Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.) Biological nanostructures associated to iberulites: a SEM study. J.L. Díaz-Hernández1, P.J. Sánchez-Soto2 and A. Serrano-Delgado3. 1 IFAPA Camino de Purchil, Granada, Área de Recursos Naturales, Consejería de Agricultura, Pesca y Medio Ambiente, Junta de Andalucia, Spain. E-mail josel.diaz@juntadeandalucia.es 2 Instituto de Ciencia de los Materiales de Sevilla (ICMS), CSIC-Universidad de Sevilla, Centro de Investigaciones Científicas Isla de La Cartuja de Sevilla, c/Américo Vespucio 49, 41092 Sevilla, Spain. E-mail: pedroji@icmse.csic.es 3 Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF), CSIC-Universidad de Sevilla, Centro de Investigaciones Científicas Isla de La Cartuja de Sevilla, c/Américo Vespucio 49, 41092 Sevilla, Spain. E-mail: aurelio@ibvf.csic.es Scanning Electron Microscopy (SEM) is a reliable technique by which geological objects can be screened with magnification ranges between that of optical microscopy and the nanoscale. It is, therefore, suitable for the study of iberulites, which are pinkish mineral microspherules, formed and structured in the troposphere, and composed of complex mineral associations whose phases have diverse hygroscopic properties. These mineral phases are mainly silicates, carbonates, sulphates, halides, oxides and phosphates. Other minor but not less important compounds are the biological constituents. The sources of such heterogeneous materials are the soils of North Africa which, once dispersed into the atmosphere (plumes), undergo physico-chemical transformations resulting in the formation of iberulites. Here we report a microscopy research study that analyzes the biological nanostructures associated to iberulites. A prior selection of iberulite samples was carried out using optical microscopy. Because the material forming iberulites comes from the neighbouring aerosols, their biological constituents should be, a priori, qualitatively similar. However the processes undergone by the iberulites, and their properties, can cause some differences resulting from the role played by these microenvironments as a physical support that facilitates microbial survival and spreading. Dust plumes can have high relative concentrations of remains of composited organisms (plants and diverse microorganisms) which signify additional loads of viable, yet dormant, biological specimens (bacterial and fungal spores, algal cysts, plant pollen grains) which move with these plumes. These studies, together with future metagenomic data, will improve our knowledge of the biodiversity of microbial communities integrated in aerolites, a particular class of microenvironments with global ecological projection. In addition, they will contribute to establish well-founded strategies for environmental prevention and public health in connection with this issue. Keywords aerosols; biological nanostructures; iberulites; Saharan plumes; SEM 1. Introduction European air quality guidelines reflect great concern about so-called particulate matter, which is a heterogeneous mixture of solid particles and liquid droplets found in the atmosphere. This concern is founded on the increased knowledge of the impact these particles have on agriculture, the environment and human health [1, 2]. The main sources of particles are natural (sea salts and soil mineral particles) and anthropogenic activity (fuel combustion from automobiles and power plants, industrial processes and wood burning). Although some atmospheric dust constituents can have a beneficial effect as organic or inorganic nutrients [3, 4], others may represent potential risks (salts, microorganisms) [5]. Dust travels through the troposphere from frequently distant source areas to recipient areas, often with very different features. This type of dust transport mainly occurs during the spring and summer either as intensive events (plumes) or as more discreet phenomena, caused when hot air masses are driven towards more northern latitudes from North Africa, where the Sahara desert and its surrounding vicinity represent the biggest dust producing area in the world (170-760 109 kg yr-1 [6]). The main recipient areas of this dust flow are the circum-Mediterranean areas. The specific values of dust deposit in these areas range from 79g m-2 yr-1 in the Canary Islands [7] to 23g m-2 yr-1 in south-eastern Spain [8]. Because the Iberian Peninsula can receive this flow both directly and via the Atlantic Ocean, it is one of the most ideal zones in the world for the study of these processes. Dust transport processes the matter involved in the flow by particle-size selection, abrasion, wetting-drying cycles and chemical interaction between heterogeneous phases. The aerodynamic processing of these particles is of particular interest since it is the cause of their aggregation, resulting in the formation of iberulites (Fig. 1) [9]. Biological material [10] is processed in the same way as the other particles, but the dusty environment can often be aggressive [11, 12] and specimens can be partially destroyed, resulting in taphocenosis consisting of more or less disperse biological remains, each usually over 1µm in size. Biological remains larger than this are normally broken, except in the case of external skeletal structures of organic and inorganic compositions (microbial cell walls, algal frustules, protistan tests, loricas of small animals, etc.) that in many cases are remarkably resistant to physico-chemical © 2012 FORMATEX 154 Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.) stress. This could explain the abundance of these types of remains in iberulites, as described in this study (see below). Although physical processes may be inefficient in the case of submicrometer and nanometer sized biological particles (e.g., small prokaryotic microbes and viruses), survival of these microbial specimens may also be endangered during transport by physico-chemical agents. In this sense, many studies of atmospheric dust samples have determined the broad spectra of the biological content [13, 14], while others establish that microbes adhered to dust can live for centuries and, in the case of Saharan dust, easily survive transport across the Atlantic [15, 16]. Fig. 1 SEM image of a group of iberulites. Observe the spherical shape, the range of sizes, granular aspect and the presence and shape of the vortexes (arrows). Two halves of a split iberulite can be seen in the middle-lower area and show that the interior consists of aggregated mineral particles. Each iberulite is built around a granular core and has a thin smectite rind; the maximum development of the rind is near the vortex. This zone has a smooth appearance, while the other side is more granular. Mineral particles (mainly silicates, carbonates, sulphates, halides, oxides and phosphates) are weakly linked, as we can see by the detached mineral particles around the iberulites. Based only on SEM analysis, this chapter includes a selection of the most representative biological nanostructures found inside or in connection with iberulites included in Saharan dust events in European environments. This research was conducted from an interdisciplinary viewpoint. 2. Procedures 2.1 Sampling The samples (iberulites and aerosols) were collected at a monitoring station located in the south-eastern Iberian Peninsula (Granada Depression, 37º10’N-3º31’W, 640m asl). The area has a mean annual temperature of 15.1ºC, with mean precipitation of 357mm distributed over 52 rainy days throughout the year (data from Granada airport, Spanish Agencia Estatal de Meteorología, AEMET). For the sampling we followed the methodology described by Díaz-Hernández and Miranda-Hernández (1997) [8]. The passive collectors used were 50mm deep, circular porcelain trays (143mm radius) for measurement of dust deposition rates. Sampling was generally carried out once a week in order to obtain operational quantities for analysis and to associate these samples to specific atmospheric conditions as determined by satellite information and AEMET synoptic maps. To date we have accumulated over 300 samples characterizing this flow of matter over a wide range of formational circumstances. For the purposes of this chapter we examined a small selection of the iberulites contained in these samples. Each image has a numerical italic label indicating year-month-day of collection. 2.2 Optical observations Sample trays were examined using an Olympus SZ-10 stereo microscope with oblique illumination to determine whether the sample contained any iberulites. Samples were stored in labelled glass vials (2cc), and the iberulites were carefully separated by hand from the surrounding matter (dust) using a very fine pin to avoid contamination or breakage of the sample. 2.3 Scanning electron microscopy Samples and equipment were set up according to the specific aim of the analysis [17, 18]. SEM examination of the samples was conducted using a JEOL JSM-5400 scanning electron microscope and chemical analysis under the microscope using an Oxford Link energy dispersive ISIS X-ray detector (EDX) with Si (Li) detector and thin Be window. The filament for the electronic beam source was W at 20kV maximum. After initial examination under optical microscope, samples were fixed with graphite adhesive and gold coated using an Emitech © 2012 FORMATEX 155 Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.) 550 Sputter Coater. It should be noted that gold coating provides better resolution and is more frequently used in SEM studies than carbon coating, although some interferences between element peaks can be avoided using the latter. Figure 1 shows a typical SEM image of a group of iberulites. 3. Results and discussion The following paragraphs describe the features of the specimens obtained in our samplings, ordered by size. 3.1 Plant material This type of biological material is the largest in size and is sometimes visible to the naked eye at almost 1mm. Iberulites can frequently be found formed around this material. The vegetal filaments occasionally conserve their original tubular structure, or spiral endings (Fig. 2A, C). Iberulites can often have black speckles on their surface corresponding to remains of burnt plant material. Another type of material in this category is pollen, a fine powder-like material formed by microscopic grains produced by the anthers of seed plants. The quantity and type of pollen grains vary widely and constitute a particular subject beyond the scope of this chapter. However, it is relevant to note that although they vary considerably in size (from 10 to 100µm approximately), only smaller specimens actually become associated to iberulites during assemblage and transport. They were collected mainly in spring, mixed with local specimens (Fig. 2G, H). However iberulites can include pollen grains from the same source as the mineral load (Fig. 2E). Globular pollen grains sometimes adhere to iberulites as aerial floats (Fig. 2F). Generally speaking, this material is of interest because it is a type of biological remains that can support other microbial species. Moreover, pollen grains release extensively allergenic proteins into the surrounding media, so their characterization is important to properly evaluate a possible role of iberulites as a promoter of allergic diseases. Fig. 2 SEM images of different plant material related with iberulites. Plant filaments: A) Long plant filament with a spiral end. B) Detail of filament insertion in the iberulite: this area facilitates recrystallization phenomena (neoformation of minerals). C) Tubular appearance of the plant filament and aggregation of the mineral particles around it. D) Iberulite deformed by an interior filament. Pollen: E) Pollen grain enclosed in an iberulite, probably from source area. F-G) Two cases of pollen grains adhered to the mineral surface of iberulites. H) Several types of pollen grains possibly corresponding to local plants (Olea and Pinus, among others). 3.2 External skeletons of eukaryotic microorganisms These biological nanostructures are frequently observed by SEM, even in some cases as main structural components of iberulites. We have observed remains of three different major types of external skeletons based on their composition: siliceous, chitinous and calcified. The biological sources of these structures are mainly eukaryotic microorganisms (photosynthetic or heterotrophic protists, lower metazoa). Biological remains of higher plants or higher metazoa are rare but can also be found (see below). © 2012 FORMATEX 156 Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.) Siliceous skeletons Siliceous skeletons constitute an important group because of their comparatively high frequency [19, 20], and are represented by siliceous diatom frustules with an elaborate nanostructure In fact, their nanostructures are important in diatom taxonomy. Diatoms make up an extensive group of photosynthetic unicellular eukaryotes (photosynthetic protists or microalgae) containing about 100,000 species. They belong to the so-called Heterokonts (or Stramenopiles) supergroup, division Chrysophyta, class Bacillariophyceae, and form an important part of the phytoplankton at the base of the marine and freshwater food chains [21]. The characteristic feature of diatoms is their intricately patterned, glasslike (siliceous) but rigid cell wall, or frustule, which often has rows of tiny holes, known as striae. Attending to the main structural features of the frustule, they are classified into two main orders, namely Centrales (radial symmetric centric diatoms) and Pennales (bilaterally symmetric pennate diatoms). Centric diatoms dominate marine plankton communities and pennate diatoms are often found in benthic marine and fresh water communities. Fig. 3 SEM images of several types of external siliceous skeletons (frustules) from photosynthetic microalgae (diatoms). A) Remain of the siliceous skeleton of a diatom. B) Remains of the siliceous frustule of a centric diatom (Melosira-like). C) Remains of the broken frustule of a centric diatom (Melosira-like). D) Remains of frustules from centric diatoms, probably of marine origin. This biological material is so abundant that it makes up the basic physical structure of that particular iberulite, suggesting a colony of diatoms as the source. This type of material may be useful for future metagenomic studies. Inset presents an EDX spectrum indicating the Si-rich elemental composition of this material. E) Remains of centric diatoms enclosed in an iberulite, viewed across the vortex. The observed SEM morphologies of the frustules mainly correspond with perforated micro-plates or tubules (Fig. 3A-C). The siliceous nature makes them very robust and they can withstand harsh conditions; the typical nanostructures of diatoms give them great lightness facilitating aerial transport. They are generally included inside the iberulites, sometimes in great quantities (Fig. 3D). They are quite resistant to physico-chemical stresses and even complete frustules can be found (Fig. 3E). The siliceous nature of these structures was confirmed by spectral EDX analyses, showing Si as a major component (Fig. 3D, inset). Sometimes, diatom frustules are so abundant that they become the structural basis of iberulites. This is not unexpected, as siliceous diatom tests can be important structural components of microbial mat ecosystems. In any case, all frustules found in this study have radial symmetry and so are clearly from centric diatoms, probably of the genera Melosira (Eukaryota; stramenopiles; Bacillariophyta; Coscinodiscophyceae; Coscinodiscophycidae; Melosirales; Melosiraceae) or Thalassiosira (Eukaryota; stramenopiles; Bacillariophyta; Coscinodiscophyceae; Thalassiosirophycidae; Thalassiosirales; Thalassiosiraceae). The source of these microalgae could be various Saharan areas or the Atlantic Ocean, but since they are common components of iberulites, regardless of the trajectories of the dust plumes, we believe they came from desert soils. The fact that we only found remains of centric diatoms and not pennate diatoms in the iberulites examined here agrees with © 2012 FORMATEX 157 Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.) the geological histories of these dust particles generated by accretion of aerosols whose materials originated in soils, continental aquatic masses and/or the surface of the sea. Chitinous skeletons External chitinous skeletons are a group of biological nanostructures mainly from microbial sources and are quite frequently found in iberulites. Fungi are the main source of these chitinous nanostructures. They form a large group (ca. 60,000 species) of absorptive heterotrophic eukaryotes including microorganisms such as yeasts and molds. Some are saprophytes, while others are parasites or form symbiotic associations such as lichens [22]. It is relatively frequent for iberulites to contain hyphae of fungal mycellia (Fig. 4A) and unicellular fungal spores with rough surfaces richly decorated in diverse patterns (Fig. 4B-E). The outer walls of fungal spores, made of chitin networks and glucomannans, are rigid structures very resistant to physico-chemical stress, which protect the inner, yet viable, dormant cells. These structures aid reproduction by efficiently dispersing fungal species, due to their remarkably aerodynamic dragminimizing shapes. Fungal spores release biochemical compounds such as proteins including allergens. These biological particles are a well-known cause of allergy and asthma when inhaled, and allergy to these spores is an important cause of severe seasonal diseases. Thus, identification of fungal specimens associated to iberulites is a critical issue to properly understand their possible relevance for public-health affairs. Fig. 4 SEM images of frequent biological nanostructures of fungal origin. A) Fragment of a fungal hypha from a mycellium with spores. Circular structures at the hyphal surface could be small protospores, in the process of being generated. B) Possible fungal spore (Basidiomycete, allergenic mold spore). C) Fungal oblong spore (allergenic mold spore) possibly a Cladosporium conidiospore. D) Fungal disc-shaped spores possibly of a Basidiomycete fungus (basidiospore). E) Possible fungal spherical spore of an Ascomycete fungus (allergenic mold spore). F) Possible fungal spherical spore. On some rare occasions, chitinous nanostructures from arthropods (metazoa) can be found, corresponding to different parts of insect exoskeletons (Fig. 5A). Calcified skeletons External calcified skeletons are another group of biological nanostructures observed in iberulites by SEM, although less frequently. They are plates, tests or loricas of microbial origin, mainly from unicellular eukaryotic specimens (protists), which are either photosynthetic (Coccolitophores, such as the Haptophycean microalga Emiliania) or heteretrophic (such as testate amoebae). Many of these microscopic shells are largely composed of calcium carbonate adsorbed to a proteinaceous matrix. It is necessary to use mapping methods to detect them due the calcitic composition of many iberulite grains. © 2012 FORMATEX 158 Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.) Fig. 5. SEM images of infrequent nanostructures of diverse biological origin. A) Probable extremity of an insect (arthropod) included in an iberulite. B) The quite large size (ca. 50µm) of this probably calcified fusiform lorica or test-like remains suggests either a lobose testated amoeba (Arcellimidae) or a small loricated rotifer (lower metazoan), as the possible biological source. B1) Detail of the operculum of that fusiform structure. An unusual fusiform structure of a quite big size (ca. 50µm) which is probably a calcified lorica or test was found (Fig. 5B and B1), its features suggest a single-celled testate lobose amoeba (Arcellimidae, Rhizaria), or a small loricate rotifer (multicellular lower metazoan), as the possible biological source. Lobose amoebae are distributed worldwide in both freshwater and marine habitats and are especially common in soil. They are unusually large unicellular eukaryotic microorganisms (up to 100µm) and many are symbiotrophic in animals [23]. 3.3 Bacterial cells and bacterial spores Bacteria and Arqueae are single celled prokaryotes, with no organelles and no defined nucleus. They are quite similar in size (usually 1-2µm) and shape. These microorganisms are two major domains of life, making up the major fraction of biomass on the planet. They are not found frequently in iberulites, but when they are they appear in association with eukaryotic cell remains. Figure 6 shows assorted biological nanostructures of possible prokaryotic origin, as observed by SEM. Fig. 6 SEM images of microbiological nanostructures of possible bacterial origin. A) Helical nanostructures (2-3 µm) suggesting bacterial spores of unusual forms. B) Frustule remains of centric diatoms, probably of marine origin. B1) Detail of previous image showing possible rod-shaped bacterial cells (bacilli) associated to diatoms (arrow). The cluster of helical or spiral specimens shown in Fig. 6A is particularly interesting, with sizes (2-3µm in diameter) that fit well with those of bacterial cells. They may be bacterial spores with unusual helical forms. It should be noted that spiral spores have been reported for a number of Gram + bacteria (Actinomycetales) [24]. Chains of possible rodshaped bacterial cells (bacilli) associated to frustules of centric diatoms are presented in Fig. B1-B. 3.4 Viruses Because of their small nanometre-scale sizes and biochemical composition, viral particles are not expected to be present in iberulites as free particles, except when parasitizing other biological structures. They were not observed by SEM in these samples, although small virus-like particles have previously been reported in these habitats [7]. © 2012 FORMATEX 159 Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.) 4. Concluding remarks Scanning Electron Microscopy (SEM) is suitable for the study of iberulites, a type of mineral microspherules formed and structured in the troposphere, and composed of complex mineral associations whose phases have diverse hygroscopic properties. Other minor but no less important compounds are the biological constituents. These heterogeneous materials come from the soils of North Africa which, when dispersed into the atmosphere as plumes, undergo physico-chemical transformations resulting in the formation of iberulites. This microscopy study analyzes the biological nanostructures associated to iberulite samples previously selected using optical microscopy. Because the material forming iberulites comes from surrounding aerosols, their biological constituents should be a priori qualitatively similar. However properties and processes undergone by the iberulites can cause some differences resulting from the role played by these microenvironments as a physical support facilitating microbial survival and spreading. Dust plumes can have high relative concentrations of remains of composite organisms (plants and diverse microorganisms) representing additional loads of viable, yet dormant, biological specimens (bacterial and fungal spores, algal cysts, plant pollen grains) moving with these plumes. These additions mean that the circum-Mediterranean regions where the dust is deposited are both extensively fertilized with minerals and inoculated with Saharan desert microorganisms. The biological nanostructures examined by SEM in iberulites showed limited biodiversity, probably resulting from the demanding physico-chemical conditions associated with the formation and transport of these particular microenvironments. They consist of rigid structures of microbial origin -mainly mineralized external skeletons from photosynthetic and heterotrophic protists (Centrales and Pennales orders), and fungal spore walls- and from higher plants, mainly filaments and pollen grains. Most of them should be remains of non-viable specimens, but in some cases the dormant cells may still be alive. Dead or alive, these specimens could be a source of biological compounds (i.e., allergens) that may become serious issues for environmental and public health affairs. Present and future research, including metagenomic data, will increase our knowledge of the biodiversity of microbial communities contained in iberulites, a particular class of microenvironments with global ecological projection. In addition, they will contribute to establish well-founded strategies for environmental prevention and public health in connection with this issue. Acknowledgements The experimental contribution and support by Dr. MC Jiménez de Haro is gratefully acknowledged. This research was partially supported by Regional Government of Andalusia, Spain (Consejería de Innovación, Ciencia y Empresa, PAIDI groups CVI-261 (IBVF), TEP-204 (ICMS) and CTS-946 (IACT)). References [1] Cook AG, Weinstein P, Centeno JA. Health effects of natural dust—role of trace elements and compounds. Biological Trace Element Research. 2005;103:1-15. [2] Dowd SE, Maier RM. Aeromicrobiology. Academic Press, San Diego, CA; 2000. [3] Soderberg K, Compton JS. 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