We are investigating whether cryptoendolithic microorganisms are able to colonize diverse substrates through biogenic weathering. This first part of the study involves the cultivation and characterization of microbial consortia from Antarctic sandstone habitats

Blackhurst, R. L.

Genge, M. J.

Grady, M. M.

Jadbubansa, P.

Roberts, D.

English

Open Research Online (The Open University)

Open Research OnlineThe Open University’s repository of research publicationsand other research outputsCryptoendolith colonization of diverse substrates (1):cultivation and characterizationConference or Workshop ItemHow to cite:Blackhurst, R. L.; Roberts, D.; Jadbubansa, P.; Genge, M. J. and Grady, M. M. (2005). Cryptoendolith colonizationof diverse substrates (1): cultivation and characterization. In: 36th Lunar and Planetary Science Conference, 14-18Mar 2005, Houston, Texas, USA.For guidance on citations see FAQs.c© [not recorded]Version: [not recorded]Link(s) to article on publisher’s website:http://www.lpi.usra.edu/meetings/lpsc2005/pdf/1067.pdfCopyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyrightowners. For more information on Open Research Online’s data policy on reuse of materials please consult the policiespage.oro.open.ac.ukCRYPTOENDOLITH COLONIZATION OF DIVERSE SUBSTRATES (1) : CULTIVATION AND CHARACTERIZATION R. L. Blackhurst1,3, D. Roberts2, P. Jadbubansa2, M. J. Genge1, and M. M. Grady3 1Department of Earth Science and Engineering, Imperial College London, Exhibition Road, London SW7 2AZ, UK; 2Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5AB; 3Department of Min-eralogy, The Natural History Museum, Cromwell Road, London SW7 5AB. rebecca.blackhurst@imperial.ac.uk.   Introduction: Cryptoendoliths are microorganisms that have found refuge from the hostile environmental conditions of the Dry Valleys of Antarctica in the in-terstitial spaces of porous rocks. The rock substrates provide conditions that are suitable for life which con-trast sharply with the abiotic rock surface [1]. En-dolithic communities are thought to have evolved when changes in environmental conditions rendered rock surfaces inhospitable (e.g. owing to glaciation) and the organisms withdrew into protective rock niches [2]. These microbial habitats are thought to represent the last footholds of life in a gradually dete-riorating environment [1], and as such serve as models for certain exobiological scenarios of the putative Mar-tian biosphere.  The multi-coloured layers under the surface of the rocks (fig.1.) are produced by filamentous fungi and unicellular green algae, which also form symbiotic lichen associations [2].   Figure 1: Cryptoendolithic community layers in the profile of a sandstone            Previous Work: Cryptoendolithic lichens interact with the substrate they inhabit by producing oxalic acid which has a two-fold affect: (1) they leach iron-bearing minerals from their inhabited zone and re-deposit them a few millimetres below causing the sub-strate here to appear a darker red; and (2) oxalic acid also acts to dissolve the cementing substance between the crystals in the colonized zone leading to the cyclic exfoliation of the surface crust [3]. Our previous work has investigated the mineralogical and geochemical characteristics of a suite of colonized and un-colonized sandstones in order to determine any further microbe-substrate interaction. A major, minor and trace element study was undertaken utilizing ICP-AES and ICP-MS techniques. The samples were collected by a British Antarctic Survey expedition to Terra Nova Bay and the McMurdo Base during the Antarctic summer of 1995-1996 [4]. The results showed that significant disparity in elemental concentrations was evident be-tween colonized and un-colonized samples. However, mineralogical analysis revealed that the differences were likely to be a primary feature of the different rock types rather than a result of cryptoendolith action. There were distinct differences in mineralogical matur-ity between the samples and each fell into one of three categories; mature (colonized), intermediate (colo-nized), and immature (un-colonized). To establish whether the differences had a biologi-cal or mineralogical control we conducted a further study involving the comparison of the different layers within individual samples (e.g. colonized and un-colonized). Several layers can make up these rocks, a siliceous crust, colonized upper lichen zone, a micro-algal zone, a red layer formed of re-deposited iron compounds and the deeper rock substrate, which is presumably unaltered. (fig. 1). It is therefore easy to differentiate between layers that are colonized and those that are not. Chemical and mineralogical analy-ses were conducted on each of the layers within sam-ples of different mineralogical maturity. The results of both the chemical and mineralogical study revealed that compositional differences occurred between colo-nized and un-colonized layers within the same samples (e.g. fig. 2.). These results indicate that cryptoendoliths may weather the layers they inhabit through their pro-duction of oxalic acid. This may be in order to access essential nutrients and/or perhaps to increase the habi-tability of the layer.  crust  lichen layer  re-deposited iron  unaltered rock substrate  Figure 2: Major elemental concentrations within separate layers in mature colonized sample EB1  00.050.10.150.20.250.3Al Ca Mg Mn Na K Fe TiElementConcentration Wt %EB1 CrustEB1 ColonizedEB1 RedEB1 Pink           Lunar and Planetary Science XXXVI (2005) 1067.pdfCurrent Analysis: Most lithobionts (rock-inhabiting microbes) have been classified into types, however intermediary forms exist e.g. Ostreobium sp., a green algae that inhabits corals. These organisms can behave as euendoliths (rock-borers) and penetrate carbonate structures but also colonize pre-existing structural cavities [5]. Though cryptoendolithic communities are reported not to penetrate the substrate by solubilization and favour the colonization of rocks which have a pre-existing porous structure they have the means for min-eral dissolution. If the essential porosity is not avail-able can cryptoendoliths behave like euendoliths and inhabit more diverse substrates then weather them to improve their habitability?  Our current study is investigating the extent of the weathering capabilities of cryptoendolithic communi-ties to determine whether they are used in colonization e.g. does the ability to weather rock substrates equal the ability to colonize more hostile substrates? As pio-neering colonists of diverse substrates in extreme envi-ronments, their role as analogues of Martian microor-ganisms would be greatly enhanced. The study in-volves cultivation of microbial consortia from Antarc-tic sandstone habitats and then their re-colonization of a variety of selected substrates. These substrates will include a range of rock types including sandstones of varying mineralogical maturity and porosity, basalts, fractured granites and a Martian meteorite.  Cultivation of Cryptoendoliths: Samples were flame sterilized to eliminate exterior contamination. Each sample was then cracked open using a flame-sterilized chisel. Grains from individual layers (e.g. colonized and un-colonized) from the interior of each sample were scraped onto Petri dishes containing BG-11 agar and PYGV agar using a flame-sterilized blade. Whole pieces of the rocks taken from the interiors of larger samples were place in sterile jars containing liquid media BG-11 and SNAX both commonly used to cul-tivate cyanobacteria.  Petri plates and jars were incu-bated at 4oC under constant illumination. After one week under these conditions fungal mycelia with spores were observed to be growing from around sand-stone grains scraped from the “colonized” layers of several samples (fig. 3. a). Fungal growth occurred on both BG-11 and PYGV agars. Spherical structures growing out from around grains indicated the growth of bacteria colonies derived from the grain surface (fig.3. b). The colonies were prepared on a wet slide and observed under a phase-contrast microscope. They consisted of rod-shaped bacteria which were motile and could be seen to be dividing by binary fission. Observed bacterial colonies and fungal mycelia were separated and placed on agar slants to isolate pure cul-tures. After eight weeks bacterial colonies were also observed growing out from several rock samples im-merse in both the BG-11 and SNAX media. We are currently verifying the identity of the cul-tured microbes and characterizing the constituents of the cryptoendolithic communities using both culture-based and culture-independent molecular phylogenetic analyses. This is being conducted by examining small sub-unit (SSU) rRNA genes amplified from commu-nity DNA using the techniques outlined in [6] and [7].  Figure 3: Cultured microbes (x 6.5) (a) fungi grow-ing from around sandstone grain; (b) bacteria colony emerging from around grain          (a)  (b) Discussion: In cultivating these cryptoendolithic communities potential problems arise, like contamina-tion. Steps to limit contamination have been taken throughout the study e.g. sterilization and DNA se-quencing will allow characterization of the microbes that have been cultured. Another problem may be in only successfully cultivating parts of the cryptoen-dolithic community. The microbes may only re-colonize as a consortium. To date we have not ob-served any of the cultured fungi or algae growing to-gether in lichen association. If the microbes re-colonize “out of symbiosis” then they may not interact with the substrate in the same way as true cryptoen-dolithic consortia.  Implications: If cryptoendolithic organisms, culti-vated in this study, are shown to be able to colonize samples by biogenic weathering then they are likely to be able to adapt relatively rapidly to changing envi-ronmental conditions without adaptive evolutionary changes. Such ability has important implications for the survival of micro-organisms close to the Martian surface.  References: [1] Friedmann E.I. and Ocampo-Friedmann R. (1984) Origins of Life, 14, 771-776 [2] Friedmann E.I. (1982) Science, 215, 1045-1053 [3] Sun H.J. and Friedmann E.I. (1999) Geomicrobiol. J. 16, 193-202 [4] Edwards H.G.M. et al., (1997) J. Ra-man Spectrosc., 28, 685-690. [5] Golubic S. (1981) J. Sed. Pet., 51, 0475-0478 [6] Hugenholtz P. et al., (1998) J. of Bacteriology 180, 366-376 [7] de la Torre J.R. (2003) App. Environ. Microbiol., 69, 3858-3867  Lunar and Planetary Science XXXVI (2005) 1067.pdf

Cryptoendolith colonization of diverse substrates (1): cultivation and characterization

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Cryptoendolith colonization of diverse substrates (1): cultivation and characterization

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