2.1

Water

Water is second most abundant molecule (only H₂ molecules are more abundant) in the Universe.. Water is also an absolute requirement for all known forms of life. This is largely because the H2O molecule possesses a unique suite of physical and chemical properties. The water molecule is polar and it has an angular shape due to the paired bonding of the electrons shared between the oxygen and hydrogen atoms. Because of the charge separation and the shape of the dipole water molecule, the oxygen atom has a partial negative charge and the hydrogen atoms a partial positive charge. This makes water molecules behave like tiny magnets. Without this property, water would be a gas at room temperature. This also makes water a good solvent ideally suited for supporting organic chemistry and capable of dissolving most biological molecules. Water has a high dielectric constant ensuring solubility of polar species and flexibility for biological structures stabilized by electrostatic interactions. It also has a high heat capacity and an astonishingly rich phase diagram with fifteen crystalline phases observed in laboratory experiments and eight others predicted theoretically. Water remains in liquid state over a wide range of temperatures. It is now well known that there exists a vast diversity of living organisms that inhabit deep-sea hydrothermal vents at temperatures well above 100 C.

It is also known that carbonaceous meteorites contain ~3%-20% indigenous water, extraterrestrial amino acids, nucleobases and other life-critical biomolecules and it is generally accepted that the parent bodies of the CI1 and CM2 carbonaceous meteorites are water-bearing asteroids or comets.^1,2 Water is the dominant volatile on comets and the icy moons of Jupiter and Saturn. To the great astonishment of many scientists, recent observations by the Spitzer Telescope and the Dawn Mission provided evidence that water ice and organic molecules also exist on the asteroids 24 Themis and 65 Cybele at the outer edge of the asteroid belt (Fig. 1.a).^3,4 Data from the ESA Herschel Space Observatory indicates that water vapor, possibly from ice volcanism, is being spewed into space from Ceres, the largest asteroid (also considered a dwarf planet) of the solar system (Fig. 1.b).^5,6. The ESA/Rosetta/NavCam image (Fig. 1.c) shows water jets being spewed from the comet 67P/Cheryumov-Gerasimenko. It has been shown that the surfaces of asteroids and the nuclei of comets are jet-black (albedo ~ 0.04) and these dark bodies can be heated by absorbed sunlight to temperatures well above the melting point of pure water ice (273 K) even at distances beyond the orbit of the planet Mars. Cracks within the surface would allow the water vapor to rapidly vaporize and vent directly into space. However, if these avenues of escape are impeded as the water re-freezes sealing the surface cracks, pressures within cavities could become high enough to allow liquid water pools and films to exist for periods of time beneath the surface of the asteroid or the comet’s crust. During such periods, cyanobacteria, sulfur bacteria, archaea, diatoms, algae or other microorganisms might find conditions suitable for growth. Some of these microorganisms have doubling times of only a few hours even at low temperatures, which would permit sizeable populations to develop in water-bearing asteroids or cometary nuclei in only a matter of days or weeks.

Fig. 1.

Illustration of water ice and water vapor being spewed a. from the asteroid 65 Cybele and b. the asteroid or dwarf planet Ceres and c. Rosetta/NavCam image showing water jets from the comet 67/P Cheryumov-Gerasimenko. Image Credits: a. Gabriel Pérez, Instituto de Astrofìsica de Canarias, Spain; b. Pennington Planetarium; c. ESA/Rosetta/NavCam/Emily Lackawalla

When salts are present, water remains unfrozen at temperatures far below zero. Microorganisms thrive in the frigid hypersaline Don Juan Pond of Antarctica, cryopegs deep beneath the Siberian permafrost and tidal pools of Southern Patagonia.⁷ Water molecules can form a three-dimensional hydrogen bonded network which facilitates its participation in a wide variety of life-critical biochemical processes. When water freezes a slight expansion of hydrogen bond angles results in the solid phase having lower density than the liquid phase and consequently ice floats. This is important to life, because lakes, rivers and oceans freeze from the surface down rather than the bottom up and the icy surface layer provides insulation from sub-zero atmospheric temperatures. This allows large bodies of liquid water to exist deep beneath the surface of frozen lakes, seas or the polar ice caps of Earth. For this reason, liquid water oceans or pools may be present beneath the frozen crusts of the icy moons of Jupiter and Saturn or the frozen surfaces of comets and asteroids. Water is so essential to life that “Follow the Water” was adopted as the central strategy of the NASA Astrobiology program searching for evidence for life elsewhere in the Cosmos.

While it is a widely held belief that large bodies of liquid water are necessary for life, this assumption is incorrect. It has long been known that cryptoendolithic lichens and purplish red cyanobacteria (the Red Gloecapsa Community) colonize very thin films of liquid water within the photosynthetic regimes (upper layers) of sandstones in the polar regions of Earth.⁸ Virtually all cells of prokaryotic and eukaryotic organisms have the ability to be freeze-dried and stored for long periods of time at liquid nitrogen temperatures (lyophilization). Abyzov et al. ^9,10 established that microorganisms remain alive in a super-long state of anabiosis within ancient layers of the central Antarctic ice sheet above Lake Vostok. Many bacteria and diatoms use special enzymes (ice active substances) to locally melt ice and remain alive in the solid ice of glaciers, polar ice sheets, cryopegs at temperatures below –10 C.^11-13 The novel lactic acid bacterium, Carnobacterium pleistocenium isolated from a frozen thermokarst pond in Fox Tunnel. Alaska can grow at −5 C.^15,16

2.1

Energy Sources

Living organisms employ a variety of mechanisms to obtain the energy necessary to carry out metabolism, growth, replication and for the myriad of other vital processes associated with the biochemical machinery of life. The different mechanisms used to harvest energy make it possible to divide organisms into broad groups:

Autotrophs grow by carbon fixation - the process by which living organisms convert inorganic carbon from carbon dioxide into the array of organic compounds and biomolecules needed for life. Carbon and Nitrogen are unique in that they cannot be used by living organism until after they have been fixed by other life forms. Inorganic atmospheric CO₂ is converted into organic carbon via complex metabolic pathways found only in living organisms. There are presently six known autotrophic carbon fixation pathways.

Photoautotrophs perform carbon fixation via the process of photosynthesis with photons as the energy source. By far the most important autotrophic carbon fixation process is oxygenic photosynthesis. In the late 1940’s, Melvin Calvin and his team from the Radiation Laboratory of the University of California, Berkeley first described the photosynthetic process. They used radioactive Carbon-14 tags and green algae to decipher the complex metabolic process in which enzymes, proteins and chlorophyll pigments contained in the stroma of chloroplasts use the energy of sunlight to produce sugars and other carbohydrates and release oxygen from water and CO₂. Calvin received the Chemistry Nobel Prize in 1961 for the discovery of this fundamental process of life which is now known as the Calvin Cycle.⁸ During the first stage of the carbon fixation process the Ribulose-1,5-Biphosphate Carboxylase/Oxygenase enzyme (RuBisCO) incorporates carbon dioxide into the five-carbon sugar ribulose biphosphate (RuBP). The life-critical biomolecule RuBisCO is found in the chloroplasts of cyanobacteria, algae, diatoms and other plants and may well be the most abundant protein in Earth. The coenzyme Adenosone Triphosphate (ATP), the energy carrier in all living cells, is spent as the energy source and NADPH is consumed as the reducing power to add high energy electrons in the last step of the electron chain of light reactions of the complex photosynthetic process in which sugars are produced from CO₂.

Five other autotrophic carbon fixation cycles are presently known in living organisms. These include reductive citric acid cycle (Arnon-Buchanan Cycle) which occurs within anoxygenic photosynthetic bacteria (e.g;, the green sulfur bacterium Chlorobium thiosulfatophilum); the reductive acetyl Co-A pathway (Wood-Ljungdahl pathway); and the pathways used by the anaerobic and microaerobic Archaea and Crenarchaeota.¹⁹

Carbon fixation can also occur by chemosynthesis in the absence of light. Chemotrophs are organisms that acquire energy by the oxidation of electron donors in their environment. Chemoorganotrophs obtain carbon and energy by consuming organic molecules as food. Chemolithotrophs extract carbon and energy from inorganic molecules. Chemotrophs can be either autotrophic or heterotrophic. Chemoautotrophs can perform carbon fixation and obtain their energy from chemical reactions using molecular hydrogen, methane, hydrogen sulfide elemental sulfur, etc. Oxygen is released as a by-product as the organisms break up the carbon dioxide molecules to synthesize all necessary organic compounds. Most chemoautotrophic life forms such as thermoacidophiles, halophiles, methanogens, sulfur oxidizers, sulfur reducers and archaea thrive in extreme environments like deep crustal rocks and deep-sea hydrothermal vents. Chemoheterotrophs are unable to fix their own carbon and can use sulfur or other inorganic energy sources or organic sources-lipids, proteins or carbohydrates. Many organisms are Organotrophs, and obtain their energy and biomolecules by consuming organic matter manufactured by the metabolic process within the cells of other living organisms.

2.2

Biogenic Elements

The diversity and complexity of life on Earth is so great and their range of habitats so broad that almost all of the 92 naturally occurring chemical elements on Earth may play a role in some of the different organisms from time to time. But there is a subset of approximately 20 of these elements that are absolutely essential for all known life forms on Earth and therefore have been called “biogenic elements”. Water and all of the life-critical biogenic elements are found in carbonaceous meteorites. The ratios of biogenic elements in biological remains change through diagenetic process over geological time periods. Ratios of the biogenic elements carbon, oxygen and nitrogen provide a powerful tool for distinguishing between modern biological contaminants and indigenous microfossils in terrestrial rocks and meteorites.²⁰

There are six Major Biogenic Elements (C, H, O, N, P, S) that are absolutely essential for all living organisms and abundant in all cells. There are five Minor Biogenic Elements (Ca, Cl, Mg, K, Na) that are also life-critical but typically present in cells in relatively small quantities and several Trace Biogenic Elements (Si, Mn, Fe, Cu, Zn, As, Ni, I, V) which are present in very small quantities but essential for the chemical reactions and metabolic pathways upon which life depends. These six major biogenic elements make up more than 97% of the matter of all living cells. The first four are by far the most abundant as they are present in the water, and all of the carbohydrates, amino acids and proteins found in every living cell. Phosphorus and sulfur are present in much smaller quantities in living organisms. Phosphorus is essential for life-critical biomolecules such as DNA, RNA, ATP (adenosine triphosphate), NADP (nicotinamide adenine dinucleotide phosphate) and many life-critical proteins and enzymes. Sulfur is essential for the amino acids methionine and cysteine and therefore a key component of most proteins. Nitrogen comprises almost 78% of our atmosphere in the form of diatomic nitrogen (N₂) molecules held together by a strong triple bond. Atmospheric nitrogen is a relatively inert molecule that is completely useless to life until it has been converted by the process of nitrogen fixation.²¹ Abiotic nitrogen fixation can occur in nature when the enormous energy of lightning strikes breads the triple bond and forms nitrates. Nitrogen fixation can also be achieved by synthetic mechnisms c(e.g., Haber-Bosch, cyanimide or electric arc processes) which usually require very high temperatures. Biological Nitrogen Fixation is by far the dominant mechanism for nitrogen fixation on Earth. Biological Nitrogen Fixation occurs when cyanobacteria, bacteria or archaea accomplish the reduction of the N2 molecule to ammonia, nitrates, or nitrogen dioxide. Cyanobacteria play the dominant role in nitrogen fixation on Earth. This is typically achieved via the nitrogenase enzyme that is found in several genera of heterocystous cyanobacteria (e.g. Nostoc, Calothrix, etc.). However, biological nitrogen fixation can also be performed by several species of non-heterocystous cyanobacteria and other diazotrophic microorganisms of the families Azotobacteracea, Rhizobiaceae, and Actinomycetaceae. Biological nitrogen fixation occurs in the heterocystous cyanobacteria, via the nitrogenase enzyme contained within the thick-walled specialized nitrogen fixing cells known as heterocysts. The heterocysts have hyaline, refractive walls that provide well-protected centers in which the nitrogenase enzyme can function. The heterocysts produce three additional cell walls, including one with glycolipid that forms a hydrophobic barrier to oxygen to protect the nitrogenase enzyme, which oxygen inactivates. To provide further protection, the heterocysts themselves lack photosystem II (therefore producing no oxygen) and any of these organisms also up-regulate glycolytic enzymes and produce proteins that scavenge for any remaining oxygen.²²

2.3

Biomolecules

Biomolecules are typically considered to be organic molecules that are produced by living organisms. They include relatively small organic molecules and large biomolecules like RNA, DNA and proteins. Many small biomolecules like amino acids, can be produced by abiotic processes such as Strecker or Miller-Urey synthesis.²³ However, when these small molecules are found on Earth, they apparently resulted result from biology as they exhibit properties (e.g., homochirality and biological fractionation of stable isotopes) that make distinguishable from abiotic molecules. Small biomolecules are often produced by metabolic pathways or enzymatic activity as primary or secondary metabolites. These include proteinogenic amino acids, sugars such as ribose (C₅H₁₀O₅), deoxyribose (C₅H₁₀O₄) and nitrogenous nucleobases guanine (C₅H₅N₅O), cytosine (C₄H₅N₃O), adenine (C₅H₅N₅), thymine (C₅H₆N₂O₂), and uracil (C₄H₄N₂O₂). In the canonical Watson-Crick base pairing of DNA, Guanine always pairs with Cytosine and Adenine pairs with thymine (replaced by uracil in RNA). These small biomolecules are “life-critical” as they the fundamental building blocks for the formation the large biomolecules that are essential to all known living organisms.

2.3.1

Chiral Amino Acids and Sugars

Amino acids comprise the basic building blocks of all proteins and enzymes. Several hundred amino acids are known, but only small subset of some twenty or so “proteinogenic” amino acids are the monomers for a host of complex protein polypeptide chains that are essential to life for every organism on Earth. Each of the amino acids consist of four clusters of atoms branching out in three-dimensional space. Amino acids are are composed of an amine (-NH₂) functional group linked to a core central α-carbon atom bonded to a hydrogen atom, a carboxylic acid (-COOH) group, and a variable-R side chain. The chemical composition of the variable side chain (-R) uniquely defines each distinct amino acid. The location in three-dimensional space of the -R side chain determines the chirality. The location in three-dimensional space of the -R side chain determines the chirality. Chiral molecules are asymmetric and occur in non-superposable mirror images. The term “enantiomers” refers to the two stereoisomers that are related to each other by reflection.

Fig. 2.

Spatial configuration of the L- and D- enantiomers of the amino acids Alanine (CH3NH2COOH) and Phenalyalanine C₆H₅CH₂CH(NH₂)COOH. (C-Grey; H-White; O-Red; N-Blue).

In 1815 the French chemist Jean-Baptiste Biot demonstrated that some organic molecules rotated the plane of polarization of polarized light.²³ Chiral molecules have optical activity are termed “dextrorotary” D- or “laevorotary” L-based on whether they rotate the plane of a beam of polarized beam in a “right handed” or clockwise (D-) manner or in a “left handed” or counterclockwise (L-) direction. Since the L- and D- enantiomers are mirror images of one another they are not superimposable in space in a manner analogous to your right and left hands. (The term “chiral” is from the greek word for “hand”.) This phenomenon of chirality was first explained on a molecular basis by Louis Pasteur.^24,25 In 1848 Pasteur suggested that the origin of chiral biomolecules might hold the key to the origin and nature of life. All amino acids found in proteins have the same absolute steric configuration as L-glyceraldehyde, with the sole exception of the Glycine. Glycine, the smallest amino acid, has a single hydrogen atom for the -R side chain. It is symmetric and therefore is achiral. In addition to amino acids, sugars and many other biomolecules are asymmetric and exhibit chirality.

There are many different hypotheses for the pre-biotic formation of chiral molecules.²⁶ These include the action of: A. Linearly polarized light from neutron stars; B. Circularly polarized light from faint stars or planet suaces; C. Inclusion polymerization in chiral clays; D. Parity violation in weak interactions (Vester-Ulbricht hypothesis); E. High pressure acting on racemic mixtures; F. Asymmetric crystals or inorganic surfaces acting as catalysts; G. Soia asymmetric autocatalysis reaction.^27-32 Experiments have shown that most these are amplification processes produce very low yields of L-amino acids or D-sugars and none have found shown to produce enantiopure or homochiral assemblages as are found in biology.

2.3.2

Homochirality and Life

Homochirality of asymmetric biomolecules plays a crucial role in life and it is one of the most distinctive biochemical signatures of Life. It is also one of the most mysterious, since the manner of the origin of homochirality is not at all understood. In every living organism, essentially all amino acids in proteins are the L- enantiomer and all of the RNA and DNA molecules have exclusively right-handed pentose sugars (D-ribose and D-deoxyribose). After the organism dies, the homochirality of amino acids and sugars (which represents a strong signature of life) begins a slow diagenetic process toward racemization. In a racemic mixture the two enantiomers are present in equal numbers (D/L=1.0). The phenomenon of homochirality is crucial for proper folding of the enormously complex life-critical macromolecular biopolymers, such as the right-handed helical RNA and DNA biomolecules that encode the genetic instructions; pigments that harvest and store energy; and proteins and enzymes that catalyze complex metabolic reactions which produce the multitude of biomolecules essential for living organisms.

Proteins are only synthesized by ribosomes within living cells by a strictly regulated and error-checked multi-step gene expression process known as translation. During translation, ribosome complexes within the cytoplasm bind to the messenger RNA (mRNA) and produce a specific amino acid polypeptide chains that subsequently folds into an active protein.³³ Protein molecules can be astonishingly large. Titin, the largest known protein, has a mass of ~3 megadaltons and it is composed of over half a million atoms (C₁₆₉₇₂₃H₂₇₀₄₆₄N₄₅₆₈₈O₅₂₂₄₃S₉₁₂₀) folded in an exquisite package that incorporates some 34,350 amino acids.³⁴ (By definition a Carbon 12 atom has a mass of 12 Daltons.) Proteins are polypeptides that are composed of chains of amino acids, and therefore they contain large amounts of nitrogen. Nitrogen is also present in the nucleobases of the DNA and RNA molecules. Nitrogen atoms are shown as blue spheres in the ball and stick models of the B-DNA dodecamer of 5′ subunit of the DNA molecule (Fig. 3.a.) and the crystal structure (Fig. 3.b.) of 14 kilodalton fragment of the alpha-subunit of phycoerythrin pigment of the Cyanobacterium Phormidium tenue.

Fig. 3.

Ball and Stick model of a. portion of 5′ subunit of DNA molecule and b. crystal structure of fragment of of Phycoerythrin pigment from Cyanobacterium Phormidium tenue. (C-Grey; H-White; O-Red; N-Blue; P-Orange; S-Yellow).

After death of the organism, the amino acids in proteins begin a long slow process of being destroyed by a variety of biological and biogeochemical processes. Nitrogen is initially removed from biological materials by the process of deamination in which the amine group is removed from amino acids and converted into ammonia. In living organisms, deaminase enzymes usually catalyze the deamination so the components of unneeded amino acids can be recycled or oxidized for energy or incorporation into other biomolecules. Amino acids and proteins within cells are often re-used after consumption by organotrophs. After burial, these biomolecules are usually broken down and the organic nitrogen molecules re-converted back into N2 molecules and returned to the atmosphere. This is the crucial component of the Nitrogen cycle, by which the nitrogen atoms sequestered in amino acids, proteins and other organic nitrogen molecules in living organisms are slowly converted (by bacterial denitrification; anaerobic ammonium oxidation or by the breakdown of nitrogen bearing rocks) back into atmospheric dinitrogen (N₂) molecules. This process of the breakdown of amino acids and the removal of organic nitrogen from living cells can be very slow, especially at low temperatures. The U. S. Army Cold Region Research and Engineering Laboratory (CRREL) Permafrost Tunnel near Fox, Alaska is a direct window into the Pleistocene. The CRREL Fox Tunnel contains a frozen record of Pleistocene life. Some of the metazoans (such as tardigrades and moss) are still viable after having been frozen in the permafrost for many millenia. Field Emission Scanning Electron Microscope (FESEM) images are shown for: Fig. 4.a living Pleistocene moss¹⁷ from the Fox Tunnel (grown in NASA/NSSTC Astrobiology Laboratory after being frozen for 32,000 years) and Fig. 4.c. the guard hair of a Woolly Mammoth. Strong nitrogen peaks are shown in EDAX spectra: Fig. 4.b. for edge of leaf (~4% atomic) of Pleistocene sedge (Carex sp.) and guard hair of Woolly Mammoth (11.6% atomic). The square spot on the image is beam damage to the mammoth guard hair by the 11 keV electron beam from the EDAX spot data analysis. (This type of beam damage was commonly encountered during studies of living or even long dead cyanobacteria or other biological materials but was almost never seen during EDAX analyses (even at 30 keV) of filamentous microfossils in the carbonaceous meteorites also indicates they are not modern bio-contaminants.

Fig. 4.

Image of a. living Pleistocene moss that was grown in the NSSTC Astrobiology Laboratory after being frozen in the permafrost of the Fox Tunnel and b. EDAX spectrum of torn edge of Pleistocene sedge shows 4% (atomic) nitrogen. c. FESEM image of mammoth guard hair showing beam damage spot and d. EDAX data with 11.6% (atomic) nitrogen peak.

Table I provides a compilation of the relative abundance of amino acids in terrestrial cyanobacteria and bacteria, a Miocene fly entombed in amber and a Cretaceous Hadrosaur for comparison with amino acids detected in CM2 (Murchison and Murray) and CI1 (Orgueil and Ivuna) carbonaceous meteorites. (Amino acids marked “−” were not detected in these terrestrial fossils or carbonaceous meteorites.) The most abundant amino acids (weight%) in the cyanobacterium Microcystis sp. and bacteria (E. coli and Salmonella) were GLU, ASP, ALA, GLY and LEU, all of which are present at levels above 8%. The next most abundant amino acids in the living cyanobacteria are THR, SER, VAL, ILEU and PRO, which are all at levels above ~5%. Glycine (GLY) is by far the most abundant protein amino acid in the Murchison and Murray (CM2), Orgueil and Ivuna (CI1) carbonaceous meteorites and the next most abundant are ALA, GLU and ASP. However, the protein amino acids LEU, THR, SER, VAL, ILEU and PRO, which are abundant in all life on Earth, are detected only at trace levels or are totally absent in the CI1 and CM2 carbonaceous meteorites.

TABLE I

Amino Acids in Living Bacteria, Terrestrial Fossils and Carbonaceous Meteorites

Protein Amino Acids	Living Bacteria	Fossils	Carbonaceous Meteorites
Microc ystis	E. Coli	Salm. pull	Salm. senf	Fty/Amber	Hadr osaur	Murchison CM2	Murray CM2	Orgueil C11	Ivuna C11
Wt %	Mol/ALA	Mol/ALA	Mol/ALA	Mol/GLY	Mol/GLY	Nmol/g	ppb	ppb	PPb	Ppb
L-Alaninc ALA	10.3	1.00	1.00	1.00	0.37	0.53	15.3	956	647	69	157
D-Alanine ALA	-	-	-	-	-	720	617	69	82
Arginine ARG	4.4	0.51	0.48	0.52	-	-	-	-	-	-	-
L-Aspartic Acid ASP	12.0	1.01	1.00	1.00	0.23	0.77	8.5	342	65	54	146
D-Aspartic Acid ASP	-	-	-	-	-	100	51	28	30
L-Glutamic acid GLU	12.3	1.14	1.11	1.14	0.57	0.67	18.2	801	261	61	372
D-Glutamic Acid GLU	-	-	-	-	-	537	135	15	8
Glycine GLY	8.7	0.93	1.02	0.96	1.00	1.00	45.8	2919	2110	707	617
Histidine HIS	1.0	0.18	0.21	0.19	-	-	-	-	-	-	-
Isoleucinc ILEU	5.0	0.55	0.51	0.55	-	-	-	-	-	-	-
Leucine LEU	8.2	0.83	0.78	0.78	-	-	1.9	-	-	-	-
Lysine LYS	4.4	0.56	0.59	0.56	-	-	-	-	-	-	-
Methionine MET	1.9	0.31	0.37	0.23	-	-	-	-	-	-	-
Phenalyalanine PHE	3.8	0.34	0.33	0.33	-	-	-	-	-	-	-
Proline PRO	4.9	0.25	0.26	0.28	-	-	13.5	-	-	-	-
Serine SER	6.6	0.41	0.48	0.43	0.56	0.91	4.7	-	-	-	-
Threonine THR	6.6	0.48	0.50	0.48	-	0.41	-	-	-	-	-
Tryptophan TRY	-	0.05	0.05	0.04	-	-	-	-	-	-	-
Tyrosine TYR	3.4	0.12	0.15	0.08	-	-	-	-	-	-	-
Valine VAL	6.5	0.73	0.66	0.75	-	0.24	8.6	-	-	-	-
Non-Protein Amino Acids
α-Aminoisobutyric AIB	-	-	-	-	-	-	-	2,901	1,968	39	46

The most abundant non-protein amino acids reported in several studies of carbonaceous meteorites are α-aminoisobutyric acid (AIB), Isovaline (IVA) and γ -Aminobutyric Acid (GABA). It has been suggested that the presence of non-protein amino acids in the meteorites proves that the meteorite amino acids were formed by abiotic processes. This hypothesis is not sustainable. Even though these amino acids are not used in proteins, it is an error to conclude that this means they must be abiotic. The amino acids IVA and AIB can be formed on Earth by the diagenetic alteration of ancient biological materials and γ-Aminobutyric Acid is synthesized by lactic acid bacteria and other microorganisms.³⁵

As Engel et al.³⁶ have pointed out, only 12 of the 20 life-critical protein amino acids are not found in the carbonaceous meteorites providing evidence that these meteorites have not been contaminated by modern microorganisms. There is no doubt that the Miocene fly and Cretaceous Hadrosaurs are undeniably biological and must have had all protein amino acids while living. They are missing several of the same amino acids that are also absent in the carbonaceous meteorites. Furthermore, several of these missing amino acids (Threonine, Leucine and Isoleucine) are abundant in living cells and should have been detectable if the meteorites were contaminated by living or recent microorganism. Once formed in living organisms, many amino acids and proteins are extremely stable over long periods of time. Ancient proteins have recently been detected in remains of a fly in Miocene Amber, in Cretaceous Hadrosaurs³⁷ (80 million year old duckbilled dinosaurs) and preserved within organic tubes found in fossils of the Ediacaran Sabellidites cambriensi from the Upper Ediacaran (~540 mya) period.³⁸ Loss of amino acids and the nitrogen atoms that they contain from dead cells certainly does not occur on timescales of years, decades, centuries or even millennia. Therefore, the absence of detectable nitrogen by EDAX studies of microfossils of cyanobacteria, diatoms or bacteria found embedded in the rock matrix of carbonaceous meteorites indicates these recognizable biological remains are not recent microbial contaminants that entered the meteorites after they arrived on Earth.

2.3.3

Pigments and the Remote Search for Evidence of Extraterrestrial Life

The process of photosynthesis requires large, complex, nitrogen-rich biomolecules known as pigments. The major pigments of photoautotrophic organisms are:

Chlorophylls--greenish pigments that contain the stable ring shaped porphyrin molecule, around which electrons can move freely. The porphyrin ring can gain or lose electrons easily and thus can provide energized electrons to other molecules. This is the fundamental mechanism used by chlorophyll to harvest the energy of sunlight.

Chlorophyll a, the most important chlorophyll molecule, is present in all photosynthetic cyanobacteria, algae and plants. It passes its electrons on to molecules that manufacture sugars. Chlorophyll b is found primarily in green algae and plants. However, some genera (e.g. Prochloron, Prochlorococcus and Prochlorothrix) of cyanobacteria use chlorophyll b for photosynthesis as they lack phycobilisomes, which are the light harvesting antenna of photosystem II that contain the phycobilin proteins. Chlorophyll c occurs in dinoflagellates and the eukaryotic supergroup known as the chromista. The green chlorophyll pigments of the halophilic green alga Dunaliella salina can be clearly seen in an image from the International Space Station (Fig. 5.a) in ponds surrounding the periphery of the hypersaline Owens Lake, CA.³⁹

Fig. 5.

Diverse colors of pigments in microorganisms. a. ISS image of Owens Lake, Ca showing green chlorophyll and red carotenoid pigments of Dunaliella salina; b. red spores of snow algae Chlamydimonas nivalis in Siberia; c. bloom of marine diatom Thalassiosira sp.; d. red phycoerythrin pigments in conical stromatolites of cyanobacteria Phormidium sp.on floor of Lake Untersee, Antarctica. (Photo Credits: a. NASA/JSC; b. NOAA; c. Richard B. Hoover; d, Dale Andersen)

Carotenoids--red, orange or yellow carotene containing pigments composed of two small six carbon rings linked by a chain of carbon atoms. Carotenoids are insoluble in water and attach to membranes within cells. They are unable to transfer the energy gained from sunlight directly into the photosynthetic pathway but must transfer their absorbed energy to chlorophyll molecules and are thus termed “accessory pigments.” Red and orange carotenoid pigments present in spores of the halophilic green alga Dunaliella salina and a novel carotenoid-like pigment found in Salinobacter ruber and halophilic archaea that cause the deep red color of hypersaline evaporite lakes such as Owens Lake can also be seen in Fig. 5.a.^39,40 Red carotenoid pigments are also seen in spores of the psychrophilic red snow algae Chlamydomonas nivalis (Fig. 5.b) collected in 1999 by the author from a snow bank during the International Siberian Astrobiology Expedition.⁴¹ Fucoxanthin is another accessory pigment that gives brown color to photosynthetic golden-brown algae known as diatoms. Figure 5.c is a NOAA image showing red and brown pigments in a large bloom of the marine planktonic diatom Thalassiosira sp. off the coast of Volusia County, Florida on July 25, 2010.

Phycobilins-blue or red water-soluble pigments that are the metabolic product of certain porphyrins. They serve as the light harvesting bilane photoreceptor pigments found in cyanobacteria and the chloroplasts of red algae, glaucophytes and cryptomonads. Phycocyanin is the phycobilin pigment that gives the blue-green color to many genera and species of cyanobacteria. Phycoerythrin is the red pigment that gives color to the eukaryotic red algae (Rhodophyta) and several of the red colored species of Cyanobacteria, such as Phormidium tenue and the nitrogen-fixing heterocystous strains of Anabaena.³⁹ Some non-heterocystous red cryptoendolithic species from Antarctica (such as Gloecapsa spp.) have c-phycoerythrin pigments as well as phycoerythrocyanin.⁴⁰ Figure 5.d is an image of large, laminated conical stromatolites that rise to a height of 0.5 growing on the floor of the perennially ice-covered Lake Untersee, Antarctica dominated by Phormidium species that contain red phycoerythrin pigments.

The Voyager 1 and Voyager 2 spacecraft obtained color images of the Jovian satellites Europa revealed large brownish areas and intersecting linear features. Higher resolution with better color rendition were then obtained from the Galileo Spacecraft. Figure 6 shows Galileo images of a. the Thrace region;⁴³ b. Minos Linea region⁴⁴ and c. the Conomara Chaos region⁴⁵ of Europa. The blue and white regions are consistent with blue ice and white ice crystals/snow commonly seen in images of glaciers or polar ice sheets on Earth. The red and brown colors have been attributed to “likely contaminants in the ice” ⁴⁴ or “minerals, such as salt, sulfur compounds, and possibly organic compounds that were previously sprayed on the surface from the ocean below.”⁴⁵ However, for this hypothesis to be the correct, then the ocean far beneath the thick ice crust of Europa would have to be blood red and filled with water-soluble red minerals, which are unknown in any of the oceans or seas of Earth.

Fig. 6.

Color images of Europa from the Galileo Spacecraft showing: a. Europa with enlargement of Thrace; b. Minos Linea; and c, Conomara Chaos regions exhibiting brown, orange, and red colorations consistent with pigments of diatoms and cyanobacteria. Image Credits: a-c. Arizona State University/NASA.

In 1986, Hoover et al.^46-48 pointed out that diatoms and other algae are the dominant eukaryotic life forms that inhabit the glaciers, sea ice and polar ice sheets of Earth. They suggested that these pigmented microorganisms were ideally suited to grow and carry out photosynthesis on the ice and in thin films of liquid water in cryptolithic regimes, cryoconite pools or larger bodies of water within comets, Europa or other icy moons of the Solar System. Cyanobacteria and archaea are the dominant types of prokaryotic life forms that inhabit cryptolithic and cryoconite environments and live in glaciers, ice sheets and sediments beneath perennially ice-covered lakes of Earth. The remains of filamentous and heterocystous cyanobacteria have been found in carbonaceous meteorites. These organisms could carry out photosynthesis and the life-critical functions of the carbon- and nitrogen-fixation on comets, water-bearing asteroids or icy moons of the Solar System just as they do on Earth.^49,50 Based upon these observations, the hypothesis has been advanced that the diverse orange, brown and red colors observed on the surface of Europa might be produced by photosynthetic pigments (Chlorophylls, Carotenoids and Phycobilins) contained within algae, diatoms, cyanobacteria, bacteria and archaea. This hypothesis is directly testable by remote observations to search for evidence of the distinctive fluorescence characteristics of these large complex pigment biomolecules.

Occasionally, large bodies of water on Earth do turn red, but this is not due to red minerals. Instead, “red tides” are caused by carotenoid pigments within the dinoflagellate Karenia brevis (=Gymnodinium brevis) of other phytoplankton that have undergone bloom conditions.⁵¹ Many red cryptoendolithic and heterocystous nitrogen-fixing cyanobacteria contain large quantities of phycoerythrin.^52,53 However, not all cyanobacteria are blue-green, brown, orange or red. Some of the typically red cyanobacteria are almost black to provide protection of spores or increase their photosynthetic efficiency. These include the nitrogen-fixing coccoidal Gloeocapsa magma which is responsible for black or dark green stains on roofs as it produces black UV resistant spores in strong sunlight. There are also black filamentous cyanobacteria (e.g., Phormidium corallyticum and Trichodesmium spp.) responsible for coral black band disease.⁵⁴ The complex conformational configurations of the large folded biomolecules of pigments involved in photosynthesis are strongly fluorescent at specific wavelengths and exhibit distinctive relative absorption and emission spectral signatures.^55-57 These response characteristics are not mimicked by minerals or the simple organic molecules that can be formed by abiotic mechanisms. These properties have made it possible to investigate cyanobacteria, diatoms and other algal blooms in lakes, seas and oceans of Earth from space observatories. Therefore, the fluorescence properties of photosynthetic pigments could be used to directly test the hypothesis that the colorations of Europa, asteroids and other solar system bodies are in some cases the result of microorganisms rather than abiotic minerals. These studies could provide direct support of this hypothesis and the remote detection of the complex photosynthetic pigment biomolecules only associated with metabolic energy harvesting pathways of life, would provide clear, convincing and unambiguous evidence for life elsewhere in the Cosmos.

3.1

Biomolecules in Carbonaceous Meteorites

Carbonaceous meteorites contain extraterrestrial water and carbon as well as all of the other life-critical biogenic elements. Most of the carbon contained within carbonaceous meteorites is in the form of Insoluble Organic Matter (IOM) similar to Kerogen ^58,84 with nominal bulk composition⁸⁵ of C₁₀₀H₇₀O₁₂N₃S₂. The carbonaceous meteorites also contain a vast array of suite of organic chemicals and biomolecules. Schmitt-Koplan et al.⁸⁶ analyzed polar and apolar solvent extracts of uncontaminated, freshly fractured interior fragments of the Murchison meteorite. Using Fourier transform ion cyclotron resonance/mass spectrometry (FTICR/MS) and other advanced instrumentation methods they reported the detection of 46,696 elemental compositions corresponding to 14,197 distinct elemental formulas and suggested that several million different chemical compounds might be present in carbonaceous chondrites. The great majority of these organic compounds may well have been formed by abiotic processes on the parent bodies. However, many of the carbonaceous meteorites also contain evidence for microfossils (Table III) as well as non-racemic and extraterrestrial protein amino acids, nucleobases and large biomolecules that represent the diagenetic breakdown products of photosynthetic pigments which have no known abiotic production mechanisms. Although these complex biomolecules are often attributed to the post-arrival terrestrial contamination, the missing amino acids, nucleobases and other life-critical biomolecules that have never been detected in interior fragments of these meteorites invalidates the recent bio-contamination hypothesis. Table IV summarizes many biomolecules found in carbonaceous meteorites along with the missing biomolecules that should be present if the meteorites were contaminated by modern microorganisms. The biomolecules that are missing in the carbonaceous meteorites are just as important as the ones that have been found. They provide clear and convincing proof that the interior portions of these stones that were analyzed have not been contaminated by recent microorganisms.

TABLE IV.

Biomolecules Present and Absent in Carbonaceous Meteorites

As seen in Table IV, only 8 of the 20 life-critical protein amino acids are present in the carbonaceous meteorites investigated by several different scientific teams. With the passage of time the homochirality of amino acids degrades as they become more racemic in dead microorganisms. They also decompose and disappear and their nitrogen is slowly lost and returned to the atmosphere as part of the nitrogen cycle. Simulation experiments by Vallentyne¹⁰⁴ evaluated the relative stability of protein amino acids in fossil shells. He reported the following general trend with regard to the stability of many of the amino acids that constitute proteins:

where the least stable (Italics) is Serine and the most stable (Bold) is Glutamic acid. The unstable acids are also absent in carbonaceous meteorites. The amino acids Serine, Threonine and Phenalynine are relatively abundant in living cyanobacteria and bacteria (Table I) but they are absent in carbonaceous meteorites and ancient fossilized remains. There is no known mechanism by which these meteorites could be contaminated by modern biology without including all 20 protein amino acids, DNA, RNA, all nucleobases, sugars, pigments and other biomolecules into the stones.

Biomolecular evidence for ancient photosynthetic microorganisms inhabiting the parent bodies is also provided by the detection of porphyrins, pristane and phytane in several of these stones. Pristane (2,6,10,14-Tetramethylpentadecane) is a C19 isoprenoid hydrocarbon that is the diagenetic breakdown product of phytol.¹⁰⁵ The long phytol chain is a side chain of the chlorin ring component of the photosynthetic pigment chlorophyll. Pristane is possibly the most abundant acyclic isoprenoid in the biosphere and represents a petroleum biogeochemical marker for ancient aquatic environments.¹⁰⁵ Phytane (2,6,10,14-Tetramethylhexadecane) is the C20 acyclic isoprenoid hydrocarbon that is associated with the membranes of thermophilic archaea. Pristane and phytane are both considered to products of the diagenesis of the phytyl side chain of the chlorophyll molecule.¹⁰⁶ Porphyrins are water-soluble nitrogenous biological pigments that are found in living organisms. These heterocyclic compounds are derived from the pyrrole ring and contain a central metal atom - Fe in heme and Mg in chlorophylls. Pristane, phytane and vanadyl porphyrins are found on Earth in ancient sediments, coal and petroleum.^107,108 Hodgson and Baker^94,96,109 reported spectroscopic evidence for pigments in the Orgueil meteorite that are indistinguishable from the vanadyl porphyrins present in bitumen, asphaltene and crude oil. They also reported that the Orgueil sample they studied did not contain chlorins, which are present in all recent biological deposits.¹¹⁰ The presence of pristane, phytane and vanadyl porphyrins is indicative of photosynthetic organisms that died millions to hundreds of millions or billions of years ago on the meteorite parent body. The absence of photosynthetic pigments provides further evidence that these meteorites have not been contaminated with modern cyanobacteria or diatoms.

Several independent early researchers reported the detection of heterocyclic nitrogen compounds such as purines, pyrimidines and triazines in carbonaceous meteorites.^99-101 More recent studies have established that several carbonaceous chondrites do contain the nitrogenous nucleobases Adenine, Guanine and Uracil and stable carbon isotope data clearly establishes that the nucleobases in carbonaceous meteorites are extraterrestrial in origin.^102,103,110 However, none of these different investigations have resulted in the detection of the nucleobases Cytosine or Thymine. By Watson-Crick base pairing rules of DNA, Adenine always pairs with Thymine and Cytosine pairs with Guanine. (In RNA, the complementary base to Adenine is Uracil rather than Thymine.) The missing nucleobases, Cytosine and Thymine are unstable. At 0 C, the Cytosine converts to Uracil by spontaneous deamination (releasing ammonia) with a half-life of only 17,000 years.¹¹¹ Thymine is much more stable, but it still has a half-life of only 1.3 million years at 0 C and pH 7.0, and it converts to Xanthine, which is present in carbonaceous meteorites.^103,110 If the microfossils found in carbonaceous meteorites are the remains of organisms that died millions of years ago, it would not be surprising for the meteorites to contain no detectable levels of Cytosine and Thymine. However, if the stones were contaminated by living microorganisms within the past several centuries, they should have both Cytosine and Thymine. The absence of Cytosine and Thymine provides clear and convincing proof that interior regions of these carbonaceous meteorites have not been invaded by living cyanobacteria or other terrestrial microorganisms since they arrived on Earth.

3.2

Microfossils in Carbonaceous Meteorites

The search at NASA/MSFC for evidence for microfossils in carbonaceous meteorites was initiated in 1996 immediately after McKay et al.¹¹² reported the discovery of PAH’s, biogenic magnetites and possible nanofossils in the Mars meteorite ALH84001. Environmental and Field Emission Scanning Electron Microscope (ESEM and FESEM) studies of uncoated, freshly fractured, interior fragments of carbonaceous meteorite resulted in the detection of large microfossils of cyanobacteria.¹¹³ Similar findings were reported for SEM studies of carbonaceous meteorites conducted by Rozanov and coworkers at the Paleontological and Microbiological Institutes, Russian Academy of Sciences, Moscow.¹¹⁴ These independent (and subsequent joint) studies resulted in the detection of the remains of a large number of diverse cyanobacteria, acritarchs, hystrichospheres, diatoms and rare unidentified protists. Many of the larrge, complex microfossils found exhibit the sizes and detailed morphologies of known genera and species of cyanobacteria and diatoms.^{20,50,115-120} EDAX studies at NASA/MSFC measured relative elemental compositions. In general, the spot analyses and 2-D X-Ray Element Maps revealed that microfossils were typically enriched in carbon and often contain several of the more abundant elements (Si, Fe, Mg, S, O) present in the particular meteorite rock matrix. In the Orgueil meteorite, EDAX data shows many of the filaments have highly carbonaceous sheaths and contain Mg, S, and O. This suggests they were infilled with water-soluble magnesium sulfates after they died and the internal cells were lost. This is not the case for cyanobacterial filaments found in Murchison. It is difficult to account for these composition differences by the modern bio-contamination hypothesis. Since the mineral grains of the Orgueil meteorite disaggregates upon contact with liquid water, the infilling of the filaments with magnesium sulfate must have occurred on the parent body before the stones landed or Earth. Exposure to water after landing would have destroyed the stones. Furthermore, the microfossils in the meteorites are heavily depleted in nitrogen as compared with living or dead organisms on Earth.

Nitrogen is one of the most abundant elements in the Solar System, but it is severely depleted in meteorites.^121,122 Nitrogen is rarely present in microfossils in the meteorites at levels (>0.5% atomic) high enough to be detected by EDAX. However, EDAX studies at NASA/MSFC have shown that modern or even dead dells typically have nitrogen levels of 2% to 20% (atomic) and the absence of nitrogen in meteorite microfossils rules out contamination.

Figure 7 shows images of microfossils of cyanobacteria in a. Murchison b. Orgueil and c. Polonnaruwa meteorites. The Murchison filaments of Fig. 7.a. are consistent with species of cyanobacteria that grow together in polar environment. These include the multiseriate Microcoleus chtonoplastes and Phomidium species and tapered filaments of Calothrix sp. with apical cell and calyptra; and escaped hormogonium with cell wall constrictions; b. Orgueil meteorite long filament with thick sheath and emergent trichome (Lyngbya sp. morphotype) c. Polonnaruwa meteorite with cells of the filamentous planktonic diatom Aulacoseira ambigua showing rimoportulae and ringleiste structures.

Fig. 7.a.

Trichomic filamentous cyanobacteria in a. Murchison meteorite showing morphotype of Microcoleus sp.; tapered Calothrix sp. filament with apical cell and calyptra and an escaped hormogonium; b. Orgueil meteorite with long Lyngbya sp. filament with thick sheath and emergent trichome; c. Polonnaruwa with filamentous planktonic diatom Aulacoseira ambigua. Photo Credits: a.&b. Gregory Jerman and Richard B. Hoover, NASA/MSFC; c. N. C. Wickramasinghe, University of Buckingham