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Journal of Cosmology, 2011, Vol 13, xxx.
JournalofCosmology.com, March, 2011

Fossils of Cyanobacteria in CI1 Carbonaceous Meteorites:
Implications to Life on Comets, Europa, and Enceladus

Richard B. Hoover, Ph.D.
 

NASA/Marshall Space Flight Center, Huntsville, AL


Abstract

Environmental (ESEM) and Field Emission Scanning Electron Microscopy (FESEM) investigations of the internal surfaces of the CI1 Carbonaceous Meteorites have yielded images of large complex filaments. The filaments have been observed to be embedded in freshly fractured internal surfaces of the stones. They exhibit features (e.g., the size and size ranges of the internal cells and their location and arrangement within sheaths) that are diagnostic of known genera and species of trichomic cyanobacteria and other trichomic prokaryotes such as the filamentous sulfur bacteria. ESEM and FESEM studies of living and fossil cyanobacteria show similar features in uniseriate and multiseriate, branched or unbranched, isodiametric or tapered, polarized or unpolarized filaments with trichomes encased within thin or thick external sheaths. Filaments found in the CI1 meteorites have also been detected that exhibit structures consistent with the specialized cells and structures used by cyanobacteria for reproduction (baeocytes, akinetes and hormogonia), nitrogen fixation (basal, intercalary or apical heterocysts) and attachment or motility (fimbriae). Energy dispersive X-ray Spectroscopy (EDS) studies indicate that the meteorite filaments are typically carbon rich sheaths infilled with magnesium sulfate and other minerals characteristic of the CI1 carbonaceous meteorites. The size, structure, detailed morphological characteristics and chemical compositions of the meteorite filaments are not consistent with known species of minerals. The nitrogen content of the meteorite filaments are almost always below the detection limit of the EDS detector. EDS analysis of terrestrial minerals and biological materials (e.g., fibrous epsomite, filamentous cyanobacteria; mummy and mammoth hair/tissues, and fossils of cyanobacteria, trilobites, insects in amber) indicate that nitrogen remains detectable in biological materials for thousands of years but is undetectable in the ancient fossils. These studies have led to the conclusion that the filaments found in the CI1 carbonaceous meteorites are indigenous fossils rather than modern terrestrial biological contaminants that entered the meteorites after arrival on Earth. The δ13C and D/H content of amino acids and other organics found in these stones are shown to be consistent with the interpretation that comets represent the parent bodies of the CI1 carbonaceous meteorites. The implications of the detection of fossils of cyanobacteria in the CI1 meteorites to the possibility of life on comets, Europa and Enceladus are discussed. Keywords: Origins of life, CI1 meteorites, Orgueil, Alais Ivuna, microfossils, cyanobacteria, comets, Europa, Enceladus


1. INTRODUCTION

The CI1 carbonaceous chondrites are the most primitive of all known meteorites in terms of solar elemental abundances and the highest content of volatiles. Carbonaceous chondrites are a major clan of chondritic meteorites that contain water, several weight % Carbon, Mg/Si ratios at near solar values, and oxygen isotope compositions that plot below the terrestrial fractionation line. The CI1 classification indicates the meteorites belong to the CI (Ivuna Type) chemical group and are of petrologic Type 1. The CI1 meteorites are distinguished from other carbonaceous chondrites by a complete absence of chondrules and refractory inclusions (destroyed by aqueous alteration on the parent body) and by their high degree (~20%) of indigenous water of hydration. The aqueous alteration took place on the parent bodies of the CI1 meteorites at low temperature (<50 oC) and produced hydrated phyllosilicates similar to terrestrial clays, carbonates and oxides magnetite Fe3O4 and limonite Fe2O3 . nH2O. Sparsely distributed throughout the black rock matrix are fragments and crystals of olivine, pyroxene and elemental iron, presolar diamonds and graphite and insoluble organic matter similar to kerogen.

The CI1 carbonaceous chondrites are extremely rare. Although over 35,000 meteorites have been recovered there are only nine CI1 meteorites known on Earth (Table I). Five of them were observed falls: Alais, Orgueil, Ivuna, Tonk and Revelstoke) and the other four (Y-86029, Y-86737, Y980115 and Y-980134) were collected in 1986 and 1998 from the blue ice fields of the Yamato Mountains by Antarctic Expeditions of the National Institute of Polar Research, Japan. The great rarity of the CI1 stones is undoubtedly due to the fact that they are friable micro-regolith breccias. All five CI1 meteorites known before 1986 were collected soon after they were observed to fall. The particulates of the CI1 meteorites are cemented together by water soluble evaporite minerals such as epsomite (MgSO4.7H2O) and gypsum (CaSO4.2H2O). The fact that these stones disintegrate immediately after they are exposed to liquid water was observed during the initial studies of the Alais meteorite (Thénard, 1806; Berzelius, 1834, 1836) and the Orgueil stones (Leymerie, M. 1864). These stones are destroyed and disaggregate into tiny particles as the water soluble salts that cement the insoluble mineral grains together in the rock matrix dissolve (Hoover, 2005).

Although ejecta from the moon and Mars are associated with several known meteorites, the parent bodies for vast majority of all meteorites on Earth are the asteroids. The high water content, D/H ratios, and the evidence of extensive aqueous alteration of the CI1 carbonaceous meteorites indicate their parent bodies were either water-bearing asteroids or comets.

1.1. Chemical, Mineral and Morphological Biomarkers in CI1 Carbonaceous Meteorites. A number of biominerals and organic chemicals (that are interpreted as biomarkers when found in Earth rocks) have been detected in CI1 carbonaceous meteorite. These include weak biomarkers such as carbonate globules, magnetites, PAH’s, racemic amino acids, sugar alcohols, and short chain alkanes, alkenes and aliphatic and aromatic hydrocarbons that are produced in nature by biological processes but can also be fomed by catalyzed chemical reactions such as Miller-Urey and Fisher-Tropsch synthesis. However, the CI1 meteorites also contain a host of strong biomarkers for which there are no known abiotic production mechanisms. These include magnetites in unusual configurations (framboids and linear chains of magnetosomes), protein amino acids with significant enantiomeric excess, nucleobases (purines and pyrimidines), and diagenetic breakdown products of photosynthetic pigments such as chlorophyll (pristine, phytane, and porphyrins), complex kerogen-like insoluble organic matter and morphological biomarkers with size, size range and recognizable features diagnostic of known orders of Cyanobacteriaceae and other prokaryotic microfossils. Table II provides a chronological summary of chemical, mineral and morphological biomarkers found by many independent researchers who have studied carbonaceous meteorites since 1806 when research began shortly after the fall of the Alais CI1 carbonaceous meteorite.

1.2. The Alais CI1 Carbonaceous Meteorite. Alais was the first carbonaceous meteorite known to science. Two thunderous detonations were heard across southern France at 5:30 P.M on March 15, 1806. Two soft, black stones that emitted a “strong odor of bitumen” were then observed to fall in small villages near Alais, Languedoc-Roussillon, France – (44o 07’N, 4 o 05’E). One black stone of 2 kg mass landed in the small village of St. Eteinne de Lolm and a 4 kg stone landed near Valence, France and broke a branch from a fig tree as it fell. Louis Jacques Thénard (1806), the reknowned Professor of Chemistry at the College de France in Paris, conducted the first study of the Alais CI1 carbonaceous meteorite. He realized that these stones were different from all other meteorites since they had the appearance of solidified clay. Thénard reported that “when the stones were placed in water they disintegrated immediately and gave off a strong clay-like odor.” He found the Alais meteorite stones contained 2.5% carbon and oxides of iron, magnesium, and nickel.

The Alais stone was subsequently analyzed by Jöns Jacob Berzelius, the distinguished Swedish organic chemist and mineralogist. Berzelius discovered the elements silicon, selenium, thorium and cerium. He obtained a small fragment of the Alais meteorite from the French mineralogist Lucas. Berzelius (1834, 1836) was initially astonished to find this stone contained water and almost discarded it as being contaminated. In the English translation provided by Nagy, (1975, p. 45) he says: “I was so suspicious, because this meteorite contained water that I was about ready to throw my sample away. However, fortunately before I discarded the sample, I reread the record and found certain data which completely agreed with the meteoritic origin of the stone. This intrigued me so that I then carried on the investigation with great interest. The question arose in my mind; does this carbonaceous earth contain humus or a trace of other organic substances? Could this give a hint to the presence of organic formations on other planets?” Berzelius was the first scientist to recognize that the Alais meteorite consisted mostly of clay-type minerals and he confirmed the observation first reported by Thénard that the Alais stones were destroyed by liquid water: "These stones are different from all other meteorites because they look like solidified clay and because when they are placed in water they disintegrate and give off a clay-like odor.” Berzelius concluded that the Alais meteorite contained a portion of metallic iron and nickel (12%) that was attracted to a magnet as well as indigenous extraterrestrial water and carbon, saying: “some organic matter and 10 per cent of a salt which contained no iron, being a mixture of sulphates of nickel, magnesia, soda, potash and lime with a trace of sulphate of amnonia.” When he heated the sample it turned brown and “gave off a tarry odor.” Berzelius reported “in water it disintegrates instantaneously to a greyish-green powder which has an odor reminding one of fresh hay.” He also found it to contain carbon dioxide and a soluble salt containing ammonia.

1.3. The Orgueil CI1 Carbonaceous Meteorite. The Orgueil meteorite is one of the most extensively documented and thoroughly investigated of all known meteorites. At 8:08 P.M. on May 14, 1864 a brilliant fireball illuminated a large region of southern France and thunderous explosions were heard as the blue-white fireball streaked across the sky, turned a dull red color and produced a long thin white smoke trail (Jollois, 1864; d’Esparbés, 1864). The weather was nice on this spring evening in the south of France. Soon after the explosions were heard, a shower of stones fell within an 18 km east-west scatter ellipse between the villages of Orgueil, Campsas and Nohic (Tarn-et-Garonne). The main fall occurred near the village of Orgueil (43o 53’ N; 01o 23’ E) and villagers collected over 20 jet-black stones immediately after the fall. Many of the Orgueil stones had complete fusion crusts and a few were quite large (one with mass ~11 kg). The Orgueil bolide was so spectacular that many villagers at St. Clar thought they were surrounded by flames. The Marquise de Puylaroque (1864) reported that her house looked like “the interior of a furnace” and she heard a rumbling noise that sounded like firearms and lasted for 2-3 minutes. The detonations were so violent that some villagers thought the event was an earthquake (Bergé, 1864). Eyewitness reports from all over the region were sent to M. Le Verrier, Director of the Imperial Observatory, and to the eminent geologist Academician G. A. Daubrée. These accounts were published immediately (Daubrée, 1864) and English translations have subsequently been made available (Nagy, 1975). The observations of the fireball and timing of the detonations made it possible to set the upper limit for the altitude at which the bolide exploded at 30 km and to conclude that the main part of the meteoritic mass continued to move in its orbit after the explosion leaving “only a few minor pieces of its pre-terrestrial body” (Daubrée and Le Verrier 1864).

M. Leymerie (1864) examined a 211g stone that fell in Campsas and reported that the interior of the Orgueil stones exhibited a “stunning difference” as compared with ordinary stony meteorites: “The broken surface reveals a dark charcoal colored substance so soft that it can be easily cut with a knife. One can even write with the fragments on a piece of paper. The knife cut creates smooth and shiny surfaces which is an indication of fine, paste-like matter. Fragments placed in water disintegrate immediately.” This astonishing observation that the Orgueil stones immediately disintegrated into minute particulates when they came in contact with cold water was independently confirmed by Cloëz (1864a) and Pisani (1864) exhibiting a behavior similar to that of the Alais meteorite. Cloëz (1864a) correctly recognized that the Orgueil meteorite was a breccia composed of microscopic particles cemented together by magnesium sulfate and other water soluble salts. When these salts dissolve in water, the tiny particulates that constitute the Orgueil meteorite are released from the matrix. He found that the Orgueil stones also disintegrate in alcohol, but that the dispersed particles released were not as finely divided and that the disintegration in alcohol takes place more slowly than in water. These very important observations were recently confirmed at the NASA Astrobiology Laboratory using video optical microscopy and Environmental Scanning Electron Microscopy (ESEM) methods. Small samples of Orgueil meteorite stones were placed on a sterile silicon wafer and exposed to a droplet of sterile de-ionized water at 20 oC. Immediate profuse effervescence was observed and within a few minutes the samples were completely disaggregated into an assemblage of micron-sized particulates. Immediately after the water had evaporated a white residue was found on the silicon wafer substrate around the meteorite mineral grains and particulates. Energy Dispersive X-Ray Spectroscopy (EDS) analyses established that the residue was composed primarily of magnesium, sulfur, and oxygen, which is consistent with magnesium sulfate (Hoover, 2005a; Hoover, 2006a).

The Orgueil silicate minerals are more properly designated as serpentine rather than peridotite. The dominant mineral (62.6 %) of the Orgueil meteorite is Chlorite [(Fe, Mg, Al)6(Si, Al) 4O10(OH)8] of the clay phyllosilicates mineral group. The other major minerals of Orgueil include: 6.7% Epsomite (MgSO4.7H2O); 6% Magnetite (Fe3O4); 4.6% Troilite (FeS), 2.9% gypsum (CaSO4.nH2O) and 2.8% Breunnerite (Fe,Mg)CO3. Epsomite forms white veins in the meteorite and is a significant evaporite mineral that helps cement together the meteorite particulates into the stones. In 1868, Pierre Marcellin Berthelot The famous French chemist who had shown in 1860 that all organic compounds contain C, H, O, and N experimented with hydrogenation to explore the organic chemistry of the Orgueil CI1 meteorite. He found complex hydrocarbons in Orgueil that were analogous to carbonaceous substance of organic origin on Earth (Berthelot, 1868).

It is now well established that the total organic content of carbonaceous meteorites consists of 90-95% polymer-type organic matter that is insoluble in common solvents. Nagy (1975) reported that this substance is “structurally not unlike coal or the aromatic-type kerogen that is the insoluble organic matter encountered in terrestrial sedimentary rocks.” The complex polymer-like organic matter similar to kerogen in the carbonaceous meteorites is clearly indigenous and constitutes an important biomarker. In terrestrial rocks, Kerogen, isoprenoids, pristine, phytane and other biochemical fossils have long been considered valid biomarkers. Kaplan (1963) reported that the Ivuna CI1 meteorite contained significantly larger quantities of pristane and phytane (diagenetic breakdown components of chlorophyll) than the Orgueil and Alais meteorites. These types of geochemical biomarkers comprise a standard tool for petroleum exploration as they are stable of geologically significant time periods (~ billions of years). On Earth they are undeniably biological in origin. Diagenesis and catagenesis processes that alter the original biochemicals is usually minimal and the basic carbon skeleton remains intact. For this reason, although functional groups (e.g., -OH, =O, etc.) may be lost, the chemical structure derived from biological origins of these stable fossil biomolecules remains recognizable.

1.4. The Ivuna CI1 Carbonaceous MeteoriteThe Ivuna CI1 carbonaceous meteorite fell near Ivuna, Mbeya, Tanzania (8° 25'S, 32° 26'E) in southeast Africa at 5:30 P.M on December 16, 1938. Approximately 705 gms were recovered shortly after the stones were observed to fall. Clayton (1963) investigated carbon isotope abundances in the carbonates of both the Ivuna and Orgueil meteorites and found the Ivuna carbon isotopic values to be virtually identical to those of Orgueil. The δ13C value for these meteorites was approximately +60 per mil, which is dramatically different from the abiotic or biotic terrestrial carbon. This provides conclusive evidence that the meteoritic carbon is extraterrestrial in origin and cannot be associated with terrestrial bio-contaminants. The Alais, Ivuna and Orgueil CI1 meteorites have also been found to contain chiral amino acids, nucleobases, pristine and phytane, spectacular magnetite framboids and platelets, and the well-preserved and mineralized remains of diverse filaments interpreted as the mineralized remains of cyanobacteria and other trichomic prokaryotes. Amino acid analyses using HPLC of pristine interior pieces of the Orgueil and Ivuna meteorites resulted in the detection of β-alanine, glycine, and γ-amino-n-butyric acid (GABA) at concentrations ranging from ≈600 to 2,000 parts per billion (ppb). Other α-amino acids such as alanine, α-ABA, α-aminoisobutyric acid (AIB), and isovaline are present in trace amounts (<200 ppb). Carbon isotopic measurements of β-alanine and glycine and the presence of racemic (D/L ≈ 1) alanine and β-ABA have established that these amino acids are extraterrestrial in origin. The amino acid composition of the CI1 meteorites is strikingly distinct from that of the Murchison and Murray CM2 carbonaceous meteorites. This indicates that the CI1 meteorites came from a different parent body than CM2 meteorites, possibly from an extinct comet.

Claus and Nagy (1961) studied the Ivuna and the Orgueil CI1 meteorites and found a large number of forms that they originally interpreted as indigenous microfossils. After intense criticism, they subsequently designated them as “organized elements” so as to not make any judgement as to their biogenicity. Since they used standard palynological methods to dissolve the rock matrix in acids for extracting the insoluble kerogen-like bodies, any unseen pollen contaminants on the exterior surfaces of the meteorite would remain intact and be concentrated in the acid-resistant residue they analyzed. They failed to recognize a pollen grain and erroneously included an image of it in their original paper. This resulted in their work being discredited and it is still widely believed that all of the the “organized elements” they described were either abiotic mineral grains or pollen. Subsequent work by Rossignol-Strick and Barghoorn (1971) revealed that the “organized elements” type microstructures were in fact not pollen grains and were indigenous to the meteorites, but their forms are too simple to make any decision regarding whether they are abiotic or biogenic in origin.

1.5. The Tonk and Revelstoke CI1 Carbonaceous Meteorites Although the mineralogy and petrology of the Tonk and Revelstoke CI1 meteorites have been carried out, no data has yet been published on the organic chemistry, amino acids and other possible chemical or morphological biomarkers that may be present on these meteorites has ever been published. This is undoubtedly due to the fact that only a tiny amount of the Tonk (7.7 g) and Revelstoke (1 g) meteorites was recovered. After the Tonk meteorite fell, it spent two years at an unknown location in India (Christie, 1913). The Revelstoke meteorite fell on a frozen lake in Canada. It remained on the ice for almost two weeks before the stone was recovered (Folinsbee et al., 1967). Both the Tonk and Revelstoke meteorites have been found to contain hydrated magnesium and calcium sulfates (Christie, 1913; Endress et al., 1994). Larson et al. (1974) performed thermomagnetic analysis of all five CI1 meteorites known at the time and found that the predominant phase of magnetite in the Revelstoke meteorite was essentially Nickel-free Fe3O4. This was in contrast to the other four CI1 meteorites known at the time, which all contained magnetite with nickel at <6%. Based on their studies of saturation moments, the weight percentage of magnetite for the CI1 meteorites was reported to be: Alais (5.3 ± 0.4%); Orgueil, (11.9 ± 0.8%); Ivuna, (12.2 ± 0.9%); Tonk, (9.4 ± 0.6%) and Revelstoke, (7.2 ± 0.5%).

2. MATERIALS AND METHODS

Samples used in this study were:

Ivuna CI1 Carbonaceous Meteorite

DuPont Meteorite Collection, Planetary Studies Foundation, Chicago

1 stone: (0.1 gm). Courtesy: Dr. Paul Sipiera

Orgueil CI1 Carbonaceous Meteorite

Musée Nationale d’Histoire Naturelle, Paris

1 stone S219: (0.5 gm) Courtesy: Dr. Claude Perron

2 stones: (0.6 gm & 0.3 gm) Courtesy: Dr. Martine Rossignol-Strick

DuPont Meteorite Collection, Planetary Studies Foundation, Chicago

2 stones: (0.4 gm & 0.1 gm). Courtesy: Dr. Paul Sipiera

EDS elemental analysis of the Orgueil filaments indicated that many of them were carbonized sheaths that infilled with magnesium sulfate minerals. To evaluate the morphology, chemical composition and size and size range of abiotic mineral structures that might have similar compositions, samples of native fibrous epsomite were obtained and studied using the Hitachi FESEM and the FEI Quanta ESEM and FESEM. The biotic mineral samples investigated were:

Modern Cyanobacteria

Plectonema (Lyngbya) wollei–Lake Guntersville, Al

Richard B. Hoover - Collected in May, 2004 (Living Environmental Sample)

Lyngbya (Leptolyngbya) subtilis – Lake Michigan,

Courtesy: Ann St. Amand, Phycotech, Inc. (Fixed Environmental Sample)

Oscillatoria lud –UTex Collection LB 1953

F. T. Haxo - Deposited October, 1972 in UTex Collection as C-43 (Living Axenic Culture)

Arthrospira platensis – Carolina Biological Supply (Living Axenic Culture)

Calothrix sp.–Little White River, Oregon,

Courtesy: Ann St. Amand, Phycotech, Inc. (Fixed Environmental Sample)

Living Microbial Extremophiles

Carnobacterium pleistocenium str. FTR-1T; Fox Tunnel, Alaska

Richard B. Hoover-Coll. May, 2000 - Pleistocene ice (Living Axenic Culture; Type Strain)

Dried Herbarium Material

Bangia quadripunctata, Lyngbye–Collected 5/20/1816 Hoffman Bang, Hals Peninsula Courtesy: Dr. Walter van den Bergh, Henri van Heurck Museum, Antwerp, (Dried Type)

Fossilized Cyanobacteria

Upper Archaean (Lopian) tufa-genic rocks (2.8 Ga) of Northern Karelia

Courtesy: Dr. Alexei Yu. Rozanov, Paleontological Institute, RAS, Moscow

Hair and Tissues of Egyptian Mummies (2 Kya & 5 Kya) and Mammoths (32 Kya & 40 Kya)

Lyuba Mammoth Tissue & Stomach Milk Samples Courtesy: Dr. Daniel Fisher, Univ. Michigan

 

Cambrian Trilobites

Perinopsis interstricta & Perinopsis pygidia – Cambrian (505 Mya) Wheeler Shale, Utah

Coll. Richard B. Hoover

Instruments used in this study were:

ElectroScan Environmental Scanning Electron Microscope (ESEM)

• Secondary Electron Detector (SED); Water vapor (10 Torr vacuum) 90-100,000X

• Noran EDS (Z> Boron)

Hitachi S-4100 Field Emission SEM (FESEM)

• Cold cathode field emission electron gun; 20 - 300,000X;

• Secondary Electron Detector (SED) and Backscattered Electron Detector (BSED);

• KEVEX EDS - Lithium Drifted Silicon detector (Z>Boron)

 

Hitachi S-3700N Variable Pressure Scanning Electron Microscope

• Tungsten emitter electron gun; 5 - 300,000X; SED & BSED;

• 4 Pi EDS - Silicon Drifted Silicon Detector (Z>Boron)

 

FEI Quanta 600 (FESEM and ESEM)

• Simultaneous SED and BSED images; 5 - 300,000X

• 4 Pi EDS - Lithium Drifted Silicon detector (Z>Boron)

 

In order to minimize the possibility of the detection of coating artifacts or recent contamination by terrestrial biological materials, the study was confined to investigations of uncoated, freshly fractured, interior surfaces of the meteorites. All tools, sample holders and stubs were flame sterilized. Lunar dust samples and silicon wafers were used as negative controls. To further protect the samples from biological contamination, the meteorites were stored in sealed vials at -80 oC and after preparation, electron microscopy stubs were kept in sealed containers in dessicator cabinets or in the freezer. The fusion crust and old cracks in the stones were carefully avoided. The meteorite samples were placed in the instrument chamber (with the fresh fracture surface up) a pumped down immediately after the stones were fractured. All solvents, acids or other liquids were strictly avoided. Acids and solvents were used in early studies by other workers h to extract “acid resistant microfossils” from the host rock. However, this method sometimes resulted in the inadvertent contamination of the sample with acid resistant modern pollen grains that could have been on the external surface or in old fissures within the stones. It should be noted that only one seriously Murchison sample was found to be contaminated with fungal filaments (in old cracks in the fusion crust) and not a single pollen grain has been encountered during extensive studies of carbonaceous meteorites carried out since 1996 at the NASA/Marshall Space Flight Center. While fingerprints, pollen grains, fungi, bacteria, laboratory oils and other contaminants could be encountered on the fusion crust or exposed old cracks and fractured surfaces of meteorites, the hypothesis that the interior surfaces carbonaceous meteorites are seriously contaminated by modern bacteria, fungi, or pollen is simply not consistent with the observational results. For this reason, it is extremely important that studies of biomarkers such as amino acids, nucleobases, complex organic chemicals and microfossils should be rigorously restricted to freshly fractured interior portions of the stones.

3. OBSERVATIONAL RESULTS AND INTERPRETATIONS

Field Emission Scanning Electron Microscopy (FESEM) studies of the interior surfaces of freshly fractured CI1 carbonaceous meteorites carried out at NASA/MSFC resulted in the detection of a diverse suite of large and complex filamentous microstructures embedded in the matrix of carbonaceous meteorites. Energy dispersive x-ray analysis of these structures reveals that these filaments are permineralized with minerals rich in magnesium and sulfur. Most of the filaments are encases within a carbon-rich external envelope. Images and EDS elemental data for several selected filaments are presented. To increase readability, the interpretation for each set of images is presented immediately after the Observational Results section for each Figure.

3.1. 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

3.1.1 Interpretation and Discussion of Images and EDS Data of the Orgueil Filaments. 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.

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 http://www.cyanodb.cz//Phormidium/Phormidium.jpg 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.




Figure 6.a. EDS data on Nitrogen content of filaments in Ivuna and Orgueil meteorites compared with modern fungal contaminant in Murchison and living, dead and fossil cyanobacteria, mummy/mammoth hair, trilobites & 2.7 Gya cyanobacteria; b. Mammoth Hair with beam damage at EDS spot & strong Nitrogen peak (N 11.9% atomic).

Figure 6.a is a compilation of the nitrogen level measured by EDS for a number of the filaments encountered in Ivuna and Orgueil CI1 carbonaceous meteorites compared with modern and ancient terrestrial life forms. The meteorite filaments are typically severely depleted in nitrogen (N < 0.5%) whereas life forms on Earth have nitrogen levels from 2% to 15%. Nitrogen is encountered at detectable levels even in the hair/tissue of mummies from Peru (2Kya) and Egypt (5Kya) and the hair and tissues of Pleistocene Mammoths (40-32 Kya). Fig. 6.b is a FESEM image the Guard Hair of a 32,000 year old Pleistocene mammoth collected by Hoover in the Kolyma Lowlands of NE Siberia. The square spot in the image is EDS beam damage (10 KeV electron beam) during spot analysis, which revealed a strong peak (11.94% atomic) at the nitrogen Kα line between the Carbon and Oxygen lines. Even though the biological material was 32,000 years old, the nitrogen of the proteins was still present. Similar results have been obtained for cyanobacterial filaments found in the stomach milk of the 40,000 year old baby mammoth “Lyuba” and for pre-dynastic (5,000 year old Egyptian mummies. However, truly ancient biological materials (e.g., Cambrian trilobites from the Wheeler Shale of Utah (505 Mya) and 2.7 Gya filamentous cyanobacteria from Karelia) have nitrogen levels below the limit of detection with the FESEM EDS detector. These results provide definitive evidence that the filaments encountered in the CI1 carbonaceous meteorites are indigenous to the stones and are not the result of microbiota that invaded the stones after they arrived on Earth in 1864 or 1938. Hoover (2007) has discussed the use of Nitrogen levels and biogenic element ratios for distinguishing between modern and fossil microorganisms as a mechanism for recognizing recent biological contaminants in terrestrial rocks and meteorites.

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