Methylamine and ethylamine, the degradation products of glycine and alanine, are detected in increasingly high abundances with age in ancient sulfate samples up to ~40Ma. The comparison of these concentrations to their parent amino acids allow for the calculation of glycine and alanine decarboxylation rates and are assumed to be indicative of these rates in sulfate matrices. Because sulfate minerals have been detected in high abundance on Mars, these rates are extrapolated to Mars’ average surface temperatures to offer an estimate of the rates that might be expected on Mars. Chapter 4 analyzes modern sulfate samples from Southern Australia for amino acids and extrapolates these concentrations to equivalent bacterial concentrations . The low abundances of methylamine and ethylamine in these modern samples show consistency with previous findings . Chapter 5 focuses on a new analog to the Martian hematite blueberries that have been detected on Mars in the Meridiani Planum region. These ironstones are ubiquitous in San Diego county, and by analyzing samples from various deposits,nft hydroponic system they are dated using amino acid racemization based on the measured D/L-ratios and a sample calibration method. Similar concretions on Mars would show good preservation in such environments despite the fact that they’re iron rich.
The ironstone formation seems to be mediated by enhanced precipitation as well as the possibility of bacterially induced mineralization within these deposits. Chapter 6 investigate an extreme environment deep within a South African subterranean gold mine. These filtrates show extremely low bio-densities and is of interest because it might represent the extremely low levels of biomass and extremely slow turnover times that push the limits of analytical sensitivity. A simple steady-state model corroborates the long turnover times that have been reported for these samples. Two well known Mars analog sites comprise the next two chapters, rock samples from the Antarctic dry valley Deserts and surface soils from the Atacama Desert, Chile , which have been suggested to be the best terrestrial Mars soil analog . The fact that life can persist in these extreme climates is impressive enough, however the extremely cold samples from Antarctica show amazing preservation while the opposite is observed in the Atacama Desert surface and near-surface samples. The inferred cell counts based on total amino acid abundances within cryptoendolithic microbial life agree well with previous bio-density enumerations in similar environments. The Atacama Desert shows extremely degraded organic material at the surface and shows drastic variations in amino acid distributions and diagenetic state as a function of depth and surface micro-environment habitability. One of the premier instruments for advanced in situ Mars life detection experiments is the Urey – Mars Organic and Oxidant detector. Chapter 9 focuses on research and development of the instrument over the past 3 years, specifically the optimization of the extraction system for Mars exploration. Both laboratory and field experiment data is discussed in detail and exhibits efficient extraction of target bio-molecules, specifically amino acids and total organic carbon.
The use of sublimation as a second-stage extraction method for extract purification and concentration is evaluated for recoveries using various mineral matrices.The astrobiological studies have broad implications for the search for life on Mars over the next decade. Two missions will be launched within the next 6 years: NASA’s Mars Science Laboratory and the ESA’s ExoMars . The studies herein validate the importance of targeting amino acids in life detection studies, emphasize their importance as an indicator of bio-density, and demonstrate the potential for sequestration within Mars mineral deposits. Not to be dismissed is the fact that the detection limits of the Urey Mars Organic Detector are many orders of magnitude greater than necessary for the detection of amino acid biomarkers in some of the most uninhabitable places in the world such as the Atacama Desert. Sulfate minerals are highly abundant on Mars and our studies suggest that they can offer enhanced preservation on the Martian surface based on the estimated rates observed for in situ diagenetic reactions.Bacterial bio-density can be quantified by a number of traditional cell staining methods. Many of these methods target intact DNA, such as 4′,6-diamidino-2-phenylindole , acridine orange , or ethidium bromide. These methods target only intact nucleoid containing cells . Other staining methods, such as trypan blue, target cell membranes and will react with both living and dead cells. Cell staining can lead to erroneous bio-density calculation due to human error and interfering medium . Analyses of individual cellular molecular components can be used to accurately determine cell concentrations in natural samples. Two of these methods are cell enumerations based on total adenosine triphosphate and phospholipid fatty acid analyses, and these have been shown to be accurate at determining cell concentrations in natural samples . ATP is a nucleotide and a ubiquitous component of bacterial life while the majority of phospholipids and fatty acids are present as components within microbial cell walls. One unique aspect of PLFA analyses is that they can distinguish between different types of bacteria depending on the distribution of the target molecules. Similarly, any biomarker can be used to quantify bacteria such as amino acids or nucleobases.Quantifying total hydrolyzable amino acids yields accurate determination of the total protein content in bacterial colonies.
Traditional wet chemistry extraction and analytical methods for amino acid quantification involve acid hydrolysis followed by desalting, pre-column derivatization using a fluorescent chiral adduct, separation by reverse-phase high performance liquid chromatography , and quantification by fluorescence detection against standards of known concentration . Most amino acids are stable during traditional acid-hydrolysis methods as only the peptide bonds within proteins are cleaved during this treatment. The exception is tryptophan which is completely destroyed during acid hydrolysis while arginine, tyrosine, threonine, serine, methionine, and cysteine are degraded to a small degree during longer hydrolysis times .These degradation mechanisms involve the conversion of the carboxamide side groups to carboxyl groups through the incorporation of water and liberation of ammonia.Ortho-phthaldialdehyde/N-acetyl-L-cysteine was first used to derivatize amino acids by Aswad and later applied to amino acid quantification in geological samples . The fluorescent derivatizing chiral adduct is made by combiningOPA and NAC into an alkaline borate buffered solution to form a cyclic fluorescent derivative .This fluorescent chiral adduct reacts with primary amines to form fluorescent derivatives . The fluorogen reacts with all primary amines, so it targets all 20 protein amino acids except for proline. Highly specific fluorescence detection is accomplished at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. This highly specific derivatization allows for low interference during quantification.In order to determine the amino acid composition and distribution of a typical bacterial culture,hydroponic nft system and to test the THAA method for cell enumeration, samples of cultured E. coli cells were run through traditional wet chemistry extraction and analytical protocols. This allowed for determination of the most important targets for the search for amino acid bio-signatures derived from bacterial proteins in the study of geological samples and for the purposes of life detection.Cultured E. coli cells were obtained and added to a sterilized crushed serpentine medium. Cell bio-densities were measured by traditional methods on the inoculated sample and a procedural blank growth medium that did not contain E. coli cells. The OD460 of the E. coli growth medium was measured to be 0.65 which corresponded to 6.5 x 109 E. coli cells in 10 ml of LB growth medium with a 5% measurement error. Because physiological variation may changes in cell size, capsule formation, or aggregation, small differences in the conversion between OD and total cell counts may be observed. For this reason, the total number of E. coli cells as determined from the OD reading was independently confirmed by measuring the mass of a solid E. coli pellet generated by overnight growth and centrifugation of a volume of LB medium identical to that used to inoculate the serpentine. If it is assumed that the E. coli cells were homogenously mixed into the 0.5-g crushed serpentine sample, a concentration of 1.3 x 1010 cells/g for the serpentine inoculated with E. coli was inferred. ~200 mg of the inoculated serpentine cell media and a serpentine growth medium blank were hydrolyzed and desalted according to the procedures of Zhao and Bada . The sample was vapor-phase hydrolyzed under 6M doubly-distilled HCl for 24 hours in a flame-sealed test tube after flushing with nitrogen. The hydrolyzed residue was loaded onto an equilibrated desalting column of ~2.5 mL of BioRad AG50W-X8 resin in a pasteur pipette. The sample was rinsed with ~6 column volumes of doubly-distilled water before eluting the amino acid fraction with 3mL of ~3M doubly-distilled ammonium hydroxide .
These fractions were concentrated on a vacuum centrifuge under 60°C heat into 1.5mL mini-eppendorf vials. These residues were resuspended into 100µL of ddH2O for derivatization and analysis by RP-HPLC. The OPA/NAC fluorescent derivative was prepared with chemicals purchased from Sigma. 4 mg of OPA was first dissolved into 300µL of methanol, and 250µL of borate buffer was added to the solution, followed by the addition of 435µL of double-distilled water. The last step is the addition of 15µL of 1M NAC solution . This derivatizing solution has a final concentration of ~0.03M OPA and ~0.015M NAC before they react. The final concentration of the cyclical derivative in the OPA/NAC solution is 0.015M OPA/NAC. The reaction between OPA/NAC and primary amines has been demonstrated to be linear over large concentration ranges for reaction with amino acids and biogenic amines . 10µL aliquots of diluted fractions of the desalted hydrolyzed E. coli extracts and procedural blanks were first dried down on a vacuum centrifuge at room temperature with 10uL of borate buffer to remove any residual ammonia from the NH4OH carried through from the desalting stage. These residues were brought up in 20µL of ddH2O and derivatized for 1 minute with 5µL of the 0.015M OPA/NAC solution. After this pre-column derivatization, the samples were separated by RP-HPLC and quantified with a fluorescence detector. The RP-HPLC setup utilizes an Hitachi L6200 Intelligent HPLC pumps, rheodyne sample injectors, coupled with a Phenomenex Luna-C18 RP-HPLC column and a Shimadzu fluorescence detector . Data sampling and analysis, including automatic and manual Gaussian peak integrations, were performed using Thermo Scientific Grams/AI software. Sample peak intensities were quantified against 100-1000x diluted commercial standards of known concentration , and the trace amounts of D-enantiomers ratio-normalized against racemic laboratory standards of similar concentration. The HPLC conditions included a stationary-phase buffer, 50mM sodium acetate solution with 8% methanol, with methanol as the mobile phase. Two gradient protocols were necessary to resolve the 16 amino acids extracted and purified by these methods . The traditional amino acid separation protocol developed over the last 20 years has been used in a variety of studies including those on natural samples , meteorites , and hydrolyzed bacteria . This RP-HPLC protocol was primarily developed to well separate the primary amino acids present in geological samples as well as their enantiomers. A slower methanol elution gradient was developed that was necessary to resolve the coeluting peaks of glycine and arginine, and to better resolve threonine as a shoulder of glycine and tyrosine from alanine. The trace amounts of D-enantiomers did not interfere with the peak separation as the D/L-enantiomer ratios were too small to be significant. This is expected of any extant bacterial community and the hydrolysis not harsh enough to cause a significant degree of racemization.The OPA/NAC fluorescent derivatizing reagent tags only primary amines, so proline does not react, tryptophan is completely destroyed during hydrolysis in 6N HCl for 24 hours, and asparagine and glutamine degrade to aspartic acid and glutamic acid during acid-catalyzed hydrolyis, respectively. This results in the derivatization of 16 of 20 total protein amino acids.Aspects of this study have been investigated before. Similar procedures were utilized by Glavin et al. to determine the amino acid composition in similarly treated fraction of hydrolyzed E. coli cultures, however they analyzed a more limited set of amino acids during their experiments. More recently, the recoveries of adenine from sublimed E. coli colonies were used to enumerate bacterial bio-density , so this study provides verification of procedures for THAA determination and accurate enumeration of cell bio-density.