored by light and scanning electron microscopy in backscattered electron mode. A total of 14 taxa were recognized as constituents of the eukaryotic assemblages. The eukaryotic communities were dissimilar at the different sampling sites. The lowest diversity was found at the most extreme locations, in terms of pH and heavy metal concentrations. The biofilms were mainly formed by species from the genera Dunaliella and Cyanidium. Two genera of filamentous algae, Zygnemopsis and Klebsormidium, were principally responsible for the variability in the cell number throughout the year. These species appear in June to decrease almost completely between October and November. In contrast, the number of heterotrophic flagellates and ciliates remained constant throughout the year. The microcolonization sequence showed an initial accumulation of amorphous particles composed of bacteria and inorganic grains of minerals. By the end of the second month, the organic matrix was also populated by fungi, bacteria, and a few eukaryotic heterotrophs such as amoebae and small flagellates. Diatoms only showed significant colonization in regions where mycelial matrices were first established. Flagellated green algae such as Dunaliella or Chlamydomonas as well as Euglena were also present at the very beginning of the biofilm development, although in low numbers (<100 cells cm^–2). After the flagellated cells, sessile species of algae such Chlorella or Cyanidium appeared. Filamentous algae were the last species to colonize the biofilms. Most of the naturally grown biofilms were found to be structures composed of different species organized in different layers separated, probably by extracellular polymeric substances, although more analysis should be done in this regard. The possible implications of the biofilm structure in the adaptation to this extreme habitat are discussed.