Ian D. Bull

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.

Detection and classification of atmospheric methane oxidizing bacteria in soil.

Well-drained non-agricultural soils mediate the oxidation of methane directly from the atmosphere, contributing 5 to 10% towards the global methane sink. Studies of methane oxidation kinetics in soil infer the activity of two methanotrophic populations: one that is only active at high methane concentrations (low affinity) and another that tolerates atmospheric levels of methane (high affinity). The activity of the latter has not been demonstrated by cultured laboratory strains of methanotrophs, leaving the microbiology of methane oxidation at atmospheric concentrations unclear. Here we describe a new pulse-chase experiment using long-term enrichment with 12CH4 followed by short-term exposure to 13CH4 to isotopically label methanotrophs in a soil from a temperate forest. Analysis of labelled phospholipid fatty acids (PLFAs) provided unambiguous evidence of methane assimilation at true atmospheric concentrations (1.8–3.6 p.p.m.v.). High proportions of 13C-labelled C18 fatty acids and the co-occurrence of a labelled, branched C17 fatty acid indicated that a new methanotroph, similar at the PLFA level to known type II methanotrophs, was the predominant soil micro-organism responsible for atmospheric methane oxidation.

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.

The origin of faeces by means of biomarker detection

One of the defining characteristics of a biomarker is the retention, during diagenesis, of structural characteristics indicative of its original biogenic source (Peters and Moldowan, 1993). Since the 1960s an increasing interest has developed in the use of environmental biomarker compounds to monitor levels of human and animal waste introduced to marine and terrestrial environments; the relative stability of these compounds enabling both spatial and long-term temporal studies to be realized (e.g., Walker et al., 1982; Vivian, 1986; Evershed and Bethell, 1996 and references therein). The specificity of particular biomarkers for different faecal sources makes their use particularly attractive especially in circumstances where deducing the origin of sewage constituting a pollution event is crucial. Research into organicmatter in the marine and terrestrial environments over the last 20 years has led to the development of two main groups of organic compounds found in faecal matter that can be used to monitor sewage, namely: 5b-stanols and bile acids. This communication provides an overview for researchers not necessarily familiar with molecular methods of sewage monitoring. The two compound classes will be considered separately, and then their integration and future development discussed.

Organic geochemical studies of soils from the Rothamsted classical experiments-IV. Preliminary results from a study of the effect of soil pH on organic matter decay

Total lipid extracts and solvent insoluble organic matter in soils from the Park Grass Exper- iment at Rothamsted Experimental Station, Harpenden, U.K. were studied to determine the eAect of pH on the preservation/degradation of plant derived biomolecules. Analyses involved high temperature- gas chromatography (HT-GC), HT-GC-mass spectrometry (HT-GC-MS), GC combustion-isotope ratio MS (GCC-IRMS) and flash pyrolysis-GC (Py-GC) and Py-GC-MS. The plots selected for study have pH values ranging from 3.7 to 7.3, with acidic soils exhibiting two distinct horizons (i.e. humic rich top layer and mineral soil). The total lipid extracts of the soil samples with low pH exhib- ited higher relative abundances of long-chain (>C20) organic acids believed to be derived largely from oxidation of plant lipids. The vegetation signature in the low molecular weight fraction is only retained in the humic rich top layer. The signal in the mineral layer is believed to derive primarily from previous vegetation. Compound specific stable carbon isotope (d 13 C) measurements of long-chain n-alkanols are considered to reflect diAerences in the rate of incorporation of plant lipids into the humic top layer re- lated to the grass species dominating the standing vegetation. In the soil samples of low pH, lignin con- tributes to the high molecular weight fraction of the humic layer. In contrast, the mineral layer of the same soil shows little evidence of intact lignin, but is instead dominated by amino acid pyrolysis pro- ducts, probably deriving from (degraded) polypeptides. The pyrolysates of the mineral soils of high pH yield a distribution of products similar to that found in the deeper layer of the low pH samples but with evidence of lignin derived moieties. Overall, soil pH was found to have a significant eAect on the preservation of higher plant derived biomolecules including ligno-cellulose. # 1998 Elsevier Science Ltd. All rights reserved

Forest contraction in north equatorial Southeast Asia during the Last Glacial Period

Today, insular Southeast Asia is important for both its remarkably rich biodiversity and globally significant roles in atmospheric and oceanic circulation. Despite the fundamental importance of environmental history for diversity and conservation, there is little primary evidence concerning the nature of vegetation in north equatorial Southeast Asia during the Last Glacial Period (LGP). As a result, even the general distribution of vegetation during the Last Glacial Maximum is debated. Here we show, using the stable carbon isotope composition of ancient cave guano profiles, that there was a substantial forest contraction during the LGP on both peninsular Malaysia and Palawan, while rainforest was maintained in northern Borneo. These results directly support rainforest “refugia” hypotheses and provide evidence that environmental barriers likely reduced genetic mixing between Borneo and Sumatra flora and fauna. Moreover, it sheds light on possible early human dispersal events.

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.

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.

Organic geochemical evidence for the origin of ancient anthropogenic soil deposits at Tofts Ness, Sanday, Orkney

Lipid biomarker components of soils constituting three Orcadian archaeological fossil soil profiles were analysed. The combined assessment of lipid distributional and compound specific stable carbon isotope data enabled the identification of grass turves as the most probable material used in the formation of the anthropogenic soil deposits. Appraisal of 5β-stanol components indicated a faecal input to one of the soils which, on considering distributional evidence, was ascribed a human/porcine origin. Additional study of polar bile acids from this profile revealed a distribution exhibiting a predominance of deoxycholic acid indicating the primary faecal input to be mainly derived from humans although the minor occurrence of hyodeoxycholic acid, a characteristic component of pig faeces, attested to a limited porcine input.

The application of steroidal biomarkers to track the abandonment of a Roman wastewater course at the Agora (Athens, Greece)

A subterranean, stone-built, sediment-filled culvert discovered during excavations at the Agora (Athens, Greece) was dated to the Roman period on the basis of its characteristic construction and associated finds, including coins. The location of the culvert relative to other adjacent watercourses and an ancient river bed suggested that the structure was a sewer. This was confirmed through a multi-molecular biomarker approach based on analysis of the bile acids associated with sediment filling the sewer, using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The acid fraction contained predominantly deoxycholic and lithocholic acids, while the neutral steroid fraction comprised a complex mixture of ∆ 5 sterol and 5β- and 5α-stanols, dominated by coprostanol, suggesting the presence of faecal matter of predominantly human origin. The concentrations of neutral and acidic faecal biomarkers were observed to vary in tandem, with the highest concentrations being found in the sediment at the base of the fill in the culvert. A reduction in concentration occurred with decreasing depth of the fill, with concentrations in the uppermost samples being little different from control samples of sediment taken beyond the confines of the culvert. The enhanced concentration of bile acids relative to 5β-stanols compared with fresh human faeces must reflect the enhanced diagenetic stability of the former, thereby making bile acids the possibly preferred biomarker for this type of study. The quantitative data obtained suggest that the culvert fell rapidly out of use, possibly coinciding with the Slavic incursion in AD 582‐3.