Pim F. van Bergen

Organic geochemical studies of soils from the Rothamsted Classical Experiments - I. Total lipid extracts, solvent insoluble residues and humic acids from Broadbalk Wilderness

Abstract Total lipid extracts and insoluble organic matter, i.e. solvent insoluble matter and humic acids, were studied from soil samples taken from the three adjacent plots comprising the Broadbalk Wilderness at Rothamsted Experimental Station, Harpenden, Hertfordshire, U.K. Analyses involved high-temperature gas chromatography (HT-GC) and HT-GC-mass spectrometry (HT-GC-MS) to investigate trimethylsilylated total lipid extracts and Curie-point pyrolysis-GC (Py-GC) and Py-GC-MS to investigate solvent insoluble fractions. The plots were chosen specifically for their different types of vegetation cover. Samples of the vegetation were examined in parallel with the underlying soils in an effort to follow the fate of the major plant components in soil. The application of HT-GC and HT-GC-MS allowed changes in high molecular weight lipids, particularly intact acyl lipids, such as triacylglycerols, wax esters, steryl and triterpenyl esters, to be studied in leaf and soil extracts. The total lipid extracts of the soil samples from the wooded area were dominated by the input from leaf-derived lipids. The lipid extracts of soils from the grazed and stubbed areas were markedly different from those from the wooded area, and reflected the mixed vegetation cover dominated by grass species. In marked contrast, the pyrolysis data from the insoluble organic matter and humic fractions of the soils did not reflect the composition of the lignin comprising the overlying vegetation, but rather showed evidence of amino acid moieties probably present as polypeptides. The absence of the lignin signal is possibly due to rapid diagenetic changes presumed to be influenced by the slightly alkaline pH of the soil. The ability to recover recognizable chemical signals from soil lipids has important implications for archaeological investigations aimed at revealing temporal changes in vegetation cover and/or differences in land use at specific site locations.

Biomacromolecules of Algae and Plants and their Fossil Analogues

A review of our current understanding of resistant biomacromolecules derived from present and past algae and higher plants is presented. Insight in the nature of recent and fossil macromolecules is strongly hampered by the difficulties in obtaining the material in pure and unaltered form. For the extant material, avoiding artificial condensation and structural alteration as a result of chemical isolation and purification of biomacromolecules requires constant attention. To date, several types of sporopollenin seem to occur. One type is characterised by oxygenated aromatic building blocks, in particular p-coumaric acid and ferrulic acid. The other type is thought to consist predominantly of an aliphatic biopolymer. In this review it is concluded that extant sporopollenin consists of the aromatic type, whereas the aliphatic component of fossil sporopollenin is due to early-diagenetic oxidative polymerization of unsaturated lipids. The cuticles of most higher plants contain the aliphatic biopolyester cutin. Additionally, cuticles of drought-adapted, mostly CAM plants, seem to contain the non-hydrolysable aliphatic biopolymer cutan. Only a very few algae are able to biosynthesize resistant, (fossilisable) cell walls: some Chlorophyta, Eustigmatophyta and Prasinophyta produce the aliphatic biopolymer algaenan. Some Dinophyta are also capable of producing algaenan cell walls. Additionally, some taxa produce highly resistant cyst-walls with a high proportion of aromatic moieties. For the morphologically well-preserved fossil material, contamination by organic particles other than the target taxon is hard to eliminate and can contribute to either the aliphatic or aromatic signal. Furthermore, post-mortem migration of aliphatic moieties into, and their condensation onto the macromolecule might occur, e.g. by oxidative polymerization. These phenomena hamper the evaluation of the aliphatic signature of fossil plant material and may for example explain the preservation of initially cutin-based cuticles or non-algaenan containing algae. The extent to which migration and in situ formation of aromatic moieties plays a role in modifying resistant algal macromolecules, notably under elevated temperature and/or pressure conditions, still remains an open question.

Organic geochemical studies of soils from the Rothamsted classical experiments - V. The fate of lipids in different long-term experiments.

Lipid extracts from four long-term experiments (Broadbalk Wilderness, Geescroft Wilderness, Hoosfield Spring Barley and Park Grass) were analysed using a combination of gas chromatography, gas chromatography‐mass spectrometry and gas chromatography‐combustion‐isotope ratio mass spectrometry. The lipid content of the primary organic inputs for each soil werealso analysed inorder toassess the early diagenetic fate ofthe variouscompound classespresent.SoilpH was observed to, either directly or indirectly, have a significant eAect on lipids with a relative increase in abundance of n-alkanes at higher pH (7.31) and a large relative increase in n-alkanoic and o-hydroxy acids at low pH (3.74). Triacylglycerols exhibited severe losses irrespective of pH. In an arable soil, n-alkanoic acids showed a temporal decrease in concentration whilst levels of n-alkanols remained static, the diAerence was ascribed to a more rapid turnover and possible leachate migration of the n-alkanoic acids. The phytosterol, sitosterol, was observed to rapidly diminish in soils possibly as a result of assimilation by soil dwelling invertebrates. Analysis of 5b-stigmastanol (a faecal biomarker) showed that it remainedat levels indicativeofmanuringeven after 113 years. Furthermore, analysis of 5b-stanyl esters revealed a manuringsignal even more persistent than that exhibited by the free stanols. Knowledge of the biogeochemical cycling of lipids in the soil environment will help facilitate understanding of the processes which underpin carbon cycling in soils. # 2000 Elsevier Science Ltd. All rights reserved.

Recognition of chitin and proteins in invertebrate cuticles using analytical pyrolysis-gas chromatography and pyrolysis-gas chromatography/mass spectrometry

Flash pyrolysis/gas chromatography (py/GC) and py/GC/mass spectrometry (MS) have been utilized to characterize the cuticles of invertebrates chemically. Pyrolysis products have been identified and assigned to specific cuticular components. Acetylpyridones, acetamidofuran, 3-acetamido-5-methylfuran and 3-acetamido-(2 and 4)-pyrones are proposed as characteristic pyrolysis markers for chitin. Pyrolysis products displaying ions of m/z 70, 154, 168, 194 are thought to derive from diketopiperazine structures and provide potential markers for proteins and peptides in which proline, alanine, valine, arginine and glycine are the dominant amino acids. These products, constituting specific pyrolysis markers for invertebrate cuticles, may reflect the amino acid composition of their constituent structural proteins. The source of the various pyrolysis products of proteins has been verified by pyrolysis of reference proteins, peptides and amino acid mixtures. The presence of additional pyrolysis products related directly to histidine and catechol moieties is consistent with the chemical structure and composition proposed for arthropod cuticles based on recent work utilizing solid state 13C and 15N nuclear magnetic resonance. This study constitutes the first comprehensive chemical characterization of the pyrolysis products of invertebrate cuticles and provides the basis for future investigations requiring qualitative screening for cross-linked chitin and proteins in modern and fossil cuticles and in materials, e.g. geopolymers, that may be derived from them.

Organic geochemical studies of soils from the Rothamsted classical experiments — VI. The occurrence and source of organic acids in an experimental grassland soil

Total lipid extracts (TLEs) of grass (aerial and sub-aerial, Holcus lanatus) from a plot on a long-term grassland experiment, and associated soil, along with the organic fraction of the TLE hydrolysates and the hydrolysates of the solvent extracted vegetation have been separated into fractions containing specific compound classes and analysed using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The distributions of n-alkylcarboxylic acids, o-hydroxycarboxylic acids and dicarboxylic acids in the grass and the underlying soil have been determined. Short-chain (<C20) n-alkylcarboxylic acids were designated as having derived from both aerial and sub-aerial vegetation. However, longer-chain n-alkylcarboxylic acids were ascribed to suberin as a predominant source. Moreover, o-hydroxycarboxylic acids and dicarboxylic acids observed in the soil were designated as having predominantly derived from inputs of free, extractable polyesters and suberin intimately associated with plant roots. This study indicates the importance of root material as a predominant source of aliphatic, organic acids in the soil of temperate grassland biomes. 7 2000 Elsevier Science Ltd. All rights reserved.

The oil-generating potential of plants from coal and coal-bearing strata through time: a review with new evidence from Carboniferous plants

Abstract A combination of microscopy and chemistry is used to characterize high molecular weight components of plant material which contributed to Carboniferous coals and coal-bearing strata. Material was selected from coals, paper coals, coal balls, oil-shales and shales and included cuticles from stems and leaves, periderm from stems and rooting systems, spore walls, algal cell walls and ‘resin’ rodlets. Chemical analyses were undertaken using Curie-point pyrolysis-gas chromatography (-mass spectrometry) (Py-GC-(MS)) and 13C solid state Nuclear Magnetic Resonance (NMR). Light microscopy and scanning electron microscopy were used to ensure a detailed understanding of the plant material which was analysed chemically. This study has emphasized those plants, plant tissues and organs which are known to have been dominant or major contributors to Carboniferous coals and coal-bearing sequences: arborescent lycophyte periderm (Diaphorodendron stems and Stigmaria rhizomorphs); pteridosperm cuticles (medullosan (e.g. Alethopteris) and Karinopteris), lycophyte stem cuticle (Eskdalia) and arborescent lycophyte spores (megaspores and microspores). Several algal cell walls (Tetraedron, Tasmanites and Gloeocapsomorpha) and the ‘resin’ rodlets derived from medullosan pteridosperm petioles have also been analysed. Results show that all of these elements (except resins) contain (or are dominated by) highly resistant, highly aliphatic macromolecules which have the potential to yield, upon catagenesis, n-alkanes which are found in crude oils. Resins potentially contribute cyclic hydrocarbons to crude oils. Combining all this evidence it is concluded that Carboniferous coals are oil prone and that explanations for the absence of oil-pools derived from such coals must be sought in geological or exploration factors and not in the nature of the coals themselves. A review of coal-forming floras through time, and of suggested terrestrially sourced oils and their parent coals, leads us to conclude that high molecular weight components of higher plant materials have potentially made a major contribution to oils sourced from coals and coal-bearing strata over a long period of geological time (Devonian onwards). Cell walls of freshwater algae have made a comparable contribution since the Ordovician. The oil-generating potential of each coal or organic-rich sediment will depend upon a combination of depositional environment and the floristic and chemical composition of the source vegetation and its component plants and plant parts.

Organic geochemical studies of soils from the Rothamsted Classical Experiments—II, Soils from the Hoosfield Spring Barley Experiment treated with different quantities of manure

Abstract Total lipid extracts (TLEs) were obtained from soil samples taken in the years 1882, 1913, 1946, 1965 and 1995 from three treatments of the Hoosfield Spring Barley Experiment at Rothamsted Experimental Station, Harpenden, Hertfordshire, U.K. The extracts were fractionated and molecular analyses performed using gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). In addition to the soil samples (contemporary and archived), the two primary organic inputs, barley and farmyard manure (FYM), were studied so that the composition and diagenetic behaviour of extractable lipids from the two inputs could be assessed. The major aliphatic soil lipids exhibited variable dominance with respect to the expression of barley and FYM derived lipids. Wax esters were of low abundance and too strongly affected by degradation and transesterification processes to identify a dominant input whilst the composition of soil n -alkanols was largely determined by FYM with a minor pedogenic input. n -Alkanoic acids increased in overall abundance in soils with a continual FYM input and showed appreciable degradation in soils receiving no manure. C 32 ββ hopanoic acid was detected in two plots and appeared to degrade at a rate similar to 5 β -stanols with the most likely source of this compound being the FYM. Measurements of absolute concentrations of 5 β -stanols, biomarkers characteristic of manuring, revealed that a manuring signal persisted for >120 years within the soil which had been intensively cultivated annually and had received no manure since 1871. The persistence of a manure signal in soils has important implications for archaeological studies of agricultural practices based on 5 β -stanols.

Stable carbon isotopes of wood: a clue to palaeoclimate?

Abstract Detailed stable carbon isotope and molecular investigations were undertaken on a number of archaeological and fossil wood specimens to provide insights into their use as rigorous independent palaeoclimatic and palaeoenvironmental parameters. The isotope data revealed large differences amongst the material with the oldest specimens being least depleted in 13 C. Although natural variation could account for some of the observed differences, the isotope values of the archaeological and Tertiary specimens are most probably related to the absolute abundance of polysaccharides present and the degree of lignin alteration. The molecular data, based on pyrolysis, of the Antarctic Cretaceous conifer specimens revealed only transformed lignin, with virtually no intact lignin building blocks (2-methoxyphenols) preserved, and no evidence of polysaccharides. This degree of chemical alteration is suggested here to be one of the main causes for the 13 C enriched values of these conifer specimens. These results show the importance of combining detailed molecular information on individual wood components with stable carbon isotope data for palaeoclimatic and palaeoenvironmental studies.

A qualitative study on the chemical composition of ester-bound moieties in an acidic andosolic forest soil

Abstract Solvent insoluble ester-bound moieties, released after base treatment from an acidic andosolic forest soil A-horizon from Madeira, were sequentially extracted and analysed using gas chromatography and gas chromatography–mass spectrometry. The separation of all moieties released into several pH “subfractions” allowed the various products released to be studied in detail. The distributions obtained for 2-alkanols, diols, n-alkanols, n-alkanoic acids, ω-hydroxy acids, trihydroxy acids, dihydroxy diacids and phenolic acids indicate a suberin dominated origin of ester-related moieties in this andosolic soil A-horizon. However, a small contribution from plant cutins can not be ruled out completely. Besides plant biopolyesters, the detection of iso and anteiso alkanoic acids, together with short-chain di and ω-hydroxy acids suggests an additional bacterial-derived contribution. In addition to being attached to intact biopolyesters or molecules trapped in the insoluble organic macromolecular network present in soils, we suggest that hydroxy, n-alkanoic, aromatic and diacid moieties released upon base treatment of andosolic soils may occur as Al/Fe organic complexes.

Evidence for demethylation of syringyl moieties in archaeological wood using pyrolysis-gas chromatography/mass spectrometry.

Archaeological oak (Quercus sp.) wood samples, ranging from 16(th) C. AD to 6000 BP, were studied using flash pyrolysis-gas chromatography/mass spectrometry to obtain insight into angiosperm lignin degradation. The pyrolysates revealed evidence of a number of 3-methoxy-1,2-benzenediol derivatives, methoxycatechols, directly related to 2,6-dimethoxyphenol, syringyl, moieties which are characteristic building blocks of angiosperm lignin. Mass spectra and mass chromatograms of these compounds are reported. The finding of these characteristic pyrolysis products in well-preserved archaeological wood provides unequivocal evidence that demethylation of syringyl units occurs very early in wood degradation. It is highly likely that the absence of abundant 3-methoxy-1, 2-benzenediols in degrading plant materials containing angiosperm lignin relates to the lability of these newly formed moieties.