Review of methods used in lunar organic analysis: Extraction and hydrolysis techniques

Extraction, hydrolysis and crushing procedures have proven useful in analyzing for some of the carbon-containing compounds which are present in Apollo 11 and 12 lunar samples. Three main extraction methods employed with aqueous and nonaqueous solvents were refluxing in open and closed systems, Soxhlet extraction, and sonication. With water and acids, refluxing was the method of choice. Of the various nonaqueous solvent systems used, benzene: methanol mixtures were most often selected, and sonication was favored over Soxhlet extraction. Extraction of lunar samples with water followed by acid hydrolysis of the water extract was found to be superior to direct acid hydrolysis of lunar material in the search for amino acids or their precursors. Direct acid hydrolysis of lunar materials did demonstrate however, that carbides or carbide-like compounds are present on the moon. Hydrolysis with deuterated acids and bases showed that lunar samples contain indigenous methane and ethane and confirmed the presence of carbide-like materials. Crushing experiments also showed that gaseous hydrocarbons can be released from lunar samples.     

Extralunar sources for carbon on the moon

Contrary to previous assumptions, the solar wind may not be the only source of lunar carbon, possibly contributing as little as 50 ppm carbon to the lunar surface fines. Because of uncertainties about the true composition of the solar wind, it is impossible to be definite in any calculation. Meteorites and comets, previously assumed to be negligible contributors, might account for 5–10 ppm carbon via a cycle of carbon through the lunar atmosphere. Carbon isotope ratio measurements have the potential of greatly clarifying the situation, but key data are missing.     

Influence of the solar wind on the distribution of the electric potential near the Moon’s surface

The Moon’s surface illuminated by the Sun acquires an electric charge due to photoelectron emission and interaction with the solar wind plasma. The influence of the solar wind on the nonmonotonic distribution of the electric potential near the Moon’s surface is studied in a wide range of the densities of emitted photoelectrons. It is found that, for any photoelectron density, the surface potential reaches its minimum value for a slow solar wind. Although the electron thermal velocity can exceed the solar wind velocity by several times, taking into account the directed flow velocity in the electron velocity distribution function substantially affects the value of the potential in the lunar regolith regions not enriched with hydrogen, for which the photoelectric work function under solar irradiation is significantly higher than for regions enriched with hydrogen.     

An evaluation of pyrolytic techniques with regard to the Apollo 11, 12 and 14 lunar samples analyses

Two different pyrolysis techniques have been used in the analysis of lunar fines. The first technique involved pyrolysis at 700°C under an inert atmosphere in a flowing He system at normal pressure. The products were collected at liquid N₂ temperature and then allowed to pass instantaneously into a combined capillary gas chromatograph-mass spectrometer. The second technique consisted of a vacuum pyrolysis where the sample was first degassed at 150°C and then pyrolyzed at 500°C and 1000°C consecutively. The products were again collected at liquid N₂ temperature and then they were directly introduced to the ion source of the mass spectrometer through a modified gas inlet system.An evaluation of the two techniques based on control experiments has shown that the probability of secondary reactions is greater in the inert atmosphere pyrolysis method. Pyrolysis of benzene in He under atmospheric pressure at 600°C showed the presence of small quantities of biphenyl and trace amounts of naphthalene. Biphenyl pyrolyzed under vacuum at 600, 700, 800 and 900°C by passing through a hot zone containing a quartz wool plug showed the presence of a wide range of synthesis and breakdown products as the temperature increased.These experiments have shown the importance of taking into account the factors that influence pyrolytic degradation and/or the synthesis of products. These can be diffusion effects, involving sample size, sample form, pyrolysis pressure conditions; temperature, catalytic effects from the pyrolysis vessel, contamination, perhaps other factors. Pyrolysis is an effective method of analysis if used under carefully controlled conditions. Pyrolysis of Apollo 14 lunar fines and scrapings from an astronaut's glove gave different products by mass spectrometry and showed different looking flaky materials upon scanning electron microscopy.     

On the abiotic formation of amino acids I. HCN as a precursor of amino acids detected in extracts of lunar samples II. Formation of HCN and amino acids from simulated mixtures of gases released from lunar samples

Two studies on the abiotic formation of amino acids are presented. The first study demonstrates the role of hydrogen cyanide as a precursor of amino acids detected in extracts of lunar samples. The formation of several amino acids, including glycine, alanine, aspartic acid, and glutamic acid, under conditions similar to those used for the analysis of lunar samples is demonstrated. The second study investigates the formation of hydrogen cyanide as well as amino acids from lunar-sample gas mixtures under electrical discharge conditions. These results extend the possibility of synthesis of amino acids to planetary bodies with primordial atmospheres less reducing than a mixture of methane, ammonia, hydrogen and water.     

Study of carbon compounds in Apollo 12 and 14 lunar samples

The gases released on DF dissolution of a variety of samples have been studied by gas chromatography and high resolution mass spectrometry. Results on Apollo 12 samples confirm previous observations that CH₄ and C₂H₆ are released as well as CD₄, C₂D₄, C₂D₆ and higher deuterated hydrocarbons. The yields correlate with the total carbon content of the samples and the CH₄ and C₂H₆ released may be regarded as indigenous while the deuterated products result from hydrolysis of carbide material. Dissolutions were also performed on five size fractions of sample 14240,5, ranging from >420 to <37 . The yields of CH₄, CD₄,²⁰Ne and³⁶Ar correlate with the surface area and therefore probably arise from solar wind implantation. Other deuterocarbons released include C₂D₄, C₂D₆, C₃D₆, C₃D₈ and C₄D₁₀. Preliminary pyrolysis results of these size differentiated samples confirmed the presence and surface correlation of the CH₄,²⁰Ne and³⁶Ar. Dissolution of the 14148, 14156 and 14149 trench samples showed that their carbon chemistry and solar wind exposure are very similar to that of the 14240 SESC and Apollo 11 and 12 fines of high carbon content. Other interesting components released from the soil samples by DF include D₂S, DCN and CS₂.This paper is an amplified version of the comments made by Dr Holland during the discussions at the meeting on Lunar Analysis: Significance for Exobiology, held at College Park, Maryland, October 26–28, 1971.     

Distribution and isotopic abundance of biogenic elements in lunar samples

A review of the abundance of six biologically important elements (H, C, N, O. P and S) demonstrates that they are present in trace amounts only in lunar matter analysed to date. To the endogenous lunar content, elements are contributed by solar wind irradiation and meteorite impacts. However, it is not yet possible to determine the relative importance of the three sources. Enrichment of the heavy isotopes, C¹³, O¹⁸, S³⁴ suggest that these elements may be lost from the lunar surface by hydrogen stripping (from solar wind protons) as volatile gases. The general lack of water, suggests that organic synthesis could not easily be accomplished in lunar rocks. High energy irradiation of the lunar surface may result in rapid destruction of organic matter not protected by a silicate matrix. It is apparent from present data available, that the ambient lunar surface could not support metabolism of known microorganisms.Publication No. 997 Institute of Geophysics and Planetary Physics, University of California at Los Angeles, Los Angeles, California 90024.     

Aromatic and heteroatom-containing organic compounds in the lunar samples

The data concerning the indigenous organic compounds in the lunar samples has been consistent. The Apollo 11 sample appeared to be moderately contaminated, and most investigators found known terrestrial artifacts in these samples. The Apollo 12 data indicated that perhaps benzene and toluene were indigenous to the Moon at concentrations below 100 parts per billion. Other, more complex organic molecules (in particular of aromatic structure) might also be present, but in concentrations below 1 ppb. In general, the structural types reported have been relatively simple; perhaps indicating that what little organic chemistry occurs on the lunar surface can best be described as reactions between individual atoms of carbon, hydrogen, oxygen and nitrogen.     

In situ synthesis during organic analysis of lunar samples

In the development and application of extremely sensitive analytical techniques the need for extreme cleanliness and avoidance of contamination has been generally recognized and complied with in the organic analysis of lunar samples. Much less attention has been paid to the possibility of inadvertent synthesis of certain organic molecules from indigenous smaller organic or even inorganic constituents of the lunar material. At the part-per-billion level of detectability reactions proceeding even at very low yield can lead to detectable products. A particular area of concern should be the amino acid analysis since those substances are known to be formed by condensation of ammonia, cyanide and carbonyl compounds, all potential products of lunar material upon treatment with aqueous media.     

Lunar organic analysis: Implications for chemical evolution

The lunar samples provided some material from the early period of the solar system. These samples could have preserved in them some of the stages in the evolution of carbon compounds. The analysis, so far, has not revealed with certainty a single organic compound of biological significance. However, the finding of methane, carbon monoxide, carbides, etc. could be considered in the context of cosmic evolution.     

Research for amino acids in lunar samples

Water extracts of lunar fines were analyzed for amino acids by a gas-liquid chromatographic technique whereby amino acids were converted to the N-trifluoroacetyln-butyl, esters prior to analysis. The lunar material studied included both Apollo 14 (14240 SESC and 14298) and Apollo 12 (12023) samples. The water extract of the special Apollo 14 sample (14240 SESC) was analyzed both for free and bound amino acids (hydrolysis with 6 N hydrochloric acid). In both the hydrolyzed and unhydrolyzed extracts, the amino acids were not observed above background levels.The analysis of Apollo 12 and 14 samples (12023 14298) yielded similar results. Detection limits were established at 300 pg to 1 ng for different amino acids. A large chromatographic peak with a retention temperature of 126°C was observed on analysis of sample, (12023); it was identified as oxalic acid by GC-MS. The concentration of amino acids in the Apollo 14 SESC samples processed and analyzed in the joint experiments at Ames by GLC and IEC were found to be extremely low (glycine at 3 to 4 ng g^–1). As the quantities were so minute, these identifications could not be confirmed by GLC-MS and therefore should still be considered as tentative. Other studies included the analysis of performance standards at the 2 to 6 ng level of each of 17 amino acids, and the analysis of 5 ml of H₂O containing 2 ppb of each amino acid. Recovery of amino acids added to lunar fines were conducted at the 10, 50, and 70 ng level of each amino acid with 50 to 70 mg of lunar material. The recoveries varied from as high as 80% for some of the aliphatics to complete loss of the amino acids ornithine and lysine.Contributed from Missouri Agricultural Experiment Station Journal Series No. 6255. Approved by the Director. Supported in part by grants from the National Aeronautics and Space Administration (NGR 26-004-011) and the Experiment Station Chemical Laboratories.     

Catalytic conversion of cellulose to chemicals in ionic liquid

A simple and effective route for the production of 5-hydroxymethyl furfural (HMF) and furfural from microcrystalline cellulose (MCC) has been developed. CoSO₄ in an ionic liquid, 1-(4-sulfonic acid) butyl-3-methylimidazolium hydrogen sulfate (IL-1), was found to be an efficient catalyst for the hydrolysis of cellulose at 150 °C, which led to 84% conversion of MCC after 300 min reaction time. In the presence of a catalytic amount of CoSO₄, the yields of HMF and furfural were up to 24% and 17%, respectively; a small amount of levulinic acid (LA) and reducing sugars (8% and 4%, respectively) were also generated. Dimers of furan compounds were detected as the main by-products through HPLC-MS, and with the help of mass spectrometric analysis, the components of gas products were methane, ethane, CO, CO_2, and H₂. A mechanism for the CoSO₄-IL-1 hydrolysis system was proposed and IL-1 was recycled for the first time, which exhibited favorable catalytic activity over five repeated runs. This catalytic system may be valuable to facilitate energy-efficient and cost-effective conversion of biomass into biofuels and platform chemicals.     

Potential for cometabolic biodegradation of 1,4-dioxane in aquifers with methane or ethane as primary substrates

The objective of this research was to evaluate the potential for two gases, methane and ethane, to stimulate the biological degradation of 1,4-dioxane (1,4-D) in groundwater aquifers via aerobic cometabolism. Experiments with aquifer microcosms, enrichment cultures from aquifers, mesophilic pure cultures, and purified enzyme (soluble methane monooxygenase; sMMO) were conducted. During an aquifer microcosm study, ethane was observed to stimulate the aerobic biodegradation of 1,4-D. An ethane-oxidizing enrichment culture from these samples, and a pure culture capable of growing on ethane (Mycobacterium sphagni ENV482) that was isolated from a different aquifer also biodegraded 1,4-D. Unlike ethane, methane was not observed to appreciably stimulate the biodegradation of 1,4-D in aquifer microcosms or in methane-oxidizing mixed cultures enriched from two different aquifers. Three different pure cultures of mesophilic methanotrophs also did not degrade 1,4-D, although each rapidly oxidized 1,1,2-trichloroethene (TCE). Subsequent studies showed that 1,4-D is not a substrate for purified sMMO enzyme from Methylosinus trichosporium OB3b, at least not at the concentrations evaluated, which significantly exceeded those typically observed at contaminated sites. Thus, our data indicate that ethane, which is a common daughter product of the biotic or abiotic reductive dechlorination of chlorinated ethanes and ethenes, may serve as a substrate to enhance 1,4-D degradation in aquifers, particularly in zones where these products mix with aerobic groundwater. It may also be possible to stimulate 1,4-D biodegradation in an aerobic aquifer through addition of ethane gas. Conversely, our results suggest that methane may have limited importance in natural attenuation or for enhancing biodegradation of 1,4-D in groundwater environments.     

Terrestrial contamination in Apollo lunar samples

The Apollo lunar samples were seen to offer a unique opportunity in the search for extraterrestrial organic matter without the ambiguity surrounding meteorite analysis due to their unknown contamination histories. The recognition that only a small amount of indigenous organic material was likely to be present in lunar samples combined with the extreme sensitivity of organic analysis methods made it clear that this opportunity could be realized only by carefully controlling the collection, processing, and analysis of the samples in order that they might remain free of significant levels of contamination. The contamination control procedures adopted are described and the analytical evidence obtained throughout the program on potential contamination sources is presented. The organic contaminants actually found in the lunar samples by the various investigators are summarized. It is shown that the program succeeded in providing investigators with samples containing less than 0.1 ppm total contamination.     

Characteristics of hydrogen and methane production from cornstalks by an augmented two- or three-stage anaerobic fermentation process

This paper presents the co-production of hydrogen and methane from cornstalks by a two- or three-stage anaerobic fermentation process augmented with effective artificial microbial community. Two-stage fermentation by using the anaerobic sludge and DGGE analysis showed that effective and stable strains should be introduced into the system. We introduced Enterobacter aerogens or Clostridium paraputrificum into the hydrogen stage, and C. paraputrificum was proven to be more effective. In the three-stage process consisting of the improved hydrolysis, hydrogen and methane production stages, the highest soluble sugars (0.482 kg/kg cornstalks) were obtained after the introduction of Clostridium thermocellum in the hydrolysis stage, under the thermophilic (55 °C) and acidic (pH 5.0) conditions. Hydrolysates from 1 kg of cornstalks could produce 2.61 mol (63.7 l) hydrogen by augmentation with C. paraputrificum and 4.69 mol (114.6 l) methane by anaerobic granular sludge, corresponding to 54.1% energy recovery.     

Thermophilic anaerobic microbial communities that transform cellulose into methane (biogas)

The project is devoted to the screening of active anaerobic microbial communities which produce biogas via the decomposition of cellulose in thermophilic conditions (+55°C). Twenty-four samples were isolated from different natural and anthropogenic sources that contain desired microbial organisms. Growth medium was chosen to optimize the conditions for proliferation and selection of cellulolytic and methanogenic microorganisms. During the study of biogas formation dynamics, the most productive communities that remain active during five passages were selected. The biogas composition (methane, carbon dioxide, hydrogen) was investigated by gas chromatography. On average, the methane content in the gas mixture reached 60%. Microscopic studies revealed the presence of various morphotypes of microbial cells; their ratio varied during the stabilization of communities. The significance of the research on the transformation of cellulose into biogas is discussed.     

Hydrocarbons obtained by pyrolysis of some precambrian rocks of minnesota

Drill core samples of 42 Precambrian sedimentary, igneous, and metamorphic rocks were analyzed by heating under partial vacuum at 100°C and at 400°C to release hydrocarbons and other volatile products.The core samples yielded methane in amounts ranging from traces to 3 microliters per gram, but averaged much less. By way of comparison, samples of Middle Devonian Marcellus black shale, from Pennsylvania, yielded methane in amounts up to 7ul/g.Other straight chain hydrocarbons up to C₁₁ were found in the volatile products, especially those obtained at 400°C, and benzene was a common product, also mainly in the 400°C experiments. Carbon dioxide and nitrogen appear to form a large part of the nonhydrocarbon volatiles in at least some of the samples.Spectral data indicate that the straight chain pyrolysis products of the Precambrian rocks are mainly alkenes, whereas those of the Devonian rocks, referred to above, are a mixture of alkanes and alkenes. Alkanes were however, obtained from several algae-bearing Middle Precambrian argillites. Available evidence indicates, although not conclusively, that the alkenes were contained in the rock rather than being produced from alkanes during pyrolysis.The writers believe that surface contamination in most of the drill cores was minimal owing to the low permeability of the rocks studied, and that contamination by drilling was also minimal.There is a reasonable possibility that the volatiles, if not formed from kerogen residues by the pyrolysis experiments, are in part juvenile igneous gases or are substances that were distilled out of the deeperlying rocks during intervals of folding and metamorphism, and subsequently accumulated at higher levels.     

Microbial Formation of Ethane in Anoxic Estuarine Sediments

Estuarine sediment slurries produced methane and traces of ethane when incubated under hydrogen. Formation of methane occurred over a broad temperature range with an optimum above 65°C. Ethane formation had a temperature optimum at 40°C. Formation of these two gases was inhibited by air, autoclaving, incubation at 4 and 80°C, and by the methanogenic inhibitor, 2-bromoethanesulfonic acid. Ethane production was stimulated by addition of ethylthioethanesulfonic acid, and production from ethylthioethanesulfonic acid was blocked by 2-bromoethanesulfonic acid. A highly purified enrichment culture of a methanogenic bacterium obtained from sediments produced traces of ethane from ethylthioethanesulfonic acid. These results indicate that the small quantities of ethane found in anaerobic sediments can be formed by certain methanogenic bacteria.     

Cometabolism of DDT analogs by a Pseudomonas sp

A Pseudomonas sp. capable of growth on several nonchlorinated and mono-p-chloro-substituted analogs of DDT as a sole carbon source degraded bis(p-chlorophenyl)methane and 1,1-bis(p-chlorophenyl)ethane only in the presence of diphenylethane. The products p-chlorophenylacetic acid and 2-(p-chlorophenyl)-propionic acid were not further metabolized by the bacterium. Other chlorinated analogs of DDT were found to be recalcitrant to cometabolic degradation with diphenylethane.     

Identification of furoyl-containing advanced glycation products in collagen samples from diabetic and healthy rats

The compounds resulting from the reaction of glucose with proteins (advanced glycation products) can be important markers of chronic diabetic complications. To test the possible diagnostic value of advanced glycation products containing the furoyl moiety, collagen samples from diabetic and healthy rats were analyzed by parent ion spectroscopy. In our study, we compared normal collagen, diabetic collagen and normal collagen incubated with different glucose concentrations and we employed different hydrolysis procedures (HCl and proteinase). Mass spectroscopic measurements performed on hydrolyzed samples showed that either different samples or different hydrolysis procedures produce a similar set of furoyl-containing compounds. 2-(2-Furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) which has been reported to be one of the advanced glycation products, was never found in any of the samples examined. Hence neither FFI nor furoyl-containing molecules can be considered markers of advanced glycation processes.