Thermal characterization of natural and synthetic humic substances

Thermogravimetric technique was used for the characterization of natural (humic) and synthetic (melanoidins) substances. The influence of pH on the thermal stability of humic substances was studied. A similarity in thermal behaviour of natural humic substances and of melanoidins (prepared from an excess of sugar) and the unique thermal properties of melanoidins (prepared from basic amino acids) was observed. Thermal behavior of natural and synthetic substances was compared with model compounds of sugar, peptide and kerogen types.     

Characterization of melanoidins by IR spectroscopy—I. Galactose-glycine melanoidins

A comparative IR spectral study was conducted with synthetic melanoidins which were prepared from various ratios of sugars and amino acids and a pseudomelanoidin which was prepared from sugar only. A significant resemblance was observed between the pseudomelanoidin and the melanoidin prepared from a high (9:1) sugar ratio (type 1). Another series of melanoidins (type 2) consists of those prepared from an “excess” of amino acid (9:1 and 1:1).Type 1 showed the presence of carbonyl and hydroxyl groups as well as aromatic vibrations. The same groups were detected in the spectrum of the pseudomelanoidin. These features were observed in a very low concentration in the type 2 melanoidins.Carboxylic groups are present in pseudomelanoidin and in both types of the melanoidins. However, the acid strength of this group is high in melanoidin of type 2, but low in type 1 and in the pseudomelanoidin.The nitrogen atom which is involved in the melanoidin structure is present as a tertiary amine.Heat treatment of KBr discs containing glycine, galactose and a mixture thereof revealed that galactose was converted into pseudomelanoidin; a mixture of galactose-glycine yielded the corresponding melanoidin of type 2; no reaction was observed with glycine.     

The formation and clay mineral reactions of melanoidins

Sugars and amino acids condense readily to form polymers known as melanoidins that closely resemble natural humic substances. In this study melanoidins are formed by reacting glucose with each of three amino acids: glutamic acid, valine and lysine. The basic amino acid, lysine, reacts with glucose at a much greater rate than either of the other two compounds. The chemical properties of the melanoidins are strongly influenced by the amino acid from which they are formed. The nitrogen contents, acidities and electrophoretic behavior of the polymers all reflect functional group distributions inherited from the amino acids. These chemical differences strongly influence the extent of association of the melanoidins with kaolinite and montmorillonite clay minerals. Laboratory simulations suggest that melanoidin formation may be favored in marine environments where basic amino acids should preferentially condense with sugars to form nitrogen-rich polymers that have a great affinity for clay mineral surfaces.     

Carbon-13 cross polarized magic-angle samples spinning nuclear magnetic resonance of melanoidins

Melanoidins synthesized from amino-acids and sugars in various ratios and humic substances from Hula Basin (Israel) have been investigated by ¹³C-CP/MAS NMR spectroscopy. Most spectra indicated a significant similarity between melanoidins and humics. It is suggested that the Maillard reaction plays a more significant role in the formation of humic substances than the “traditional” theory based on a lignin source. Several heterocyclic and heteroaromatic moieties (mainly furanoid, hydroxy-alkyl-furanone and hydroxy-alkyl-cyclopentenone) have been considered as major “building blocks” of humic substances and melanoidins.Aromaticities calculated from NMR spectral data of SM (“sugar” enriched melanoidins) and AAM (amino acid enriched melanoidins) coincide remarkably with the aromaticities of terrestrial and marine humics, respectively.     

Melanoidins—kerogen precursors and geochemical lipid sinks: a study using pyrolysis gas chromatography (PGC)

Melanoidins, acidic polymeric products of amino acid/sugar condensation reactions, are shown to be capable of reacting with and binding functionalized lipid molecules. Pyrolysis gas chromatography is used to monitor the inclusion of lipids (alcohols and acids) into, and to monitor the changes in composition caused by artificial diagenesis of, the melanoidins. Artificial diagenesis of the lipidcontaining melanoidins produces a material resembling kerogen. The possible role of melanoidins in the formation of kerogens is discussed as are internal transformations within kerogens during diagenesis.     

Isotopic analyses of nitrogenous compounds from the Murchison meteorite: ammonia, amines, amino acids, and polar hydrocarbons

The combined volatile bases (ammonia, aliphatic amines, and possibly other bases), ammonia, amino acids, and polar hydrocarbons were prepared from the Murchison meteorite for isotopic analyses. The volatile bases were obtained by cryogenic transfer after acid-hydrolysis of a hot-water extract and analyzed by combined gas chromatography-mass spectrometry of pentafluoropropionyl derivatives. The aliphatic amines present in this preparation comprise a mixture that includes both primary and secondary isomers through C5 at a total concentration of > or = 100 nmoles g-1. As commonly observed for meteoritic organic compounds, almost all isomers through C5 are present, and the concentrations within homologous series decrease with increasing chain length. Ammonia was chromatographically separated from the other volatile bases and found at a concentration of 1.1-1.3 micromoles g-1 meteorite. The ammonia analyzed includes contributions from ammonium salts and the hydrolysis of extractable organic compounds, e.g., carboxamides. Stable isotope analyses showed the volatile bases to be substantially enriched in the heavier isotopes, relative to comparable terrestrial compounds delta D < or = +1221%; delta 13C = +22%; delta 15N = +93%). Ammonia, per se, was found to have a somewhat lower delta 15N value (+69%) than the total volatile bases; consequently, a higher delta 15N (>93%) can be inferred for the other bases, which include the amines. Solvent-extractable polar hydrocarbons obtained separately were found to be enriched in 15N (delta 15N = +104%). Total amino acids, prepared from a hydrolyzed hot-water extract by cation exchange chromatography, gave a delta 15N of +94%, a value in good agreement with that obtained previously. Nitrogen isotopic data are also given for amino acid fractions separated chromatographically. The delta 15N values of the Murchison soluble organic compounds analyzed to date fall within a rather narrow range (delta 15N = +94 +/- 8%), an observation consistent with their formation, or formation of their precursors, by interstellar chemistry.     

A comparative study of the hydroxy acids from the Murchison, GRA 95229 and LAP 02342 meteorites

The hydroxy acid suites extracted from the Murchison (MN), GRA 95229 (GRA) and LAP 02342 (LAP) meteorites have been investigated for their molecular, chiral and isotopic composition. Substantial amounts of the compounds have been detected in all three meteorites, with a total abundance that is lower than that of the amino acids in the same stones. Overall, their molecular distributions mirror closely that of the corresponding amino acids and most evidently so for the LAP meteorite. A surprising l-lactic acid enantiomeric excess was found present in all three stones, which cannot be easily accounted by terrestrial contamination; all other compounds of the three hydroxy acid suites were found racemic. The branched-chain five carbon and the diastereomer six-carbon hydroxy acids were also studied vis-a-vis the corresponding amino acids and calculated ab initio thermodynamic data, with the comparison allowing the suggestion that meteoritic hydroxyacid at these chain lengths formed under thermodynamic control and, possibly, at a later stage than the corresponding amino acids. ¹³C and D isotopic enrichments were detected for many of the meteoritic hydroxy acids and found to vary between molecular species with trends that also appear to correlate to those of amino acids; the highest δD value (+3450‰) was displayed by GRA 2-OH-2-methylbutyric acid. The data suggest that, while the amino- and hydroxy acids likely relate to common presolar precursor, their final distribution in meteorites was determined to large extent by the overall composition of the environments that saw their formation, with ammonia being the determining factor in their final abundance ratios.     

Fate of microbial biomass-derived amino acids in soil and their contribution to soil organic matter

Soil organic matter (SOM) is important for soil fertility and for the global C cycle. Previous studies have shown that during SOM formation no new compound classes are formed and that it consists basically of plant- and microorganism-derived materials. However, little data on the contribution from microbial sources are available. Therefore, we investigated previously in a model study the fate of C from ¹³C-labelled Gram-negative bacteria in soil (Kindler, R., Miltner, A. Richnow, H.H., Kästner, M., 2006. Fate of gram negative bacterial biomass in soil – mineralization and contribution to SOM. Soil Biology and Biochemistry 38, 2860–2870) and showed that 44% of the bulk ¹³C remained in the soil. Here we present the corresponding data on the fate of amino acids hydrolysed from proteins, which are the most abundant components of microbial biomass. After 224 days incubation, the label in the total amino acids in the soil amended with ¹³C-labelled cells decreased only to >95%. The total amino acids therefore clearly showed a lower turnover than the bulk ¹³C and a surprisingly stable concentration. Proteins therefore have to be considered as being stabilised in soil in dead, non-extractable biomass or cell fragments by known general stabilisation mechanisms. The label in the amino acids in a fraction highly enriched in living microbial biomass decreased to a greater extent, i.e. to 25% of the initially added amount. The amino acids removed from this fraction were redistributed via the microbial food web to non-living SOM. All amino acids in the microbial biomass were degraded at similar rates without a change in isotopic signature. The nuclear magnetic resonance (NMR) spectra of the soils were very similar and indicate that the residues of the degraded microbial biomass were very similar to those of the SOM and are a significant source for the formation of the SOM.     

我国东部几个中新生代盆地泥质岩中的氨基酸

从现代沉积物到前寒武纪的沉积岩中都发现有氨基酸的存在。关于它们的组成、含量及其地质意义在许多文献中都曾涉及到,特别是对近代各种环境沉积中的氨基酸及其在成岩作用过程中的变化,已经积累了相当多的分析资料。本文对以渤海第三纪沉积盆地为主的我国东部地区中新生代泥质岩样品进行了氨基酸的分析,并初步讨论了这些分析结果与沉积环境的关系。     

Organic compounds in the Forest Vale, H4 ordinary chondrite

We have analyzed the H4 ordinary chondrite Forest Vale for polycyclic aromatic hydrocarbons (PAHs) using two-step laser mass spectrometry (L2MS) and for amino acids using a standard chromatographic method. Indigenous PAHs were identified in the matrices of freshly cleaved interior faces but could not be detected in pulverized silicates and chondrules. No depth dependence of the PAHs was found in a chipped interior piece. Amino acids, taken from the entire sample, consisted of protein amino acids that were nonracemic, indicating that they are terrestrial contaminants. The presence of indigenous PAHs and absence of indigenous amino acids provides support for the contention that different processes and environments contributed to the synthesis of the organic matter in the solar system.     

Analytical problems in determining peptide nitrogen in the humic fractions of three Italian soils

Peptides were released from organic matter fractions of three Italian soils (humin, humic and fulvic acids), when the samples were hydrolyzed in Ba(ON)₂-saturated solution at 105°C for 2 hr. The peptides obtained were separated using electrophoresis and paper chromatography. The presence of polypeptides in the soil organic matter was indicated by: (1) their hydrolysis by pronase; (2) the amino acids released by 6 N HCl hydrolysis; (3) The comparison of i.r. spectra of humic fractions before and after hydrolysis with 6 N HCl.Attempts at isolating the native proteinaceous compounds using electrophoresis in polyacrylamide gel failed; additionally, our attempts to hydrolyze proteinaceous components enzymatically in unfractionated soil organic matter, as well as in its fractions, before and after methylation, with pepsin, papain and pronase, were unsuccessful. Pronase demonstrated a weak proteolytic activity only at very low substrate-enzyme ratios (20 : 1) in humic and fulvic fractions and in whole phyrophosphate extract. Deproteinated substrates treated with pronase also released free amino acids, suggesting autodigestion.In humin, humic and fulvic fractions we found a total amino acid content of 40–45%, 12–24% or 1–85, respectively. Amino acid recovery from single fractions was about 70–80% of the total content in the unfractionated soil.     

The distribution and nature of amino acids and other nitrogen-containing compounds in Lake Ontario surface sediments

Five surface sediment samples (0–3 cm), two suspended sediment samples and a zooplankton sample from Lake Ontario were analysed for nitrogen-containing compounds. Amino acids, amino sugars, ethanolamine and urea were separated and characterized by ion-exchange chromatography. Free amino acids and soluble combined amino acids and amino sugars accounted for less than 0–25 per cent of the total nitrogen in the sediments. Insoluble combined amino acids and amino sugars were the most abundant nitrogen fraction in the sediments, making up from 49 to 55 per cent of the total nitrogen. Evidence is presented that asparagine, glutamine and citrulline are present in the interstitial waters and may make up part of the sediment organic nitrogen that was not characterized.The free amino acids released by the proteolytic enzyme, pronase, from the interstitial waters and sediment humic and fulvic acid extracts were determined. Pronase released 65 per cent of the soluble combined amino acids and 34 per cent of the fulvic acid amino acids as free amino acids. Enzyme activity was inhibited in the presence of the humic acid extract. The results indicate that the combined amino acids in the interstitial waters and fulvic acid extracts are intermediates between the primary aquatic detritus and the sediment humic acids. The enzyme experiments and infra-red data indicate that part of the sediment amino acids are combined through peptide linkages.     

Influence of Murchison or Allende minerals on hydrogen-deuterium exchange of amino acids

Deuterium-enriched amino acids occur in the Murchison carbonaceous chrondrite. This meteorite underwent a period of aqueous alteration with isotopically light water. With the objective of setting limits on the conditions of aqueous alteration, the exchange of the carbon-bonded hydrogen atoms of amino acids with D2O has been studied from 295 to 380 K as a function of time and meteorite/heavy water ratio. The amount of Murchison or Allende dust present has a significant effect on the rate and amount of hydrogen-deuterium exchange observed. At elevated temperatures, the alpha-hydrogens of all the amino acids studied were found to exchange with deuterium. In glycine and aspartic acid, this process resulted in total exchange of the carbon-bonded hydrogen. A completely deuterated isotopomer of alanine was produced in significant quantities only when the rock/water ratio was greater than 0.5. No exchange of carbon-bonded hydrogens was observed in the case of amino acids which do not possess an alpha-hydrogen atom. The rates of H/D exchange for amino acids observed here did not correspond to deuterium enrichment of the amino acids in the Murchison meteorite. These results suggest that H/D exchange with water had a negligible effect on the observed deuterium enrichment of amino acids found in Murchison and that the temperature at which the amino acids were exposed to liquid water was close to 273 K.     

热带东太平洋中国多金属结核开辟区深海成矿环境的氨基酸表征

为了解中国多金属结核开辟区沉积物有机质活性及深海成矿沉积环境特征，使用高效液相色谱（HPLC）法分析了表层沉积物氨基酸的组成状况。结果显示该区表层沉积物中氨基酸与氨基糖含量(干样)均较低，分别在0.30～0.59 mg·g-1及 0.10～0.17 mg·g-1之间。其中以中性氨基酸占绝对优势，占50%以上，其次为酸性氨基酸和含氢氧基氨基酸，分别占17%和11%，碱性氨基酸占9%，芳香与含硫氨基酸均在5%以下,表明深海沉积环境为氧化环境，且偏碱性条件。表征有机质活性的Asp/β Ala、Glu/γ Aba及DI值（（Glu+Phe+Leu+Ala+Val）/ (β Ala+γ Aba)）分别为1.1～1.9，0.8～2.2和1.4～2.9，表明该区有机质成熟度已很高。蛋白氨基酸/非蛋白氨基酸的比值为1.0～2.0，葡萄糖胺/半乳糖胺比值则在1.7～3.0之间变化，表明该海区有机质经过强烈的细菌改造，即海区存在强烈的细菌活动，活性较低，表征了多金属结核有机—微生物成矿的可能性。     

N-15 NMR spectra of naturally abundant nitrogen in soil and aquatic natural organic matter samples of the International Humic Substances Society

The naturally abundant nitrogen in soil and aquatic NOM samples from the International Humic Substances Society has been characterized by solid state CP/MAS ¹⁵N NMR. Soil samples include humic and fulvic acids from the Elliot soil, Minnesota Waskish peat and Florida Pahokee peat, as well as the Summit Hill soil humic acid and the Leonardite humic acid. Aquatic samples include Suwannee River humic, fulvic and reverse osmosis isolates, Nordic humic and fulvic acids and Pony Lake fulvic acid. Additionally, Nordic and Suwannee River XAD-4 acids and Suwannee River hydrophobic neutral fractions were analyzed. Similar to literature reports, amide/aminoquinone nitrogens comprised the major peaks in the solid state spectra of the soil humic and fulvic acids, along with heterocyclic and amino sugar/terminal amino acid nitrogens. Spectra of aquatic samples, including the XAD-4 acids, contain resolved heterocyclic nitrogen peaks in addition to the amide nitrogens. The spectrum of the nitrogen enriched, microbially derived Pony Lake, Antarctica fulvic acid, appeared to contain resonances in the region of pyrazine, imine and/or pyridine nitrogens, which have not been observed previously in soil or aquatic humic substances by ¹⁵N NMR. Liquid state ¹⁵N NMR experiments were also recorded on the Elliot soil humic acid and Pony Lake fulvic acid, both to examine the feasibility of the techniques, and to determine whether improvements in resolution over the solid state could be realized. For both samples, polarization transfer (DEPT) and indirect detection (¹H–¹⁵N gHSQC) spectra revealed greater resolution among nitrogens directly bonded to protons. The amide/aminoquinone nitrogens could also be observed by direct detection experiments.     

Summary and implications of reported amino acid concentrations in the Murchison meteorite

A study of literature reports of the concentrations of amino acids in extracts from the Murchison meteorite shows that many of the concentration ratios are constant. There are two possible interpretations of these ratios. One is that they are controlled by the pathways through which the amino acids formed, from which it follows that the amino acids are distributed in the same proportions throughout the meteorite. The other interpretation is that the ratios result from the analytical procedures used to extract the amino acids from the meteorite. These methods rely heavily on high-temperature (100 degrees C) aqueous extraction and subsequent high-temperature acid hydrolysis. A correlation was observed in the present study between the relative concentrations of several amino acids in the meteorite extracts and their relative aqueous solubilities at 100 degrees C (alanine, valine, leucine, isoleucine, norleucine, aspartic acid, glutamic acid and glycine). The extract solutions are dilute, and far from the saturation limits, but these correlations suggest that the sampling procedure affects directly the reported concentrations for these amino acids. Ratios of the concentration of serine to those of glycine are also constant but cannot be accounted for solely by relative solubilities, and, as suggested elsewhere, serine as well as phenylalanine and methionine may be terrestrial contaminants. Data for beta-alanine, alpha-aminobutyric acid, proline, sarcosine, alloisoleucine, beta-aminoisobutyric acid, beta-aminobutyric acid, and threonine also show constant abundances relative to glycine, but lack of solubility data at extraction conditions prohibits evaluating the extent of possible sampling bias for these amino acids. If the extraction process does not bias the results, and all extractable amino acids are removed from meteorite samples, then the properties of amino acids which control both their solubilities and their concentrations in the meteorite need to be established. The possibility of sampling bias needs to be tested experimentally before concluding that extraction is complete, and that the constant relative abundances indicate that the relative concentrations of amino acids are homogeneous in the meteorite.     

Nitrogen-containing compounds in two CR2 meteorites: N composition, molecular distribution and precursor molecules

Amino acids, amines and aldehydes were obtained from the water extracts of two CR2 carbonaceous chondrites from Antarctica and analyzed for their molecular and ¹⁵N isotopic content. These compounds were found to differ significantly from those of CM chondrites in both overall abundances and molecular distribution. The amino acids suites comprise a preponderant abundance of linear, 2-H amino acids, show rapid non-linear decrease with the compounds’ increasing chain length and include protein amino acids never identified in meteorites before, such as threonine, tyrosine and phenylalanine. The presence of tertiary amines as well as a diverse, large abundance of aldehydes and ketones also distinguishes both CR2 organic suites. The δ¹⁵N values determined for CR2 amino acids have a distribution between molecular subgroups that is opposite to the one of their δD values, with 2-H amino acids having higher δ¹⁵N and lower δD values than 2-methyl amino acids, while the opposite is true for 2-methyl amino acids. Based on theoretical data, these isotopic findings would place the formation of the two amino acid groups or their direct precursors at different ISM stages of star formation.     

Amino acid abundances and stereochemistry in hydrothermally altered sediments from the Juan de Fuca Ridge, northeastern Pacific Ocean.

The Juan de Fuca Ridge is a hydrothermally active, sediment covered, spreading ridge situated a few hundred kilometres off the west coast of North America in the northeastern Pacific Ocean. Sediments from seven sites drilled during the Ocean Drilling Program (ODP) Legs 139 and 168 were analyzed for total hydrolyzable amino acids (THAA), individual amino acid distributions, total organic C (TOC) and total N (TN) contents. The aim was to evaluate the effects of hydrothermal stress on the decomposition and transformation of sedimentary amino acids. Hydrolyzable amino acids account for up to 3.3% of the total organic C content and up to 12% of the total N content of the upper sediments. The total amounts of amino acids decrease significantly with depth in all drilled holes. This trend is particularly pronounced in holes with a thermal gradient of around 0.6 degrees C/m or higher. The most abundant amino acids in shallow sediments are glycine, alanine, lysine, glutamic acid, valine and histidine. The changes in amino acid distributions in low temperature holes are characterized by increased relative abundances of non-protein beta-alanine and gamma-aminobutyric acid. In high temperature holes the amino acid compositions are characterized by high abundances of glycine, alanine, serine, ornithine and histidine at depth. D/L ratios of samples with amino acid distributions similar to those found in acid hydrolysates of kerogen, indicate that racemization rates of amino acids bound by condensation reactions may be diminished.     

Long-living free radicals study of stepwise pyrolyzed melanoidins and humic substances

ESR measurements of stepwise-pyrolyzed melanoidins and humic substances (at various temperatures, mesh size, and pH values) furnished the following information: the melanoidin structure stabilizes the long-living free radicals in a manner similar to humic substances; the g and Ng values of melanoidins are similar to those of the humic substances, the cleavage of CC and CX (X = heteroatom) bonds increases the Ng value. Thermogravimetric curves, weight loss by stepwise pyrolysis, and ¹³C-CP/MAS NMR were found to be in good correlation with ESR data regarding the structural features of melanoidins and humic substances.     

安徽和县猿人遗址牙化石的氨基酸生物地球化学

本文从氨基酸生物地球化学的角度,对安徽和县猿人遗址牙化石中的氨基酸进行了探讨,初步获得如下结果:1.牙釉化石的氨基酸组成中,酸性氨基酸含量极高(总量不低于30%);中性氨基酸含量也较大(总量在20%左右);碱性氨基酸含量极低,均在1%以下。 2.牙釉化石的氨基酸组成基本上取决于原生蛋白质的组成模式,但由于成岩作用的影响,使牙釉化石的氨基酸组成和含量与现生哺乳动物牙釉相比,存在较大差别。3.将测得的牙釉化石中的D-别异亮氨酸/L-异亮氨酸比值,以及根据对该地点动物化石研究而估算的平均成岩温度(12.5℃-13℃),代入有关的氨基酸测年公式,求得该猿人遗址的化石年龄为23万年。