Using micro-CT to explore pore contents in the ignimbrite, a volcanic rock in the Atacama Desert with

endolithic microbial communities: the microhabitat potentially expected on Mars

Javier Alba-Tercedor

1, Carmen Ascaso

2 and Jacek Wierzchos

2

1 Department of Zoology. Faculty of Sciences. University of Granada. Campus de Fuentenueva

s/n. 18071-Granada. Spain. 2 Museo Nacional de Ciencias Naturales – CSIC, 28006 Madrid, Spain

Aims The ignimbrites are volcanic rocks formed by a single pyroclastic flow, a hot suspension of particles and gases that flow rapidly from a volcano, driven by a greater density than the surrounding atmosphere. it was suggested that the interior of volcanic rocks may be ideal substrates for microbial habitability (Bagshaw et al., 2011), for the preservation of biosignatures (Cockell et al., 2011), and even for prebiotic chemistry (Brasier et al., 2011). Recently Wierzchos et al. (2013) have shown that ignimbrites from the Atacama Desert are abundantly colonized by endolithic cyanobacteria and associated heterotrophic bacteria. As this is the first known verification of an endolithic microbial community colonizing ignimbrite rocks in an extremely dry environment. They pointed out the importance of the discovery, because the existence of a habitat capable of supporting abundant phototrophic and heterotrophic communities in an environment that precludes most life forms suggests that, if similar deposits are found on Mars these should be considered important targets in the search for life. The existence in our lab of microtomograph facilities, encourages us to undertaken a micro-CT analysis of ignimbrite’s samples from Wierzchos et al. (2003) previous study. To explore pores and their contents, without manipulations that other techniques imply.

Materials and Methods Small samples pieces of ignimbrite were fixed to the sample holder with plasticiline, with the external surface upwards (Fig. 1a). Parallel to the surface, by naked eye it is possible to see under the surface a narrow green layer (thickness ca. 1mm thick) revealing the microorganism colonization (Fig. 1b).

Figure 1: Ignimbrite’ sample, fixed with plasticiline to the holder (a) It is possible to see, under the surface a narrow green layer (b) revealing the microorganism colonization (external surface appears marked with red ink).

a b

Samples were scanned with, with the micro-CT SkyScan 1172 C:. 0.5mm aluminum filter, Source Voltage = 64KV, Source Current = 100µA, and image Pixel Size = 1.4µm. Rotation step= 0.5º, Frame average=4, 360ºof Rotation; and conducting an oversized scan into with two parts. The Bruker-Skyscan free software (

®NRecon,

®CTan,

®DataViewer, and

® CTvox) was

used to reconstruct and process images, permitting to reconstruct and to get virtual slices and volume rendering reconstructions. No stain was used.

Results In figures 2 to 5, and 7, it is possible to see the obtained results. Moreover, videos can be viewed at:

1. http://youtu.be/LvO2NxMEfQE 2. http://youtu.be/G4z4WSh9img

Micro-CT volume rendering reconstructions of the ignimbrite, in grey color, visualized with CTVox software (Fig. 2), shown similar views that those obtained previously with scanning electron microscopy. Where the ignimbrite’s characteristic cavities are clearly visible. Moreover, close to the surface, corresponding with the microorganism’s colonization layer, cavities are partially filled with them. When visualizing “virtual” cuts of the samples, by using the software DataViewer, the cavities appear clearly visible. Some of them are filled with materials, in contrast with those appearing empties (Fig. 3). This results more evident when using transfer function colors within the CTVox software, permitting to get different colors according density of material. From red to blue, when the density, and thereafter the opacity to x-ray increases (Figs.: 4 & 5).

Figure 2: A comparison between micro-CT volume rendering reconstructions of the ignimbrite (a, b,

c), and SEM-BSE (SEM in Backscattered Electron detection mode) (d). Arrows point to typical ignimbrite’s cavities, partially filled.

a

b c

d

In addition, preliminary analyses of different parameters were carried out by using the CTAn software. Here we include two CTVox’s color rendered models of the saved color coded images from a

small “virtual” subsampled cylinder core of the ignimbrite, corresponding the structure thickness (Fig. 7a) and structure separation (Fig. 7b) parameters.

Figure 3: DataViewer’s transaxial (a) and sagittal (b) micro-CT images of an ignimbrite, with cavities and material inside (details of some are enlarged and red framed). Note that number of empty cavities (b) increases closer to the surface (Ex), but this not occurs with canals.

Figure 4: CTVox’s volume rendering reconstruction of the ignimbrite. Arrow indicates the

position of the external surface of the rock (Ex). By adjusting the transfer function curves it is possible to separate the structures according to their opacity to x-ray, and thereafter softer and more transparent ones appear in red color. It is clearly visible the narrow layer, parallel and close below the surface, corresponding with higher microorganism colonization’s density (a).

500µm b

a Ex

Ex

Opacity

a

Discussion In the previous paper by Wierzchos et al. (2013) was pointed out material filling pores can be not only microbiota, but also other minerals as in form of gypsum precipitates and/or silica deposits (Fig. 6: a & b). So it is important be cautious to interpret as microbiota the micro-CT images of material inside filled cavities (pores, canals, etc...). However, silica and/or gypsum, as well other mineral deposits are denser than microbiota, and they should be more opaque to x-ray. Thus in the colored images, after to create appropriate transfer functions, there is a sequential variation of the color red to the blue in parallel to an increase in the opacity to x-rays. Thereafter, red or closer to red colors should be interpreted as softer, lesser opaque materials (Figs.: 4 & 5). In figure 4 it is clearly visible the narrow layer, corresponding with higher microorganism colonization’s density (Fig. 1b), as parallel more reddish band, close below the surface (Fig. 4: see region marked with “a”). This should indicate that the microorganism layer colonization band it is pointed out by the micro-CT, and thereafter that reddish colors should correspond with microbiota. Because mineral deposits are denser than microorganism, they should be colored different. In fact looking at different cavities it is visible that together with reddish materials there are others greenish or blue (see figures 4 & 5: b & c). In addition looking at the general shapes, microbiota have defined shaped, some of them as the cyanobacteria Chroococcidiopsis sp., already identified in the samples of ignimbrite by Wierzchos et al. (2013) have granular oval shape. In contrast mineral deposits have crystal shape or they appears amorphous, filling cavities (Fig. 6: a & b). The micro-CT volume rendering reconstructions of Fig. 5, show low dense (in red color) granular shaped structures, inside the cavities. These are more conspicuous in the big cavities appearing in the center of figures 5b and 5c. In thin micro-CT section reconstructions views (Fig. 3), some of the cavities show granulated structures suggesting, also, a possible microorganism contents. Thereafter, the obtained results with micro-CT evidenced the microorganism colonization in some of the ignimbrite pores. Hence, the relevance of this pioneer micro-CT study, as a nondestructive technique to evidence of microbial communities in endolithic life. However, the application of correlative microscopy techniques would be necessary for additional confirmation of the pore spaces content. One of these techniques proposed to use is SEM-BSE according to methodology for the study of microbial endolithic communities as used from 1994 (Ascaso & Wierzchos, 1994; Wierzchos & Ascaso, 1994). This technique can achieve high resolutions and definitely provide essential information on microbiota due to micromorphological and ultrastructural characterization of microorganisms. However, the excellent agreement between micro-CT and SEM-BSE images in solid rock samples demonstrated by Needham et al. (2013), would point out the validity of this first results, evidencing microbiota inside the studied volcanic rock by using micro-CT techniques.

Conclusion The micro-CT demonstrated to be a very promising technique to explore pore content with endolithic microbial communities. Further studies are necessary to characterize the rock structure (exploring new possibilities of analysis as in Fig. 7) and their porosity parameters which can provide information, in such extreme environment, about bioreceptivity of available rocks to endolithic microbial colonization. Moreover, correlative microscopy SEM-BSE studies will be undertaken to get a detailed characterization of pore contents and thus to calibrate the grey levels obtained with it and both grey, but especially promising will be to correlate with the colors obtained with the micro-CT technique.

Figure 5: Volume rendering reconstruction’s sections at different levels permitting to

evidence the typical oval-shaped cavities of the ignimbrite (some of them are almost completely filled with low dense materials; see arrows in b and c). Moreover, these are also inside the complex web of small canals communicating with the exterior and permitting the colonization of the rocks, and to trap and keep inside any moist permitting the endolithic life (see main text).

a

b

c

Opacity

Figure 6: SEM images with details of the ignimbrite cavities; a & b: cavities filled with gypsum and c:

cavity filled with microorganism, see the granular shape of them.

Acknowledgements We thank Bruker-Skyscan staff for fast and effective support, their patience and effectiveness, and for their constant improvements to the software and in implementing new options we requested. But, especially, for their kind answers to our constant pounding of queries and suggestions. We are especially indebted to Alexander Sasov, Stephan Boons, Xuan Liu, Phil Salmon, Jeroen Hostens, and Vladimir Kharitonov. This work benefited of the Spanish Ministerio de Educación y Ciencia (CGL2007 –61856/BOS), and the Junta de Andalucía (RNM-02654) research projects.

a b

c

Figure 7: CTVox’s color rendered models of the color coded images of a small “virtual”

subsampled cylinder core of the ignimbrite, corresponding the structure thickness (a, c) and structure separation (b) parameters obtained with CTAn. Right arrows indicate the position of the external surface of the rock (Ex). The narrow layer, parallel and close below the surface, corresponding with higher microorganism colonization’s density is patent as structure thickness (a & c: marked with red arrow).

MMiinn.. <<

a

b

Ex

Ex

> Max.

Ex

References:

1. Ascaso, C., & Wierzchos, J. “New applications of submicroscopic techniques in the study of biodegradation caused by lichen thalli. Nuevas aplicaciones de las técnicas submicroscópicas en el estudio del biodeterioro producido por talos liquénicos” Microbiología SEM, 10: 103-110. 1994.

2. Bagshaw, E.A. et al. “The microbial habitability of weathered volcanic glass inferred from continuous sensing techniques”. Astrobiology 11, 651–664, 2011.

3. Brasier, M.D., Matthewman, R., McMahon, S., Wacey, D. “Pumice as a remarkable substrate for the origin of life”. Astrobiology 11, 725–735, 2011.

4. Cockell, C.S., Cady, S.L., McLoughlin, N. “Volcanism and astrobiology: Life on Earth and beyond”. Astrobiology 11, 583–584, 2011.

5. Needham, A.W., Abel, R.L., Tomkinson, T., and Grady, M.M. “Martian subsurface fluid pathways and 3D mineralogy of the Nakhla meteorite”. Geochimica et Cosmochimica Acta 116: 96-110. 2013.

6. Wierzchos, J., & Ascaso, C. “Application of backscattered electron imaging to the study of the lichen-rock interface”. Journal of Microscopy 175: 54-59. 1994

7. Wierzchos, J., Davila, A.F., Artieda, O., Cámara-Gallego, A., De los Ríos, A., Nealson, K.H., Valea, S., García-González, M.T. & Ascaso, C. “Ignimbrite as a substrate for endolithic life in the hyper-arid Atacama Desert”. Implications for the search for life on Mars”. Icarus 224: 334–346. 2013.