Sixteen of twenty protein amino acids were separated and quantified by these methods using two separate RP-HPLC protocols . The E. coli cells were easily detectable at the concentrations examined in this study. The amino acid separations were successful using the traditional laboratory protocol developed over the last 15 years .The second gradient also showed much better separation of threonine from a shoulder of glycine and tyrosine from alanine . The traditional gradient A showed much better overall separation of the amino acids, especially in the amino acids which coeluted further downfield.The only major differences between the two datasets are the magnitudes of the glycine and alanine peaks. The distinct difference in the alanine concentrations between these data reflects different amino acid compositions of the analyzed bacterial colonies. The large difference in the glycine peak can be explained by assuming that threonine, which constitutes ~5% of the total mass of E. coli, coeluted and was quantified with glycine in the previous study . The error bar over glycine for the data gathered in this study shows the magnitude of threonine plus glycine if they coeluted, explaining the disagreement between the two datasets. Threonine, present in gradient A as a shoulder of glycine, better separates using gradient B and was more accurately determined with this program. However, using gradient A, any significant concentration of threonine present in natural samples may still be determined as it shows up as a shoulder of glycine.
The cellular bio-density may be enumerated by estimating the mass of each E. coli cell to be 1.55 x 10-13 grams/cell,nft channel the protein content of each cell to be 55% , and the quantified amino acids to be ~100% of the total protein mass. Extrapolation of the total amino acid concentrations to an equivalent cell count shows that the total bio-density is on the order of 1.4 x 1010 cells per gram of serpentine. This is in close agreement to the cell staining methods utilized in this study which showed approximately 1.3 x 1010 cells per gram of serpentine. Assuming a 5% error in each approximation, these data agree within the error limits. Data from this study show that the 10 amino acids quantified with the highest concentrations account for approximately 92% of the total mass. In agreement with data from Glavin et al. , six of these amino acids account for 70.7% of the total amino acids present in E. coli. Quantification of these 6 amino acids represents the majority of the protein amino acids and is sufficient to accurately enumerate the cell bio-densities of geological samples. Also of importance is that these six amino acids and their enantiomers all elute before 25 minutes and show good chiral resolution between D- and L-configurations using the established RP-HPLC gradient protocol . The coelution of glycine and arginine in gradient A is a non-issue because arginine is present in such low concentrations in bacterial communities . One of the most important confirmations of this study was the deficiency of D-amino acids detected in these analyses. The optimum hydrolysis procedures preserve the integrities of the amino acids as well as their chiralities. The average D/L-ratios of the amino acids asp, glu, ser, gly, and val was approximately 0.02 while alanine showed a slightly higher average D/L-ratio of 0.05. This means that the racemization percentage is well under 1% and does a good job of preserving the enantiomer signature of geological samples. Figure 2.6 shows the results from this study against data from previous studies. The distributions are very similar for all of the amino acids and show good agreement.
The major differences appear to be in the amino acids asx, glx, ala, and cys. The differences in asx and glx must represent a difference in the protein content of the E. coli strains analyzed or may be explained by different methods of quantification. As these numbers agree well with the data from Glavin et al. , this is unexplainable and may reflect the superior methods of quantification utilized in this study. The difference in ala concentrations, also observed compared to the data from Glavin et al. , perhaps shows a higher percentage in this strain of E. coli compared to others studied. Finally, the differences in cysteine concentration can be explained by the effect of hydrolysis on recovery and peak coelution. The cysteine peak overlapped with trace amounts of ammonia in the standard and hydrolyzed samples. The borate drydown step did not completely remove all of the residual ammonia left over from the desalting stage , resulting in trace amounts of ammonia coeluting with cysteine.The distribution of amino acids from microbial life has been reinvestigated in this study and found to agree fairly well with previous results. Amino acids derived from extant microbial communities are homochiral with very low abundances of D-amino acids. The distributions show the major amino acids components as alanine, glutamic acid, aspartic acid, glycine, and serine. Any detected abiotic enantiomer abundance from purely extant bacteria is due to sample processing during hydrolysis or the presence of a small amount of racemized microbial remnants present in the overall community. In the case of alanine, the Denantiomer is prevalent in low concentrations within peptidoglycan and should normally show the highest D/L-ratio. The distribution of amino acids within E. coli is assumed to be representative of a wide variety of microbial life. Merely six of the twenty amino acids account for ~70.7% of the total amino acids . These are the most important amino acids to detect in the search for biomarkers within geological samples or during future astrobiological missions. The chirality of the detected amino acids is the key to the unequivocal determination of the presence of biological material.The search for evidence of water and organic compounds, including those of possible biological origin, is one of the major goals of both the NASA and European Space Agency Mars exploration programs.
The NASA Mars Exploration Rovers and the ESA OMEGA/Mars Express have provided the best evidence to date that liquid water was once present on Mars. Abundant sulfate minerals such as gypsum and jarosite suggest that large acidic water basins were once present and that as they evaporated sulfate minerals were precipitated . Although it is unknown when these standing bodies of water existed or for what duration, they could potentially have provided an environment capable of supporting life. On the other hand, it remains uncertain whether organic compounds are present on Mars. While the Viking missions in 1976 detected no organic compounds above a threshold level of a few parts per billion in near surface Martian soils . However, it is now known that key bio-molecules such as amino acids would not have been detected by the Viking GCMS even if several million bacterial cells per gram were present . In addition, oxidation reactions involving organic compounds on the Martian surface would likely produce non-volatile products such as mellitic acid salts that also would not have been detected by Viking . Thus, the Viking results did not conclusively disprove that there are organic compounds present on the surface of Mars. The only other opportunity to analyze samples from Mars has been provided by meteorites ejected from its surface and delivered to Earth. However, contamination of these meteorites by terrestrial organic material during their residence times on Earth compromises their use in assessing whether organic compounds are present on Mars . Organic matter is often co-deposited in terrestrial evaporites, and similar deposition processes should occur on Mars if organic molecules were present in the early oceans . To our knowledge,hydroponic nft there have been no systemic investigations of organic compounds in sulfate minerals on Earth. The concentrations of organic carbon and nitrogen of several sulfate minerals are reported herein, as well as the abundance of amino acids and their degradation products. Atacama Desert gypsum deposits have been reported to be late Pliocene in age, so this near-surface soil sample, composed of a high mass percent gypsum, was assumed to be ~2 Ma . The evaporite formations from the Anza-Borrego Desert have been extensively studied. The gypsum investigated here was collected from the Fish Creek area and has been dated at 3-5 Ma . Gypsum from the Haughton impact crater, Canada, was donated by John Parnell and is assumed to date from the time of the impact crater at 23 Ma . The age of the host rock of the Panoche Valley samples is 75-65 Ma but the ages of the sulfate minerals are estimated at 40 Ma because this is when the coastal ranges were raised in this area during the Sierra Nevada uplift, which caused ocean water to withdraw and deposit evaporitic minerals in California’s Central Valley. In order to verify the geologically deduced ages of the Panoche Valley samples, strontium isotope analyses were conducted. Celestite, SrSO4, is often included in gypsum mineral matrices as a minor component, and because the Panoche Valley gypsum formed evaporitically, the Sr isotopes should be indicative of the seawater ratio at the time of formation.
The Panoche Valley gypsum 87/86Sr ratio was 0.707745 . Comparing this 87/86Sr ratio to the strontium isotope history of seawater , gives an age 40 Ma, consistent with the inferred geologic age. The modern gypsum sample is from the South Bay salt works in South Bay San Diego, California. The area is rich in marshes and tidal flats and experiences continuous evaporite formation during tidal fluctuations. Because of the poor water quality in this region of San Diego Bay, the salts typically include significant amounts of organic material. The surface of each sample was thoroughly rinsed with doubly-distilled water followed by doubly-distilled 1M HCl, then again with ddH2O. The identity of each mineral was verified by XRD analyses using a Scintag XDS-2000 powder diffractometer. Samples were analyzed for total organic carbon and nitrogen using a Costech elemental combustion C-N analyzer. Carbon and nitrogen isotopic ratios were determined with a Thermofinnigan Delta-XP Plus stable isotope ratio mass spectrometer on ~30 mg of each sample. In order to remove carbonate from the samples, they were pre-treated with an excess of 3N double-distilled HCl and dried down on a vacuum centrifuge at 45°C for 1 hour before analyses for total organic carbon and nitrogen .Amino acids were isolated by vapor-phase acid hydrolysis of ground samples followed by desalting . Amines were isolated by micro-diffusion from the powdered mineral treated with 1N NaOH, into a 0.01N HCl solution at 40°C for 6 days . Extracts were analyzed for amino acids and amines by RPHPLC using pre-column derivatization with o-phthaldialdehyde/N-acetyl L-cysteine using a Shimadzu RF-530 fluorescence detector and a Phenomenex Synergi Hydro-RP column . Quantification of amino acids and amines included background level correction using a serpentine procedural blank and a comparison of the peak areas with those of an amino acid standard. A D/L-norleucine internal standard was added to normalize amino acid recoveries from desalting and derivatization. The recovery of the amines carried through the extraction procedure was found to be near 100% using spiked procedural samples. To investigate the possible presence of modern bacterial contamination in the various minerals, total adenine concentrations were measured. Both a liquid extraction involving treatment of 1 gram of sample with 2 ml of 95% formic acid solution for 24 hours at 100°C and a sublimation extraction method at 500°C for 5 minutes were performed. Adenine concentrations were quantified by HPLC with UV absorption detection and converted to bacterial cell densities as described in Glavin et al. .The XRD results verified each mineral’s identity. The gypsum samples that were obtained from Anza-Borrego, Panoche Valley, and Haughton crater were selenite, pure gypsum in discrete layers. The South Bay gypsum sample was the most impure. The Atacama Desert soils have previously been characterized as having high gypsum content and were not characterized by XRD analysis. The organic carbon and nitrogen data are tabulated in Table 3.1. The organic carbon contents ranged from 0.12 – 0.77 mg·C/g in the 3 gypsum samples, the Atacama Desert was fairly low at 0.16 mg·C/g, while the contemporary gypsum from South Bay showed significantly higher percent organic carbon due to the sample’s origin in a highly organic included region. The nitrogen trends were similar in that the ancient gypsum and anhydrite samples ranged from 0.01-0.03 mg·N/g.