Aurion Mission

Thursday, August 11, 2011

Images and EDS Spectra of Filaments in the Ivuna CI1 Carbonaceous Meteorite.
Figure 1 provides images and Energy Dispersive X-Ray Spectroscopy elemental data for filaments found embedded in the Ivuna CI1 carbonaceous meteorite. Fig. 1.a is a FESEM image of a thin uniseriate filament that is flattened at the terminal end. The filament is cylindrical in the lower portion embedded in the meteorite rock matrix. This small, undulatory filament (diameter 0.7 to 1.0 m) is rich in C, Mg, and S and depleted in N. The filament is only partially encased within a broken and very thin carbonaceous sheath. EDS elemental data is shown for spot 1 on the thin sheath (Fig. 1.b) and for spot 3 on the nearby mineral matrix (Fig. 1.c). The sheath has higher carbon content and biogenic elements N and P are below the 0.5% detection limit of the instrument. Fig. 1.d is a FESEM image of 5m diameter X 25 m long spiral filament Ivuna with white globules that are sulfur-rich as compared with the rest of the filament and the meteorite matrix. A tuft of fine fibrils is visible at the left terminus of the filament and the terminus at the lower right is rounded. Fig. 1.e is a FESEM Backscattered Electron image of an Ivuna filament with sulfur-rich globules S and rounded terminus R that is similar in size and morphology to the giant bacterium “Titanospirillum velox”.

Fig. 1a. Ivuna CI1 meteorite filament (0.8 μm diameter) with dark lines C, partially encased in thin carbon-rich sheath.

Fig 1d. FESEM Backscattered Electron image of an Ivuna filament with N<0.5% and sulfur-rich globules S and rounded terminus R that is similar in size, morphology and internal composition to terrestrial bacteria (See e, below)

Fig 1e. Giant bacterium Titanospirillum velox. Image 1.e Courtesy: Dr. Riccardo Guerrero.

Fig. 1. a. Ivuna CI1 meteorite filament (0.8 μm diameter) with dark lines C, partially encased in thin carbon-rich sheath. b. EDS elemental data of the filament sheath at spot 1 shows typical biogenic elements Nitrogen and Phosphorus (<0.5%) and Carbon (13.1%) enriched as compared with nearby meteorite matrix (C 7.2%) at spot 3; d. FESEM Backscattered Electron image of an Ivuna filament with N<0.5% and sulfur-rich globules S and rounded terminus R that is similar in size, morphology and internal composition to (e.) giant bacterium Titanospirillum velox” with sulfur (S) globules collected from Microcoleus mat of Ebro Delta, Spain. (Scale bar = 5 μm) Ivuna Meteorite Courtesy: Dupont Meteorite Collection, Planetary Studies Foundation; Image 1.e Courtesy: Dr. Riccardo Guerrero. 3.1.1. Interpretation of Images and EDS Data of Ivuna Filaments. The flattened embedded filament shown in Fig. 1.a is interpreted as the permineralized remains of a partially uniseriate, undulatory, ensheathed trichomic prokaryote. The measured diameter (0.7 - 1.0 μm) as determined from the scale bar of this calibrated FESEM image and the detailed morphology of this Ivuna filament is consistent with some of the smaller filamentous cyanobacteria. The dark lines C near the terminus of the sheath are consistent with cross-wall constrictions that are often seen as faint transverse lines in FESEM images obtained with living cyanobacteria. In this image it is possible to see an extremely thin sheath S that is broken and covers only the upper portion of the trichome, which appears to have been completely replaced by infilling minerals.
The size and morphology of this filament is consistent with filaments of the undulatory trichomic filamentous cyanobacteria Spirulina subtilissima (filaments 0.6 - 0.9 μm diameter) and S. laxissima (filaments 0.7 to 0.8 μm diameter). These cyanobacteria have not been reported as possessing a sheath, but the sheath seen in this FESEM image is extremely thin and would be very difficult to discern in by visible light microscopy techniques. There are also very small species of the genus Limnothrix that are undulatory on nature and possess facultative sheaths. However, it shoud be pointed out that there also exist groups of ensheathed filamentous anoxygenic phototrophic bacteria (photosynthetic flexibacteria) possess a thin sheath and are capable of gliding motility. There include filamentous representatives of the bacterial Phylum Chloroflexi. The thermophilic species Chloroflexus aurantiacus has a thin sheath and trichomes as narrow as 0.8 μm. There also other bacterial photoautotrophs that oxidize hydrogen sulfide and deposit it externally as sulfur (e.g., Oscillochloris trichoides) and these trichomic filaments have diameters in the 0.8 to 1.4 μm range.
The length, diameter and spiral configuration and apparent tuft of small filaments at one pole and rounded end at the other along with the internal sulfur globules distributed along the axis of filament (Fig. 1.d) found embedded in a freshly fractured surface of the Ivuna meteorite a complex suite of features.that are very similar to those observed in SEM images of the novel bipolar lophotrichous gram-negative bacterium “Titanospirillum velox” (Fig. 1.e) which was described by Guerrero et al (1999). “Titanospirillum velox” is a very large mat-forming bacterium with 3–5 μm diameter X 20–30 μm long filaments. It was collected from a mud sample beneath a Microcoleus chthonoplastes mat in the Ebro Delta in Tarragona, Spain. “T. velox” swims very rapidly (10 body lengths/sec) with spiral motility, propelled by the lophotrichous tuft of flagella at the cell terminus. The intracellular elemental sulfur storage globules are seen as white spots in this Scanning Electron Microscope image. This extremophile was grown only in mixed culture with other bacteria, which would explain the fact that this genus and species has not yet been accepted as validly published. The Bacteriological Code rules of nomenclature requires that prokaryotic microorganisms must be isolated and grown in pure culture and the designated type stain must be deposited in two international culture collections in two different countries before the genus and species names can be validated (Tindall et al., 2006). The absence of detectable nitrogen content the Ivuna filaments provides evidence that these embedded filaments are indigenous and cannot be dismissed as a modern biological contaminant.
3.2 Images and EDS Spectra of Filaments in the Orgueil CI1 Carbonaceous Meteorite. Figure 2.a. is a low magnification (1000X) Secondary Electron Detector (SED) FESEM image of freshly fractured fragment of the Orgueil CI1 meteorite that is densely populated with several different types of embedded filaments and electron transparent sheaths. Even though the field of view shown of this image is very small (~120 μm wide) a wide variety of diverse filamentous microstructures are present. To facilitate the description, the filaments and sheaths have been numbered, and all numbers are located on the filament at the site where the EDS elemental spot data were recorded. A 2D X-ray elemental map of this region of the Orgueil meteorite is shown in Figure 2.b. The large image in the upper left corner is a Backscatter Electron Detector (BSED) image. The bright spots in this image are high Z elements where clusters and crystallites of magnetite, iron and nickel are concentrated. Other images reveal where relative concentrations Oxygen, Silicon, Magnesium, Sulfur, Iron, Nitrogen; Calcium, and Aluminum are located. The major filaments and sheaths are clearly seen as bright features in the Carbon, Oxygen, Magnesium and Sulfur maps and they appear as dark features in Silicon, Iron, and Nickel due to the relatively higher content of these elements in the underlying Orgueil meteorite rock matrix. In general, the filament and sheath structures are not discernible in the Nitrogen, Phosphorus and Sodium maps, although Filament 1 can be seen in the Nitrogen map. Empty sheath 7 is wrinkled and electron transparent with a relatively high (47%) content of Carbon. This sheath is unusual in that it is one of the few filaments found in the Orgueil meteorite to have detectable levels of Nitrogen (1%) and Phosphorus (0.8%).

Figure 2.a. Hitachi FESEM Secondary Electron Detector image at 1000 X of multiple filaments and sheaths embedded in Orgueil meteorite matrix and b. Backscatter Electron Detector image along with 2-D x-ray maps showing distribution of elements O, C, Si, N, Mg, S, Fe, P, F, Ca, Ni and Cl in filaments for comparison with SED and BSED images. Orgueil Sample Courtesy: Dr. Paul Sipiera, DuPont Meteorite Collection, Planetary Studies Foundation, Chicago Filaments 1 and 2 of Fig. 1.a are observed to have sheaths with longitudinal striations that run the length of the filaments. This is characteristic of multiseriate trichomic prokaryotic filaments in which multiple parallel oriented trichomes are enclosed within a common homogeneous sheath. These filaments are observed to be either attached to or physically embedded in the Orgueil meteorite matrix. The end of filament 1 becomes slightly wider (~10 μm) where it joins the rock matrix and it appears to contain four internal trichomes, each with a diameter ~2.5 μm. Filament 2 is considerable larger (~ 20 μm dia.) and the longitudinal striations suggest it contains ~5 trichomes, each with diameters ~4 μm/trichome. Faint transverse lines orthogonal to the long axis of filament 2 are marked C.
3.1.1 Interpretation and Discussion of Images and EDS Data of the Orgueil Filaments.
The longitudinal striations of the long filament 1 and the shorter, curved filament2 are interpreted as indicating these are multiseriate filaments consisting of a bundle of multiple parallel trichomes encased within a common sheath. If the transverse striations C of filament 2 are interpreted as represent cross-wall constrictions, this would indicate that the internal cells within each trichome are ~ 4 μm in length and hence isodiametric. Consequently, the image of filament 2 is interpreted as composed of trichomes made up of spherical or cylindrical isodiametric cells of 4 μm diameter. This interpretation is consistent with morphotypes of undifferentiated filamentous cyanobacteria of the Order Oscilliatoriacea. There are many genera and species within this very common cyanobacterial order, including the genus Microcoleus Desmazières ex Gomont (Form Genus VIII. Microcoleus Desmazières 1823) (Castenholz, Rippka & Herdman, 2001; Boone et al., 2001). Reproduction within this order occurs by trichome fragmentation and the production of undifferentiated short trichome segments (hormogonia) by binary fission of the cells in one plane at right angles to the long axis of the trichomes. The small solitary uniseriate filaments 3 and 4 may be interpreted as representing members of the genus Trichocoleus Anagnostidis, which was separated from the genus Microcoleus on the basis of cell size and morphology. Filament 4 is a 2 μm diameter hook-shape filament with a narrowed terminus. Several species of the genus Trichocoleus have filaments typically in the 0.5 μm to 2.5 μm diameter range (Wehr and Sheath, 2003, pg. 136). Energy Dispersive X-Ray Spectroscopy (EDS) spot spectral data were obtained on the meteorite rock matrix as well as on all of the numbered filaments and sheaths at positions where the numbers are located in the FESEM image.

Figure 3. Hitachi FESEM images at 1500X of a. collapsed filament 9 and helical coiled empty sheath 10 and b. 6000X image of filament 11 showing hook and calyptra or conical apical cell. c. EDS spot spectra show elemental compositions c. of loose sheath 10 (C 29.1%; N=0.7%) and d. sheath 11 (C 47.8%; N<0.5%). Figure 3.a is a1500X FESEM SED images of the collapsed Filament 9 and the hollow, flattened, twisted and folded sheath 10. Sheath 10 is 4.6 μm in diameter and it is folded at the top where the EDS spectra were taken. The flattened portion of Sheath 10 forms a spiral coil near the base where it is attached to the meteorite matrix. This is very similar to helical coiled sheath of Phormidium stagninum shown in the illustration at illustration. This type of flattened, coiled hollow sheath is often seen in other species of filamentous cyanobacteria and hence does not constitute a unique diagnostic feature. Figure 3.b. provides a higher magnification (6000X) image of Sheath 11, which is visible at the top of Fig. 2.a. Sheath 11 is a tapered and hooked form with a conical terminal cell or calyptra at the apex. It is 8.5 μm wide where it emerges from the rock matrix and it tapers to 1.5 μm diameter just after the sharp hook. Figure 3.c. is a 10 keV EDS spectrum taken at spot 10 in the fold of Sheath 10 and shows detection of low, but measurable level of Nitrogen (0.7%) and Phosphorus (0.3%) and higher levels of Iron (19%) and Silicon (14%), which are probably from the meteorite matrix beneath the this, electron transparent carbon-rich sheath. The EDS spectrum at 5 keV for spot 11 on sheath 11 as shown in (Fig. 3.d) reveals this flattened sheath to be highly carbonized (48% C atomic), This small filament appears as a bright feature in the carbon map of (Fig. 2.b) and as a dark shadow in the Magnesium and Sulfur maps as it crosses in front of large filaments more heavily mineralized with magnesium sulfate. Filament 11 is also sulfur-rich (21% S), but has Nitrogen below the level of detectability (< ~0.5%).
3.2 Orgueil Filaments with Differentiated Heterocysts. Several genera of the cyanobacterial orders Nostocales and Stigonometales use specialized cells known as “heterocysts” to fix atmospheric nitrogen. Nitrogen fixation is an unambiguously biological process that is absolutely crucial to all life on Earth. Although nitrogen comprises almost 78% of our atmosphere, it is completely useless to life in its relatively inert molecular form. The biological process of nitrogen fixation occurs by the reduction of gaseous nitrogen molecules (N2) into ammonia, nitrates, or nitrogen dioxide. Many species of several genera of cyanobacteria (e.g., Anabaena, Nostoc, Calothrix, Rivularia, Scytonema, etc.) use highly specialized cells for nitrogen fixation by encapsulating the nitrogenase enzyme in thick-walled protective heterocysts.
Cyanobacteria play the key role in nitrogen fixation on Earth and many genera and species of are capable of diazotrophic growth and nitrogen metabolism. Nitrogen fixation occurs via the nitrogenase enzyme with some other proteins involved in this complex biological process. Since the activity of the nitrogenase enzyme is inhibited by oxygen the enzyme must be protected. In many species it is contained within the thick-walled specialized nitrogen-fixing cells called “heterocysts.” The heterocysts have very distinctive, thick, hyaline, refractive walls that provide well-protected centers in which the nitrogenase enzyme, which is inactivated by oxygen, can carry out its required activity.
Heterocysts of cyanobacteria produce three additional cell walls, including one with glycolipids that form a hydrophobic barrier to oxygen. This is crucial since cyanobacteria are aquatic photoautrophs that evolve oxygen during their photosynthesis. To provide additional protection, the cyanobacterial heterocysts lack photosystem II (Donze et al., 1972). Therefore the heterocysts produce no oxygen and they also up-regulate glycolytic enzymes and produce proteins that scavenge any remaining oxygen. As early as 1949, Fogg recognized that heterocysts are formed from the vegetative cells of the cyanobacteria when the concentration of ammonia or its derivative falls below a critical level and by 1968 it was becoming clear that the heterocysts were the site of nitrogen fixation (Fogg, 1949; Fay et al., 1968; Stewart et al., 1969). Heterocysts are found in cyanobacteria of the Order Nostocales and the Order Stigonematales, but they are never found in any of the genera or species of the other three orders (Chroococcales, Oscillatoriales, or Pleurocapsales). Furthermore, heterocysts have not been observed in any the known filamentous sulfur bacteria of any other trichomic prokaryotes. Consequently, the detection of heterocysts provides clear and convincing evidence that the filaments are not only unambiguously biological but that they belong to one of these two orders of cyanobacteria rather than trichomic ensheathed sulfur bacteria or any other group of filamentous trichomic prokaryotes. The presence or absence and the location and configuration of heterocysts has for a long time been a critical diagnostic tool for the recognition and classification of many important taxa of cyanobacteria. The FESEM image of the mineralized remains of polarized filaments interpreted as morphotypes of the cyanobacterium Calothrix spp. found embedded in the Orgueil CI1 carbonaceous meteorite. Several tapering filaments (diameter ~ 1 to 2.5 μm ) and recognizable enlarged cells are seen in close proximity to each other with the smooth basal heterocyst attached to the meteorite matrix (Fig. 4.a). For comparison, a FESEM image of a living Calothrix sp. with a diameter ~ 0.8 μm and basal heterocyst from White River, Washington is shown in Fig. 4.b.

Figure 4.a. FESEM image of permineralized remains in the Orgueil meteorite of polarized tapered filaments (diameter ~ 1 to 2.5 μm) with recognizable heterocysts interpreted as morphotypes of the cyanobacterium Calothrix spp. and. b. living filament of Calothrix sp. with a diameter ~ 0.8 μ and a basal heterocyst from the White River, Washington.

Figure 5. Long sinuous, helical coiled and polarized filament with conical apex (<1.3 μm) and terminal heterocyst similar to cyanobacterium Cylindrospermopsis sp. in the Orgueil meteorite and b. short embedded filament in Orgueil compared with c. living Tolypothrix distorta grown in pure culture at the NASA/NSSTC Astrobiology Laboratory. Orgueil Meteorite Sample Courtesy: Dr. Martine Rossignol-Strick, Musée Nationale d’Histoire Naturelle, Paris Figure 5.a is a Hitachi S4100 FESEM image of helical coiled polarized filament in Orgueil CI1 carbonaceous meteorite. The filament has a conical apex (<1.3 μm)at left end and a bulbous (2.3 μm diameter) heterocyst is seen at the other terminus. This filament has size and morphological characteristics of morphotypes of cyanobacteria of species of the genus Cylindrospermopsis. Fig. 5.b. is an image of a 2.5 μm diameter filament embedded in Orgueil. This filament has a 4.7 μm diameter bulbous terminal heterocyst and is interpreted as a morphotype of cyanobacteria of the genus Tolypothrix. Fig. 5.c is an image of a morphotype of living Nostocalean cyanobacterium Tolypothrix distorta shown for comparison.
Although many modern cyanobacteria are resistant to desiccation, they do not carry out active growth and mat building when they are in a dried state. However, it has been known since 1864 that the Orgueil meteorite is a microregolith breccia, comprised of minute particulates cemented together by water-soluble salts that are readily destroyed by exposure to liquid water. Therefore, it is suggested that none of the Orgueil samples could have ever been submerged in pools of liquid water needed to sustain the growth of large photoautotrophic cyanobacteria and required for the formation of benthic cyanobacterial mats since the meteorite arrived on Earth. Many of the filaments shown in the figures are clearly embedded in the meteorite rock matrix. Consequently, it is concluded that the Orgueil filaments cannot logically be interpreted as representing filamentous cyanobacteria that invaded the meteorite after its arrival. They are therefore interpreted as the indigenous remains of microfossils that were present in the meteorite rock matrix when the meteorite entered the Earth’s atmosphere. EDS elemental analyses carried out on the meteorite rock matrix and on living and fossil cyanobacteria and old and ancient biological materials have shown that the Orgueil filaments have elemental compositions that reflect the composition of the Orgueil meteorite matrix but that are very different from living and old microorganisms and biological filaments. Recently dead cyanobacteria and living cyanobacteria and other modern extremophiles are usually damaged by exposure to the focused FESEM electron beam during EDS analysis of small spots. This beam damage behavior was not observed in the Orgueil filaments or in Devonian, Cambrian, or Archaean fossils investigated. The C/N and C/S ratios of the Orgueil filaments are similar to fossilized materials and kerogens but very different from living biological matter, providing further evidence that the Orgueil filaments are not modern biological contaminants.