Scanning Electron Microscopy of Terrestrial and Extraterrestrial Samples: A Study of Bacteria and Nannobacteria.  A. Taunton, University of Arkansas, Fayetteville AR 72701, USA.

Published in Papers Presented to the 12th Annual Summer Intern Conference, pp. 33-35, LPI, Houston.

The study of microorganisms in terrestrial rocks has exploded in the past two decades. Many details are known about biomineralization and microbial participation in the formation and destruction of sedimentary deposits. Recently the discovery of nannobacteria by [1] has raised new interest in how different types of sediments are formed or altered. His findings along with the growing abundance of information on chemolithoautotrophic ecosystems has opened up new possibilities for the appearance of life elsewhere in the solar system, especially on Mars. In this project, several terrestrial samples from extreme environments on Earth and a sample from the martian meteorite ALH 84001 were studied under a scanning electron microscope for evidence of bacteria and nannobacteria.

Nannobacteria are smaller forms of regular bacteria that range from 0.02 to 0.2 µm in diameter. Normal bacteria may become stressed from starvation, changes in chemistry, or temperature of their environment, or dehydration resulting in these dwarfed forms. However, [2] observed nannobacteria in nonstressful, normal environments, concluding these bacteria have no “large” size equivalent.

Identifying nannobacteria in a sample is difficult because of the small size of the organism. X-ray analysis is almost impossible for such a restricted area. Thus a set of guidelines determined by [2] is now accepted as a standard for recognizing nannobacteria in rock samples:

  1. Clusters of bodies separated by large unpopulated areas. This type of distribution is seen where living bacteria thrive.
  2. Bodies same size as living bacteria, including nannobacteria. Populations within a sample same as those of living bacteria. Populations can range from very well-sorted to bimodal assemblages (normal and nannobacteria) to less well-sorted mixtures--different types of bacteria or varied stages of sporulation or nutrition.
  3. Bodies similar shape to living bacteria:  smooth to somewhat lumpy surfaces with shapes of cocci, ellipses, bacilli, or long filaments. The bacteria can appear as chains of spheres or rods. Most small minerals form as minute euhedra or spheres made of tiny packed crystallites or radial fibers that give a rough microsurface.
  4. Bodies do not contain Fe, but may have Ca or Si. These are precipitates of bacteria. Fe-containing magnetotactic bacteria are not included in the guidelines proposed by [2].
  5. Bodies must not be confused with minerals or artifacts:  they must be observed at very high magnifications (at least 35000×) to verify that the shapes are spheres, ellipsoids, or rods rather than cubes.
Methods:  Five terrestrial samples and one extraterrestrial sample were observed using the JOEL 35CF and the Philips 40XL Field Emission scanning electron microscopes (SEM) at 25 kV. The five terrestrial samples consisted of Persian Gulf stromatolite, Antarctic marble, desert varnish, travertines from West Texas, and Columbia River basalts. The extraterrestrial sample was from the martian meteorite ALH 84001. All samples were freshly fractured before analysis. The travertines were lightly etched with a 1% HCl solution for one minute then rinsed with distilled water. Columbia River basalt (CRB) samples obtained from T. Stevens and J. McKinley were pretreated with high-sulfate groundwater, low-sulfate groundwater, and biomass solutions, and studied along with unaltered CRBs. All samples were mounted on graphite discs or planchettes using double-stick tape and/or C paint. Each was sputter-coated with a gold-palladium alloy for 30-90 s in order to make it conductive for SEM study. Energy-dispersive X-ray analysis was performed using the PGT System IV associated with the JEOL 35 CF-SEM when possible.

Results:  Microfossils, dormant bacteria, and nannobacteria were found in all samples. The stromatolite provided microbial fossils ranging from 5 to 10 µm. These were identified as radiolarian and diatom remains. Most were Ca rich; however, several Si-rich hollow rods were found and identified. Rods with a Ca precipitate were Scytonema myochrous. Those without the precipitate were Microcoleus [3].

Two or possibly three different types of dormant cyanobacteria were identified in the Antarctic marble. The cyanobacteria had varying sizes:  5- to 10-µm chains of 1-µm cylindrical segments, 1-µm spheres, and 1-µm rods that appeared to be dividing. The chains are possibly Anabaena cylindrica. The spheres could be Chrococcus turgidus, with the rods being the dividing phase [4]. The cyanobacteria showed a strong X-ray peak for Ca and traces of Cl, Ar, Si, and Mg.

Desert varnish from South Mountain Park, Phoenix, AZ, and Newspaper Rock, Petrified Forest, NM was analyzed. A bimodal colonial distribution of spherical bodies was discovered both on the surface of the rock and at the meridian where the varnish was first precipitated. The South Mountain Park spheres were 150 nm. The spheres in the Newspaper rock were 30-75 nm in diameter. Also found in Newspaper Rock desert varnish were tubular bodies 75-100 nm in length and approximately 20 nm in width.

Fossils of dividing and rimmed bacteria were found in the travertine from bedding plane fault in Guadelupe Pass, West Texas. The spherical bacteria had an approximate diameter of 250 nm. Identified between two pairs of dividing bacteria was what appeared to be a biofilm. The composition of the rims is uncertain. Chemical analysis of this area was not possible because the count rate was not sufficient.

Several different types of bacteria and morphological features were found in the four samples of the Columbia River basalts. These basalts were collected from approximately 5 km below Earth’s surface. No bacteria were imaged in the untreated sample; however, bacteria may exist. There was no opportunity to view this sample at a magnification higher than 30000×. Phyllosilicates were present on almost the entire surface.

The surface of the biomass sample was also coated with phyllosilicates. Segmented bodies of 250 nm to ~2 µm in length and a diameter of 20 to 50 nm were found and appeared to be formed by extremely small spheres. The bacteria were arranged in circles, chains, and knots. It is interesting to note that at every bacterial location there was a dome-shaped object of similar spheres either close to or along the bacterial body.

Four types of bacteria, abundant phyllosilicates, and a possible microbial mat were found in the low-sulfate groundwater samples. The bacteria in this sample were in about the same abundance as those in the biomass solution. Bacteria identical to those in the biomass were present (Fig. 1), along with a larger form of ~150 nm diameter and 2 µm length. The third type of bacteria was 1-2 µm in length and very clearly made of chains of ~150-nm-diameter spheres. The fourth type of bacteria found were 250-nm spheres. Budding bacteria were imaged.

In the high-sulfate solution samples, two possible types of bacteria were imaged:  1-µm spheres and an ~6-µm segmented body. The spheres could be bacteria; however, they are also similar to S particles or yeast. X-ray analysis was not performed. The segmented body was considerably longer than the previously observed bacteria and consisted of irregular segments. Again, phyllosilicates were present.

Several colonies of tubular bodies and a larger singular tubular body were imaged in ALH 84001. The colonial bodies were approximately 150-200 nm in length and 10-30 nm in diameter. The singular body was approximately 400 nm long and 50 nm in diameter (Fig. 2). Ovoids of approximately 250 nm were also found in ALH 84001, some of which appeared to be dividing.

Discussion:  Each of these samples was evidence that bacteria can survive in extreme environments on Earth, from the freezing temperatures of Antarctica to 5 km beneath the Earth’s surface. What does this say about the possibility of life on Mars? The best examples in this study would be the analogies of the environments of the travertines and the Columbia River basalts to environments on Mars.

Travertines formed from thermal springs are an excellent environment for preservable microbial communities on Earth. The springs can range from 5°C to 95°C and are most commonly found near volcanically active sites [5]. Hydrothermally active sites similar to these terrestrial hot springs are said to have existed on Mars from the early history of the planet. Mouginis-Mark [6] studied the northwestern area of Tharsis and has suggested that water release has occurred as recently as 1 b.y. ago. In his models explaining this hydrothermal activity, volcanic heat sources interact with a supply of water that may be either a liquid water source associated with basal melting of an ice sheet or an ancient subsurface ice layer on Mars.

In terrestrial hydrothermally active sites, many different kinds of microbes are found, including methanogens, acetogens, sulfur- and sulfate-reducing bacteria, and thionic denitrifing bacteria [5]. Methanogens and acetogens are anaerobic microbes that metabolize C from CO2 as their only C source for biosynthesis. Sulfur-reducing bacteria receive their energy from reducing sulfate with H producing H2S. A thermophilic archaebacteria using CO2 as its only C source oxidizes H with S, also producing H2S. Fermentation by sulfate-reducing bacteria distorts amounts of inorganic S compounds in soils. Thionic species of bacteria oxidize reduced forms of S with nitrate. In the presence of H gas, Fe-reducing bacteria use ferric iron as a source of energy. Since martian soil is very rich in S and ferric iron, these types of microorganisms are all possibilities for life on Mars.

The very recent discovery by Stevens and McKinley [7] of bacterial activity in the basalts of the Columbia River adds even more promise to the idea that life could have existed on Mars. These methanogenic bacteria get their source of H not from other microbes, which is normally the case, but from the basalt itself when its ferrous silicates react with water. Several laboratory experiments involving the saturation of basalts with high-sulfate groundwater, low-sulfate groundwater, and biomass were performed, and microbial metamorphism was found to be present in all samples. Our SEM studies imaged the actual bacteria in the samples. Thus since basalt, liquid water, and bicarbonate are believed to have been present on the martian surface, this type of bacteria could have survived.

Conclusions:  It is quite evident that microbes play a significant role in the formation and destruction of terrestrial sediments Bacteria can survive in extremely harsh environments, including those with no organic C or sunlight. The presence of these bacteria on Earth and the similarities between their terrestrial environments and past martian environments leads to the conclusion that bacteria could have existed on Mars. And, since the geothermal subsurface conditions of Mars are still uncertain today [8], these types of bacteria could be presently living on Mars. Regardless, some of the bodies in the terrestrial samples were similar to those imaged in ALH 84001 by McKay et al. [9] that were interpreted to be fossilized bacteria.

References:  [1] Robert F. (1992) GSA Abstracts with Programs, A104. [2] Robert F. (1993) JSP, 63, 990-999. [3] Monty C. L. V. and Hardie L. A. (1996) in Stromatolites, 454-455. [4] Atlas P. (1984) Microbiology, 375-376. [5] Boston P. J. et al. (1992) Icarus, 95, 300-308. [6] Mouginis-Mark P. J. (1990) Icarus, 84, 362-373. [7] Stevens T. and McKinley J. (1995) Science, 270, 450-454. [8] Meyer et al. (1995) An Exobiological Strategy for Mars Exploration. [10] McKay et al. D. (1996) Science, in press.