Occurrence and distribution of non-extractable glycerol dialkyl glycerol tetraethers in temperate and tropical podzol profiles

Organic Geochemistry 41 (2010) 833–844

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Occurrence and distribution of non-extractable glycerol dialkyl glycerol tetraethers in temperate and tropical podzol profiles Arnaud Huguet a, Céline Fosse b, Pierre Metzger a, Emmanuel Fritsch c, Sylvie Derenne a,* a

BioEMCo, CNRS UMR 7618, Université Pierre et Marie Curie, 4 Place Jussieu, Paris F-75252, France Chimie ParisTech (ENSCP), Laboratoire de Spectrométrie de Masse, 11 rue Pierre et Marie Curie, Paris F-75231, France c Institut de Minéralogie et de Physique des Milieux Condensés (IMPMC), CNRS UMR 7590, Universités Paris 6 et 7, IPGP et IRD, 4 Place Jussieu, Paris F-75252, France b

a r t i c l e

i n f o

Article history: Received 15 October 2009 Received in revised form 14 April 2010 Accepted 27 April 2010

a b s t r a c t Glycerol dialkyl glycerol tetraethers (GDGTs) are high molecular weight lipids present in the membranes of archaea and some bacteria. Isoprenoid GDGTs with acyclic or ring containing dibiphytanyl chains are known to be synthesised by archaea. In soil, another type of GDGT, which can be distinguished from tetraethers of archaeal origin by way of the branched nature of the alkyl chain, was discovered recently. Alkyl branched GDGTs were suggested to be produced by anaerobic bacteria and can be used to reconstruct past air temperatures and soil pH. Lipids in soils can take two broad forms: extractable, i.e. recoverable via solvent extraction, and non-extractable, linked to the mineral or organic matrix. The present study aimed at comparing the abundance and distribution of these two pools of GDGTs in two contrasting podzol environments: a temperate podzol 40 km north of Paris (France) and a tropical podzol from the Amazon basin (Brazil). Five samples were collected from the whole profile of the temperate podzol. Five additional samples were obtained from three profiles of the tropical soil sequence, which are representative of the transition between a latosol and a well developed podzol. For the first time, we showed that substantial amounts of non-extractable GDGTs can be released after acid hydrolysis of solvent-extracted soils, non-extractable GDGTs representing 25 ± 15% of the total (i.e. extractable + non-extractable) bacterial GDGTs and 29 ± 17% of the total archaeal GDGTs in podzol samples. This implies that extractable GDGTs can be incorporated into the organic and/or mineral matrix of soil. In addition, we observed that extractable and non-extractable GDGTs could present different distribution patterns, notably suggesting that some extractable GDGTs might be preferentially transferred to the non-extractable pool and/or might be preferentially degraded by soil microorganisms. The relative abundances of bacterial and archaeal GDGTs were compared along the temperate soil profile and the tropical soil sequence. The relative amount of bacterial vs. archaeal GDGTs was shown to be much higher in the extractable than in the non-extractable fraction in the surficial horizons of the temperate podzol and in the well-developed part of the tropical podzol, implying that extractable archaeal GDGTs could be preferentially transferred to the non-extractable lipid pool compared to extractable bacterial GDGTs in these horizons. This might be due to the fact that the types of polar head groups associated with bacterial GDGTs differ from, and are more labile than, those associated with archaeal GDGTs. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Archaea are prokaryotes which are phylogenetically distinct from bacteria and eukaryotes and possess different membrane lipids: archaeal membranes are formed predominantly by isoprenoid glycerol dialkyl glycerol tetraethers (GDGTs) with acyclic or ring containing biphytanyl chains (see Appendix A for structures), while bacterial and eucaryal membranes are generally composed of straight or branched alkyl chains linked by ester bonds to the glycerol backbone. Archaea were traditionally considered as * Corresponding author. Tel.: +33 144 273 514; fax: +33 144 275 150. E-mail address: [email protected] (S. Derenne). 0146-6380/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.orggeochem.2010.04.020

extremophiles. However, they have also been found in temperate environments of both the marine and terrestrial realms, such as marine and lacustrine water columns (e.g. Karner et al., 2001; Keough et al., 2003), sediments (e.g. Kim et al., 2008; Schouten et al., 2008), peat bogs and soils (e.g. Weijers et al., 2006a,b). Another group of GDGT membrane lipids, containing branched instead of isoprenoid alkyl chains (Appendix A), was discovered in peat deposits by Sinninghe Damsté et al. (2000). Subsequently, branched GDGTs have been shown to occur ubiquitously in soil (e.g. Weijers et al., 2006b, 2007; Sinninghe Damsté et al., 2008; Peterse et al., 2009a) and hot springs (Schouten et al., 2007), as well as in coastal and marine sediments where they derive from soil organic matter (OM) transported via rivers (Hopmans et al., 2004;

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Kim et al., 2007; Walsh et al., 2008). The branched GDGTs are produced by anaerobic bacteria (Weijers et al., 2006a). Recently, Weijers et al. (2007) analyzed a large set of soils from more than 90 different locations worldwide and showed that the relative distribution of branched GDGTs depends mainly on two environmental parameters: air temperature and soil pH. The cyclisation ratio of branched tetraethers (CBT), quantifying the relative abundance of cyclopentyl rings of branched GDGTs, was shown to correlate rather well with soil pH, whereas the methylation index of branched tetraethers (MBT), expressing the degree of methylation of branched GDGTs, was observed to depend on mean annual air temperature (MAT) and, to a lesser extent, on soil pH (Weijers et al., 2007). Several recent studies have supported the use of the MBT/CBT indices as tools for the reconstruction of continental palaeotemperatures and the determination of past soil pH (Weijers et al., 2007; Sinninghe Damsté et al., 2008; Peterse et al., 2009a,b). To date, most studies of GDGTs in soil have focussed on extractable GDGTs (e.g. Weijers et al., 2007; Peterse et al., 2009a,b). However, lipids preserved in soil can take two broad chemical forms: extractable lipids, recoverable using solvent extraction, and nonextractable, linked to the mineral or organic matrix. Non-extractable lipids can be released from soil organic matter (SOM) using different treatments, depending on their mode of occurrence and/or the nature of the structures to which they are linked. Macromolecular ester-bound moieties are classically released via alkaline hydrolysis, whereas amide- or glycosidic ether-linked moieties can be freed via acid hydrolysis (Goossens et al., 1986, 1989; Zegouagh et al., 2000). It is now established that base and acid hydrolyses have the potential for providing important and detailed biogeochemical information, notably concerning the origins and sources of inputs of lipids in soils or sediments, as non-extractable lipids often show a better degree of preservation than extractable lipids. For example, some authors paid attention to the basehydrolyzable lipid fraction of soil (Nierop et al., 2003; Otto and Simpson, 2006) and others to the various pools of fatty acids released successively from lacustrine (Stefanova and Disnar, 2000; Disnar et al., 2005) and marine (Zegouagh et al., 1996, 2000; Garcette-Lepecq et al., 2004) sediments via different chemical treatments. To the best of our knowledge, no study comparing the distribution and abundance of GDGTs in the extractable and nonextractable lipid fractions of soil samples has been carried out. Nevertheless, the extractable and non-extractable lipid pools could respond to environmental change in different ways and the information obtained from the two lipid fractions could differ. Thus, Pancost et al. (2008) recently performed hydropyrolysis on marine sediments to compare the abundances, distributions and carbon isotopic compositions of archaeal GDGTs in the extractable and non-extractable lipid fractions and notably showed that the distributions of biphytanes released via hydropyrolysis did not always match those of the extractable archaeal GDGTs. The aim of the present study was therefore to compare the distribution of extractable and non-extractable GDGTs in soil. Extractable GDGTs can occur as core lipids (i.e. ‘‘free” lipids) in the extract. They may also be present in ester- and/or glycosidic ether-bound forms (i.e. as extractable ‘‘bound” lipids). Therefore, even though extractable ‘‘free” GDGTs have been mainly investigated, they are not necessarily representative of total extractable (i.e. ‘‘free” and ‘‘bound”) GDGTs. Recently, we compared the abundance and distribution of extractable ‘‘free” and total extractable GDGTs of bacterial and archaeal origin along the whole profile of a temperate podzol from 40 km north of Paris (France), and along a tropical soil sequence showing the transition between a latosol and a well-developed podzol in the upper Amazon basin (Huguet et al., 2010a). The analysis of total extractable GDGTs was performed through acid methanolysis of the extract. We observed that the relative abundance of bacterial vs. archaeal GDGTs was higher

in the extractable ‘‘free” than in the total extractable lipid fraction (containing core lipids as well as ester- and/or glycosidic etherbound GDGTs) in all the horizons. This could notably be explained by a preferential transfer of extractable ‘‘free” archaeal GDGTs into the non-extractable lipid pool or a preferential degradation of bacterial GDGTs relative to archaeal ones. Furthermore, changes in total extractable bacterial GDGT distribution were observed along the temperate soil profile, the MBT index being higher in topsoil AE horizons (0–15 and 15–30 cm depth) than in the litter layer and deep spodic Bh and Bs horizons. One of the hypotheses to explain the variability in bacterial GDGT distribution along the soil profile is the preferential transfer of some type of extractable bacterial GDGTs to the non-extractable lipid pool. The non-extractable lipid pool therefore had to be examined to answer the questions raised during that study and to obtain complete information on archaeal and bacterial GDGTs in soils. To this end, solvent-extracted soils were submitted to acid hydrolysis (6 M HCl, 100 °C) to free GDGTs linked to the macromolecular organic matrix via amide or glycosidic ether bonds. Polyvalent cations involved in clay flocculation and microaggregation are also removed with acid treatment (Oades, 1988). As a result, acid hydrolysis allows, because of its non-specific reactivity, the release of SOM stabilized via different mechanisms, such as SOM sorbed by polyvalent cation bridges, occluded SOM and complexed SOM (von Lützow et al., 2007) and was used in this study to recover non-extractable GDGTs in the soil organic/inorganic matrix. The evolution of GDGT composition and abundance was compared in the total extractable and non-extractable lipid fractions from the two podzols previously investigated (Huguet et al., 2010a,b). Podzols are acidic sandy soils, which are probably among the most studied because of their unusual morphology. Underneath an OM-rich subsurface horizon, they are characterised by an impoverished horizon on top of horizons where OM and metals accumulate. Lipids have been reported to accumulate in these acid soils (Stevenson, 1966), implying that GDGTs can be expected to be abundant in podzols. Within the frame of this work, the distribution and abundance of non-extractable and extractable GDGTs were determined for two podzols from two contrasting climatic regions, i.e. a temperate (France) and a tropical (Amazonia) podzol.

2. Materials and methods 2.1. Sampling Soils were sampled from two different locations described previously (Huguet et al., 2010a,b). The first is on the plateau of the Ermenonville forest (40 km north of Paris, France) at an altitude of 100 m and has been the subject of several pedological studies (e.g. Lamouroux et al., 1990). It is characterised by poor and sandy soils, and covered mainly by pine, oak and beech. The sylvester pine, introduced in the 19th century, is today the leading species in this massive forest (1500 hectares). Samples were collected from the litter layer and four distinct mineral horizons at increasing depth: 0–15 cm (AE1), 15–30 cm (AE2), 70–80 cm (Bh) and 80– 90 cm (Bs horizon). A precise description of the second site can be found in do Nascimento et al. (2004). Briefly, it is on the low plateaux of the Jau National Park (upper Amazon basin, Fig. 1a) and is preserved from human activity. The samples belong to a 200 m long and 2– 3 m deep sequence along which podzol soils develop at the expense of yellow clay-depleted laterites (Fig. 1b). Podzols are located in a depression, while laterites remain in higher positions. Five organic-rich horizons were selected in three profiles (I, II, III) along the sequence (Fig. 1b). They belong either to topsoil and eluviated AE horizons or to deeper subsoil illuviated and spodic B

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Fig. 1. (a) Location of investigated site (star) on a simplified soil map of the Amazon basin. (b) Simplified representation of soil catena from a high to low point of the plateau with investigated horizons (white dots). Adapted from do Nascimento et al. (2004).

horizons, with accumulations of humus (suffix h) and/or sesquioxides (mostly Al but also Fe, suffix s). One was sampled from the topsoil of the yellow clay-depleted laterite (I AE). Two horizons (II A12, II Bhs), in the upper part of pit II, belong to weakly expressed podzols at the margin of the depression. The other two correspond to better differentiated podzol horizons, sampled at the bottom part of pit II (II Bh) and towards the centre of the depression, near the surface of pit III (III A12). Soil samples from the temperate podzol were air dried overnight immediately after sampling and submitted to solvent extraction. Samples from the tropical podzol were rapidly air dried after sampling and stored in the dark in sealed glass jars for a few days prior to extraction and analysis. 2.2. GDGT analysis Samples were extracted (3  5 min each) with a mixture of dichloromethane (DCM):MeOH (9:1, v/v) by using an accelerated solvent extractor (ASE 100, Dionex) at 100 °C and 10  106 Pa. The combined extracts were rotary evaporated under near vacuum to a residual volume and submitted to acid methanolysis to release ester- and/or glycosidic ether-bound GDGTs, as described by Huguet et al. (2010a,b). The hydrolysed extract was separated into three fractions over a small alumina column (activated for 2 h at 150 °C) using hexane:DCM (9:1, v/v), DCM:MeOH (1:1, v/v) and DCM:MeOH (1:3, v/v), respectively. The medium polarity fraction (DCM:MeOH, 1:1 v/v) was condensed using rotary evaporation, dried under N2, ultrasonically dissolved in hexane:isopropanol 99:1 (v/v) and centrifugated using an Eppendorf MiniSpin centrifuge (1 min, 7000 rpm). The supernatant, containing total extractable GDGTs (i.e. core lipids as well as ester- and/or glycosidic ether-bound GDGTs), was collected and analysed using high performance liquid chromatography–atmospheric pressure chemical ionisation–mass spectrometry (HPLC/APCI–MS). The extracted residues were subjected to acid hydrolysis. Typically, the dry residue (5 g) was refluxed for 24 h at 100 °C in aqueous 6 M HCl. After cooling, the suspension was centrifuged for 10 min at 3500 rpm and the supernatant transferred to a separa-

tion funnel. The residue was washed with organic solvents, i.e. it was sequentially extracted with water (3), MeOH/water (1:1, 1), MeOH (2) and DCM (2). Each extraction was followed by centrifugation and all extracts were collected in the separation funnel. The DCM layer was separated from the MeOH/water layer, which was again extracted with DCM (3). The DCM fractions were combined and, after evaporation of the solvent, the extracts were fractionated into apolar, medium polarity and polar fractions as described above. Non-extractable GDGT lipids were thus recovered in the medium polarity fraction. 2.3. HPLC/APCI–MS HPLC/APCI–MS was performed with an Agilent 1100 liquid chromatograph equipped with an automatic injector and coupled to a PE Sciex API 3000 mass spectrometer. GDGTs were analysed using a procedure modified from Hopmans et al. (2000) and Weijers et al. (2007). Separation was achieved with a Prevail Cyano column (2.1 mm  150 mm, 3 lm; Alltech, Deerifled, IL, USA) thermostatted at 30 °C. Injection volume was 10 ll. GDGTs were first eluted isocratically with 99% A/1% B for 5 min, where A corresponds to hexane and B to isopropanol. The following linear gradient was subsequently used: 99% A/1% B to 98% A/2% B in 45 min, maintained for 5 min, followed by 98% A/2% B to 90% A/10% B in 1 min, maintained for 10 min and then back to 99% A/1% B in 1 min, maintained for 10 min. The flow rate was set at 0.2 ml/min. Detection was performed using positive ion APCI. Scanning was performed in pseudo-single ion monitoring (SIM) mode, i.e. from m/z 1290 to 1310 for detection of the [M+H]+ ions of archaeal GDGTs (I–VI) and from m/z 1044 to 1054, 1030 to 1040 and 1015 to 1025 for the detection of the [M+H]+ ions of bacterial GDGTs VII, VIII and IX, respectively. GDGT quantification was achieved by using an internal standard (Appendix A) synthesised according to Svenson and Thompson (1998). A solution of the standard (0.02 mg/ml) was added to each sample at the end of its preparation for HPLC/MS analysis, as described by Huguet et al. (2010a,b). Based on duplicate extraction of one podzol sample and on replicates of several HPLC/MS analyses, the total process er-

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ror (i.e. extraction and HPLC/MS quantification with the internal standard) was estimated to be ca. 10% and was observed to be related to the HPLC/MS quantification. The MBT and CBT indices were calculated as follows (Weijers et al., 2007):

½IXa þ IXb þ IXc ½VIIa þ VIIb þ VIIc þ ½VIIIa þ VIIIb þ VIIIc þ ½IXa þ IXb þ IXc ð1Þ   ½VIIIb þ ½IXb CBT ¼  log ð2Þ ½VIIIa þ ½IXa MBT ¼

The roman numerals correspond to the structures in the Appendix A. Average duplicate errors for the MBT and CBT indices were 0.007 and 0.02, respectively. 3. Results and discussion 3.1. Temperate podzol, France The abundances of total extractable GDGTs recovered from solvent extraction and of non-extractable GDGTs released after acid hydrolysis of extracted samples were determined along the temperate soil profile. Archaeal (I–VI) and bacterial (VII–IX) GDGTs were present in the extractable and non-extractable lipid fractions of all samples (e.g. Fig. 2), except from the deepest horizon (80– 90 cm), where archaeal GDGTs were not detected in the nonextractable lipid pool. Archaeal GDGTs were much less abundant than bacterial GDGTs in the two fractions of all the soil samples (Fig. 3; Table 1), as observed by Weijers et al. (2006b) for extractable GDGTs in a variety of soils collected from different climatic zones. Some non-extractable GDGTs were released from the organic litter layer, although in much lower amount than from the other horizons of the profile (Fig. 3; Table 1). In order to ensure that the low amounts of non-extractable GDGTs in the litter did not result from incomplete extraction of some tetraethers, this sample was extracted using ASE (Section 2.2) and was further extracted using a modified Bligh and Dyer technique, according to the protocol recently described by Pitcher et al. (2009). No GDGTs could be detected in the Bligh and Dyer extract, showing that the lipids recovered from acid hydrolysis of the ASE-extracted peat sample were indeed non-extractable using either ASE or the Bligh and Dyer technique. Similarly, non-extractable GDGTs were also observed to represent only a small proportion (<10%) of total (i.e. extractable + non-extractable) bacterial and archaeal GDGTs in four organic-rich samples from a French peat bog (Huguet et al., 2010b). Acid hydrolysis afforded substantial amounts of non-extractable bacterial and archaeal GDGTs from the four mineral soil horizons. Extraction alone of these soils would only have released (Fig. 3). between ca. 75% and 85% of bacterial GDGTs and between ca. 60% and 80% of archaeal GDGTs [except for the deepest horizon (Bs)]. This suggests that GDGTs can be linked to the organic and/or mineral matrix of soils, thereby allowing their preservation in the soil. Since non-extractable GDGTs were detected in the litter (Fig. 3), this implies that they are present within the organic matrix of soils. Therefore, in the mineral soil horizons, part of non-extractable GDGTs may be associated with the inorganic soil matrix and may be present in the form of organometallic complexes. Another part may be incorporated within the macromolecular network of SOM during humification. The relative amount of non-extractable archaeal GDGTs was observed to decrease with depth in the mineral soil horizons (Fig. 2), whereas the relative abundance of nonextractable GDGTs compared to extractable GDGTs was relatively similar in the four horizons for bacterial GDGTs (ca. 20%).

The total amount (i.e. non-extractable and extractable lipids) of bacterial GDGTs with respect to dry soil wt decreases with depth (Table 1). Weijers et al. (2006b) analysed the extractable core lipid fraction of three soil profiles from France, Nigeria and Zaire, and observed a similar trend, i.e. a decrease in bacterial GDGT concentration with depth. Total organic carbon (TOC) content (% dry wt soil) varies along the Ermenonville profile and is about four times lower at 80–90 cm depth than in the other soil horizons and 100 times lower than in the litter layer (Table 2). When GDGT concentrations are normalized to TOC (Fig. 3), the total amount of bacterial GDGTs appears about twice higher in the topsoil AE (0–15 cm) horizon (ca. 50 lg g1 TOC) than in the underlying horizons (15– 30, 70–80 and 80–90 cm depth) and about eight times higher than in the litter layer (ca. 6 lg g1 TOC). Similarly, archaeal GDGTs are more abundant in surface (2.9 lg g1 TOC) than in the other soil horizons (0.6, 1.2 and 2.1 lg g1 TOC at 15–30 cm, 70–80 cm and 80–90 cm depth, and 0.6 lg g1 TOC in the litter respectively). Taken together, these results show that GDGTs tend to be more abundant in the topsoil AE horizon, likely because the topsoil provides the best conditions for the growth of GDGT-producing microorganisms. The distribution and the abundance of individual archaeal and bacterial tetraethers in the total extractable (Huguet et al., 2010a) and non-extractable lipid fractions (Table 1) were compared. Archaeal GDGTs containing cyclopentyl rings (II–VI) were observed to predominate among archaeal tetraethers in the nonextractable lipid fractions from the upper two AE horizons and represented between about 70% and 75% of the total archaeal GDGT concentration, as for the extractable fraction (Huguet et al., 2010a). In addition, it can be noted that GDGT VI (crenarchaeol), a specific membrane lipid of the non-thermophilic group I Crenarchaeota (Sinninghe Damsté et al., 2002), was not detected in the AE horizons, while it was the second most abundant tetraether in the extractable and non-extractable lipid fractions from the litter layer (ca. 25–30% of the total archaeal GDGTs) and was detected to a lesser extent in the non-extractable fraction from the 70 to 80 cm deep soil horizon and in the extractable fraction from the 80 to 90 cm horizon. The crenarchaeol regioisomer (GDGT VI0 ) was not present in the non-extractable or extractable fractions of the temperate podzol samples. Similarly, GDGT VI0 was present in trace amounts or was not detected in soils collected worldwide by Weijers et al. (2006b). The qualitative differences in archaeal GDGT composition between the extractable and non-extractable pools of the temperate podzol samples should be interpreted with care, because of the low abundance of archaeal GDGTs (Fig. 2; Table 1), in particular in the non-extractable fraction. Bacterial GDGT IXa, followed by VIIIa, was the most abundant tetraether in the non-extractable lipid fraction of all the samples (cf. Fig. 2 and Table 1), as observed for the total extractable fraction (Huguet et al., 2010a). Nevertheless, the proportion of these two GDGTs differed with depth and the variation was not similar for extractable and non-extractable GDGTs. In the extractable fraction, the abundance of GDGT IXa relative to total bacterial GDGTs was higher in the two AE horizons (77–81%) than in the deep spodic horizons (ca. 65%), whereas the opposite trend was observed for GDGT VIIIa. In the non-extractable fraction, the ratio of GDGT IXa over total bacterial GDGTs was higher in the 15–30 cm horizon (73%) than in the rest of the soil profile (55–66%; Table 1). Comparison between the distributions of bacterial GDGTs in the extractable and non-extractable fractions revealed some qualitative differences. Indeed, in the four mineral soil horizons, the amount of GDGT IXa relative to total bacterial GDGTs was higher in the extractable than in the non-extractable lipid fraction, whereas the opposite was observed for GDGT VIIIa (Fig. 2; Table 1). The trend is particularly marked for the 0–15, 15–30 and 80–90 cm horizons. This indicates that GDGTs preserved in soils in the form

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3.0E+06 2.5E+06

Bacterial GDGTs I

1.2E+05

II III IV

IXa

8.0E+04

Temperate podzol (15-30 cm)

4.0E+04

2.0E+06 0 13

15

17

19

21

23

1.5E+06 1.0E+06

IS

Extractable fraction

Archaeal GDGTs

VIIIa

5.0E+05

IXb

IXc

0 13 3.5E+05

18

4.0E+04

3.0E+05

I

3.0E+04

23

28

Bacterial GDGTs

III II

IXa

2.0E+04

2.5E+05

33

Temperate podzol (15-30 cm)

1.0E+04 0

2.0E+05

13

15

17

19

21

23

1.5E+05

VIIIa

IS

Archaeal GDGTs

1.0E+05

Non-extractable fraction

Intensity (cps)

5.0E+04 0 13 8.0E+06

18

7.5E+05

6.0E+06

4.5E+05

5.0E+06

28

33

Bacterial GDGTs

9.0E+05

7.0E+06

23

I

II III IV

6.0E+05 3.0E+05

IXa

V

Tropical podzol III A12 Extractable fraction

1.5E+05 0 13

15

17

19

21

23

4.0E+06 3.0E+06

Archaeal GDGTs

2.0E+06

IS

IXb IXc

1.0E+06 0 13

18

23

28

33

Bacterial GDGTs

1.0E+06

1.6E+05 1.2E+05

8.0E+05

I II

8.0E+04

III

IXa

IV V

4.0E+04

6.0E+05

0

13

15

17

19

21

23

Non-extractable fraction

IS

4.0E+05

Tropical podzol III A12

Archaeal GDGTs

IXb IXc

2.0E+05 0 13

18

23

28

33

Retention time (min) Fig. 2. HPLC/MS base peak chromatograms for temperate podzol sample from 15 to 30 cm horizon and for III A12 Amazon sample showing archaeal and bacterial GDGT distribution in total extractable and non-extractable lipid fractions.

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Archaeal GDGTs

Bacterial GDGTs Litter

0.5

5.8 Extractable Non-extractable

0.1

0.3

AE1 (0 -15)

38

11

1.7

AE2 (15-30)

18

5.2

0.4

16

5.8

0.9

1.2

0.2

Bh (70-80)

0.3

Bs (80-90)

22

0

20

40

2.1

3.7

60

80

100

0

20

40

60

80

100

Relative abundance (%) Fig. 3. Amount (lg g1 TOC, bold) and relative abundances of bacterial and archaeal GDGTs in extractable and non-extractable lipid fractions obtained via chemical treatment of French soils.

of non-extractable lipids can have a distribution pattern different from that of the extractable GDGTs. As a result, the average degree of methylation of bacterial GDGTs is lower in the non-extractable than in the total extractable lipid pool in the four mineral horizons, as confirmed through analysis of the MBT values (Table 2). Bacterial GDGTs containing cyclopentane rings VIIIb, IXb and IXc were much less abundant than the corresponding bacterial tetraethers without cyclopentyl moieties (VIIIa and IXa), as observed previously (Huguet et al., 2010a). GDGTs VIIIb, IXb and IXc were present in the extractable fraction of all temperate podzol samples. Nevertheless, GDGTs VIIIb and IXc were not detected in the nonextractable fraction of most of the samples, possibly because they were below detection threshold. The ratio of total bacterial over total archaeal GDGTs was calculated (Table 1) to compare the relative abundance of bacterial and archaeal tetraethers along the profile. The relative amount of bacterial over archaeal GDGTs was much higher in the extractable than in the non-extractable lipid fractions in the litter layer and in the surficial horizons. This suggests that extractable archaeal GDGTs may be preferentially transferred to the non-extractable lipid pool (e.g. via incorporation into macromolecules in soil) compared to extractable bacterial GDGTs at the soil surface. GDGTs can occur as core lipids (i.e. as extractable ‘‘free” lipids) as well as ester- and/or glycosidic ether-bound GDGTs (i.e. as extractable ‘‘bound” GDGTs) in soil extracts. The abundance of extractable ‘‘free” and total extractable (i.e. ‘‘free” + ‘‘bound”) GDGTs of bacterial and archaeal origin was previously determined in the different horizons of the temperate podzol (Huguet et al., 2010a). The amount of bacterial vs. archaeal GDGTs was shown to be higher in the extractable ‘‘free” than in the total extractable lipidic fraction in all the soil horizons. In the litter layer and surface horizons, this phenomenon might notably be explained by a preferential transfer of extractable ‘‘free” archaeal GDGTs to the nonextractable lipid pool, consistent with the results obtained in the present study, i.e. the enrichment of the non-extractable lipid fraction in archaeal tetraethers compared to the total extractable lipid pool (Table 1). The differences in bacterial vs. archaeal abundance in the extractable ‘‘free”, total extractable and non-extractable lipid fractions of the temperate podzol samples might notably arise from

the different types of polar head groups synthesised by bacterial and archaeal GDGTs, as recently suggested for peat samples (Huguet et al., 2010b). Polar head groups of archaeal GDGTs were shown to be either purely hexose- and phosphate-based or a mixture of both (e.g. Sturt et al., 2004; Rossel et al., 2008), whereas those of bacterial GDGTs are unknown. The types of polar head groups associated with bacterial GDGTs could differ from those of archaeal GDGTs. In addition, the different polar head groups of lipids do not necessarily display the same degree of stability. For example, Harvey et al. (1986) observed a much more rapid degradation of phospholipids than glycolipids in aerobic beach sediments, notably because the ether bonds of the glycolipids are more resistant to enzymatic hydrolysis than the ester bonds of the phospholipids (Paltauf, 1983). Therefore, the polar head groups associated with bacterial and archaeal GDGTs could have different degrees of stability. The head groups of bacterial GDGTs could be more labile than those of archaeal GDGTs, i.e. extractable ‘‘bound” bacterial GDGTs could be less resistant to degradation than archaeal ones, which could explain the reported higher relative abundance of bacterial vs. archaeal GDGTs in the extractable ‘‘free” than in the total extractable lipid fraction of the temperate podzol samples (Huguet et al., 2010a). If the types of polar head groups associated with bacterial GDGTs are more labile than those associated with archaeal GDGTs, this would imply that archaeal GDGTs remain for a longer time in the possession of their head groups than bacterial GDGTs. This might make the archaeal GDGTs more likely to get incorporated into the soil macromolecular matrix, considering that the polar head groups of extractable ‘‘bound” lipids are more reactive than the hydroxyl groups of extractable ‘‘free” lipids. Consequently, the lower relative abundance of bacterial vs. archaeal GDGTs in the non-extractable than in the total extractable lipid pool observed in the litter layer and surface horizons of the temperate podzol (Table 1) could be partly explained by the preferential incorporation of archaeal vs. bacterial GDGTs in the macromolecular matrix. Nevertheless, the latter mechanism is unlikely to occur in all soils, since the relative amount of bacterial vs. archaeal GDGTs appeared to be lower in the non-extractable than in the total extractable pool for several peat samples (Huguet et al., 2010b) and for the 70–80 cm horizon of the temperate podzol (Table 1).

Table 1 Amounts (ng g1 dry wt soil) and relative abundances (%, bracketed values) of archaeal and bacterial GDGTs in extractable and non-extractable lipid fractions from chemical treatment of French and Amazonian soils (n.d., not detected; N.B., GDGTs VIIb, VIIc and VIIIc were not detected in the podzol samples). Sample

Fraction

Ermenonville Litter Extractable

Bacteria

I

II

III

IV

V

VI

VI

72 (31.8)

24 (10.4)

38 (16.8)

26 (11.3)

n.d.

67 (29.7)

Non-extractable 22 (56.5) 3.4 (8.6) 3.2(8.1) n.d. n.d. Extractable 7.4 (27.4) 5.9 (21.9) 6.4 (23.7) 6.0 (22.2) 1.3 (4.8)

Non-extractable 4.7 (25.5) 2.6 (13.9) 4.8 (25.9) 4.7 (25.5) 1.7(9.2) AE2(15–30) Extractable 3.2 (41.6) 1.4 (18.2) 1.8(23.4) 1.3(16.9) n.d.

VIIIb

IXa

IXb

Bact./Arch.a

Total

VIIa

VIIIa

IXc

Total

n.d.

226 (100.0)

78 (3.0)

855 (33.2) 8.7 (0.3) 1528 (59.3) 84 (3.3)

23 (0.9)

2577 (100.0) 11.4

11 (26.8) n.d.

n.d. n.d.

39.1 (100.0) 5.6 (4.3) 42 (31.9) 0.4 (0.3) 79 (60.2) 27 (100.0) 5.0 (0.8) 110 (18.3) 1.4(0.2) 460 (76.5)

4.4 (3.3) n.d. 15(2.5) 10 (1.7)

131 (100.0) 602(100.0)

3.3 22.3

n.d. n.d.

n.d. n.d.

19 (100.0) 7.7 (100.0)

4.4 (2.6) 55 (31.9) n.d. 50 (13.4)

n.d. 106 (61.4) 0.1 (0.7) 299 (80.9)

3.6(2.1) 9.8(2.7)

172 (100.0) 369 (100.0)

9.3 47.9

3.5 (2.0) 11 (2.9)

Bh (70–80)

Non-extractable 1.3(30.6) 1.6 (37.2) 1.4(32.2) Extractable 4.2 (27.1) 3.2 (20.6) 4.2(27.1)

n.d. n.d. 3.0 (19.4) 0.9 (5.8)

n.d. n.d.

n.d. n.d.

4.4 (100.0) 16 (100.0)

0.5 (0.4) 26 (25.0) 2.0 (0.7) 80 (29.0)

n.d. 77 (73.4) 0.7 (0.2) 185 (67.4)

1.2(1.1) n.d. 4.2 (1.5) 2.9(1.7)

105 (100.0) 274 (100.0)

23.7 17.7

Bs (80–90)

Non-extractable n.d. n.d. Extractable 4.0 (46.7) 1.4(16.5)

n.d. 1.5(17.6)

n.d. 0.7 (8.3)

n.d. n.d.

4.6 (100.0) n.d. 0.9 (10.8) n.d.

4.6 (100.0) 8.6 (100.0)

1.4(7.4) 30 (30.4) 2.1 (2.4) 28 (31.7)

n.d. 65(65.7) 0.4 (0.4) 56 (64.0)

2.4 (2.4) n.d. 1.2(1.4) 0.1 (0.1)

99 (100.0) 87 (100.0)

21.6 10.1

Non-extractable n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.0

n.d.

n.d.

8.2 (55.4)

1 (6.7)

n.d.

15 (100.0)

II A12

Extractable 6.3 (46.0) 2.2 (16.3) 2.4(7 7.6) 1.6(11.6) Non-extractable 5.7 (27.8) 1.1 (5.6) 4.3(27.7) 1.8 (8.0) Extractable 13 (24.8) 8.8 (16.4) 11 (27.7) 18 (33.2)

1.2 (8.6) n.d. 2.4 (4.5)

n.d. 4.4 (27.5) n.d.

n.d. 14 (100.0) 3.3 (76.0) 20 (100.0) n.d. 54 (100.0)

n.d. n.d. n.d. 1.4(1.3) 6.7 (5.8) n.d.

n.d. n.d. n.d.

77 (100.0) 105 (97.2) 108 (94.2)

n.d. n.d. n.d.

n.d. 1.7(1.6) n.d.

77 (100.0) 108 (100.0) 115(100.0)

II Bhs

Non-extractable 5.0 (16.2) 4.3 (14.1) 7.6 (24.6) 10 (33.6) Extractable 5.6 (30.6) 2.7 (14.7) 3.7 (20.2) 5.6(30.7)

3.5(7 7.5) 0.7 (3.8)

n.d. n.d.

n.d. n.d.

31 (100.0) 18 (100.0)

n.d. n.d.

n.d. n.d.

50(78.6) 42(88.7)

2.5 (4.0) 8.5 (13.4) 63 (100.0) 1.9(4.0) 3.0 (6.4) 47 (100.0)

2.1 2.6

II Bh

Non-extractable 1.1 (16.9) 1.2 (18.3) 1.4(21.4) 2.9 (43.4) n.d. Extractable 20 (26.3) 6.2 (8.3) 9.3 (72.5) 22(29.1) 14 (78.8)

n.d. 1.9(2.6)

n.d. 1.9(2.5)

6.6 (100.0) 75 (100.0)

n.d. n.d. 1.1 (0.6) 5.2(2.7)

n.d. n.d.

12(73.7) 182 (94.6)

n.d. 4.1 (26.3) 16 (100.0) 2.9 (7.5) 1.3(0.7) 193 (100.0)

2.4 2.6

III A12

Non-extractable 4.2 (13.0) 2.7 (8.2) 4.3 (73.3) 13 (38.5) Extractable 26 (33.6) 9.9 (12.5) 17(27.7) 14 (78.2)

8.7 (26.9) n.d. 11.0(73.9) n.d.

n.d. n.d.

32 (100.0) 79 (100.0)

n.d. n.d.

2.5(2.7) 3.3(0.7)

n.d. n.d.

80 (86.8) 405 (79.5)

2.2 (2.4) 7.4(8.1) 41 (8.0) 60 (11.9)

92 (100.0) 509 (100.0)

2.8 6.5

Non-extractable 4.7 (29.8) 1.9 (12.2) 4.0(25.9)

2.6 (76.5)

n.d.

16 (100.0)

n.d.

1.0(1.9)

n.d.

46 (84.5)

3.4 (6.3) 3.9 (7.2)

54 (100.0)

3.5

Amazonia I AE

a

0

n.d.

2.4(75.7)

n.d.

5.6 (37.9)

2.5 (4.0) 0.4 (0.9)

5.7 5.3 2.1

A. Huguet et al. / Organic Geochemistry 41 (2010) 833–844

AE1 (0–15)

Archaea

Ratio of total bacterial GDGT concentration and total archaeal GDGT concentration.

839

840

A. Huguet et al. / Organic Geochemistry 41 (2010) 833–844

Table 2 General properties (pH, organic carbon concentration) of Amazonian and French soil samples, proxy values [methylation index (MBT) and cyclisation ratio (CBT) of bacterial tetraethers) and estimated MAT values from MBT and CBT indices (Eq. (3)] and from MBT and soil pH [Eq. (4); n.d., not detected]. Sample

a

pH

Fraction

MBT

CBT

MBT/CBT-derived MAT (°C)

MBT/pH-derived MAT (°C)

Ermenonville (France) Litter 44.3

Corg (%)

3.6

AE1 (0–15)

1.6

3.7

AE2 (15–30)

2.0

4.0

Bh (70–80)

1.7

3.8

Bs (80–90)

0.4

4.0

Extractable Non-extractable Extractable Non-extractable Extractable Non-extractable Extractable Non-extractable Extractable Non-extractable

0.64 0.64 0.81 0.66 0.87 0.75 0.70 0.68 0.65 0.62

1.41 1.41 1.53 n.d.a 1.55 n.d. 1.74 n.d. 1.73 n.d.

12.5 12.5 19.9 n.d. 22.7 n.d. 12.7 n.d. 10.5 n.d.

5.4 5.4 14.0 6.8 18.2 11.1 9.4 8.5 8.2 6.6

Amazonia (Brazil) I AE

1.6

4.8

II A12

3.5

4.8

II Bhs

4.3

4.8

II Bh

1.7

4.2

III A12

1.2

4.0

Extractable Non-extractable Extractable Non-extractable Extractable Non-extractable Extractable Non-extractable Extractable Non-extractable

1.00 0.99 0.94 0.98 0.98 1.00 0.97 0.97 0.99 0.98

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

28.3 27.8 25.4 27.3 27.3 28.3 24.1 24.1 24.1 23.7

GDGTs VIIIb and/or IXb were not detected in most of the samples, hindering determination of CBT index.

GDGTs are likely to be bound to or trapped in the soil matrix via a variety of mechanisms. Therefore, either bacterial or archaeal GDGTs might be preferentially transferred to the non-extractable pool, depending notably on the initial GDGT distribution and on the nature of the soil matrix (e.g. mineral composition, OM content). This hypothesis should be tested on a large set of soils with different characteristics and GDGT distributions, i.e. from different climatic regimes. Weijers et al. (2007) showed that bacterial GDGT distribution in soils can be used to reconstruct past air temperatures and soil pH. Thus, annual mean air temperature (MAT) was estimated from CBT and MBT indices (Table 2) using the global soil correlation proposed by Weijers et al. (2007):

MBT ¼ 0:122 þ ð0:187  CBTÞ þ ð0:020  MATÞ

ð3Þ

Cyclopentane-containing bacterial GDGT VIIIb was not detected in the non-extractable fraction of most of the samples. In the original calibration set for the CBT index, Weijers et al. (2007) omitted all soils for which GDGT VIIIb could not be detected from the dataset so as not to introduce biased CBT values. Similarly, the CBT index was not determined for all podzol samples in which GDGT VIIIb was not detected. As a result, MAT was also calculated using the following equation (Weijers et al., 2007):

MBT ¼ 0:867  ð0:096  pHÞ þ ð0:021  MATÞ

ð4Þ

MAT values [19.9 and 22.7 °C, calculated using Eq. (3); Huguet et al., 2010a] derived from the extractable bacterial GDGT distribution in the two AE horizons (0–15 and 15–30 cm depth) were found to be higher than those derived from the deep horizons (10.5 and 12.7 °C; Table 2). The latter values are in close agreement with recorded MAT at the Ermenonville site (average annual temperature 10.4 °C, minimum 6.0 °C and maximum 14.8 °C; Meteo France database). It has to be noted that similar trends were obtained when MAT was estimated from Eq. (4) instead of Eq. (3), even though MAT estimates calculated from MBT and CBT indices [Eq. (3)] were systematically higher than those derived from measured soil pH and MBT [Eq. (4)]. The discrepancy could be a result of the low abundance of cyclopentyl-containing GDGTs (Table 2) in soil samples, particularly GDGT VIIIb, which may increase the ana-

lytical error in the CBT index and thus the uncertainty in the determination of MAT from Eq. (3). Moreover, the correlation between MBT, CBT and MAT [Eq. (3)], obtained from 130 soils collected worldwide, is slightly lower (R2 0.77) than the one between MBT, MAT and pH (R2 0.82; Eq. (4); Weijers et al., 2007), which could also explain the higher MAT estimates derived from Eq. (3). MAT values derived from extractable and non-extractable GDGT fractions in the litter layer were comparable [around 5 °C, calculated using Eq. (4)] and lower than recorded MAT in the temperate podzol area. In contrast, MAT estimates derived from non-extractable bacterial GDGTs in the mineral soil horizons were generally lower than those derived from extractable tetraethers (Table 2), in particular in the AE horizons. Furthermore, a lower variability in MAT estimates was observed when air temperature was calculated from non-extractable lipids. Indeed, MAT values obtained from non-extractable GDGT distribution in the four mineral soil horizons ranged between 6.6 and 11.1 °C, in better agreement with recorded MAT, whereas MAT estimates derived from extractable GDGTs were more scattered, ranging from 8.2 to 18.2 °C. Nevertheless, the differences in MAT values between the non-extractable and extractable lipid fractions observed in this study should be interpreted with care, since the original calibration of bacterial GDGT distribution in soil to soil pH and MAT (Weijers et al., 2007) is based on the extractable lipid pool and does not target the non-extractable pool analysed here. The differences in extractable GDGT distribution between surficial and deep soil horizons (Table 1), reflected in the variability of the MAT (Table 2), could be due to the preferential transfer of some extractable bacterial GDGTs to the non-extractable lipid pool. Indeed, the association of GDGTs with the inorganic and/or organic matrix of soils could lead to modification of the relative abundance and distribution of extractable GDGTs along the soil profile. The soil matrix might also have a finite capacity for the binding of GDGTs. As a result, in the two AE horizons of the temperate podzol, GDGT VIIIa could have been transferred more rapidly and in higher relative amount to the non-extractable pool than GDGT IXa. This hypothesis could explain the large difference in MBT between the total extractable and the non-extractable pool of the two surficial horizons (Table 2). The difference in MBT values between the

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A. Huguet et al. / Organic Geochemistry 41 (2010) 833–844

two lipid pools was much smaller in the deep horizons than in the surface ones, possibly resulting from changes in SOM composition and mineralogy along the temperate podzol profile. Alternatively and/or complimentarily, despite strong similarities between their structures (Appendix A), some bacterial GDGTs might also be preferentially degraded by soil microorganisms, thereby impacting on the distribution of extractable GDGTs in the different horizons of soils. In contrast, non-extractable GDGTs are likely to be well preserved in soil because of the efficient protection provided by the soil matrix and are therefore probably less readily degraded than extractable GDGTs. We may assume that the relative distribution and abundance of non-extractable GDGTs do not vary significantly with time, whereas the opposite might occur for extractable GDGTs as a result of their degradation in soil. This may partly explain why the distribution of non-extractable bacterial GDGTs is almost the same along the temperate podzol profile, in contrast to the situation for the extractable pool. Whatever the mechanism(s) responsible for the differences in bacterial GDGT distribution between the extractable and non-extractable pools, our results show for the first time that some extractable GDGTs can be incorporated into the organic and/or mineral matrix of soil. 3.2. Tropical podzol, Amazonia Abundances of non-extractable and extractable GDGTs were compared along the tropical soil sequence (Table 1; Fig. 4). Nonextractable lipids represent between ca. 10% and 60% (avg. 32%) of total bacterial GDGTs and between 15% and 60% (avg. 36%) of total archaeal GDGTs. Therefore, a significant fraction of GDGTs exists in the form of non-extractable lipids in the Amazonian soils. Through comparison between the three surficial samples (I AE, II A12 and III A12), the lateral development of the podzol at the expense of a latosol can be followed. The total amount (non-extractable and extractable lipids) of bacterial and archaeal GDGTs normalized to TOC is much higher in pit III than in pits I and II (Fig. 4). Furthermore, the comparison of GDGT concentrations between II A12, II Bhs and II Bh allows following of the vertical evolution of the podzol in a transition area. Total concentrations of bacterial and archaeal GDGTs decrease from II A12 to II Bhs and

then increase in the deepest horizon. Generally, the evolution of archaeal and bacterial GDGTs along the sequence is comparable (Fig. 4) and tetraethers appear much more abundant in the welldeveloped podzol horizons, II Bh and III A12. The enrichment in GDGTs in the well-expressed parts of the podzol may be assigned to two main factors: (i) the higher acidity of II Bh and III A12 horizons (pH ca. 4.0) compared to the latosol and transition area (pH ca. 4.8; Table 2) and (ii) increased microbial activity, as shown by Huguet et al. (2010a). Indeed, a decrease in pH is known to favour lipid stabilisation in soil (Morrison, 1969) and could explain the accumulation of GDGTs in III A12 and II Bhs. Moreover, Weijers et al. (2007) found higher amounts of bacterial GDGTs in acid soils, suggesting that low pH ranges could favour growth of branched GDGT-producing bacteria. Comparison of the three surficial samples (IAE, II A12 and III A12) shows that the relative abundance of non-extractable bacterial GDGTs is much higher in profiles I and II than in profile III (Fig. 4), where extractable tetraethers are largely predominant (ca. 90% of total bacterial GDGTs) over non-extractable GDGTs. The high abundance of organometallic complexes (mainly with Al; do Nascimento et al., 2004; Bardy et al., 2007) in pits I and II could explain the high proportion of non-extractable GDGTs in these pits. In contrast, Bardy et al. (2007) observed a depletion in complexing elements like Al in pit III in comparison with pit II and noticed a significant drop in the content of Al–SOM complexes from pit II to pit III. This could account for the low proportion of non-extractable GDGTs in III A12. In addition, we reported previously that, in pit II, total extractable lipids represent a lower proportion of SOM in Bhs than in A12 and Bh horizons (Huguet et al., 2010a). In particular, this holds for total extractable bacterial and archaeal GDGTs, which were observed to be more abundant in II A12 and II Bh than in II Bhs (Fig. 4). Bardy et al. (2009) suggested that the low concentration of lipids in II Bhs could be due to the transfer of extractable lipids to a non-extractable lipid pool, via the formation of Al complexes. Nevertheless, the complexation of GDGTs with Al is unlikely to totally explain the low concentration of tetraethers in II Bhs than in II A12 and II Bh, since non-extractable GDGTs represent a lower proportion of total GDGTs in II Bhs than in II A12 and II Bh. The de-

Archaeal GDGTs

Bacterial GDGTs I AE

4.8

0.9

6.7

1.1

Extractable Non-extractable

II A12

3.3

II Bhs

1.1

II Bh

11

III A12

42

0

20

0.9

1.8

4.4

5.4

80

100

1.9

6.6

4. 5

60

0.2

0.4

0.4

40

0.9

0

20

1 .3

40

60

80

100

Relative abundance (%) Fig. 4. Amounts (lg g1 TOC, bold) and relative abundances of bacterial and archaeal GDGTs in extractable and non-extractable lipid fractions obtained via chemical treatment of Amazonian soils.

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A. Huguet et al. / Organic Geochemistry 41 (2010) 833–844

creased amount of extractable GDGTs in the intermediate horizon of pit II could be due to the accumulation of non-extractable GDGTs in the topsoil II A12 horizon and to the transport of these complex lipids from the II A12 to the II Bh horizon. Indeed, the transfer of colloidal and particulate OM, notably extractable GDGTs, to deep soil horizons of pit II could be favoured by the presence of sandy and very porous horizons lying between topsoil eluviated and subsoil illuviated horizons (Bardy et al., 2009). Alternatively or complementarily, archaea and branched GDGTproducing bacteria might thrive better in the II A12 and II Bh horizons than in the II Bhs horizon, leading to the much lower concentrations of extractable and non-extractable GDGTs in II Bhs than in II A12 and II Bh (Fig. 4). The relative abundance of bacterial and archaeal GDGTs was compared along the soil sequence. The ratio of total bacterial over total archaeal GDGTs was comparable in the extractable and in the non-extractable fraction for soils collected in pits I and II, but was almost two times higher in the extractable than in the non-extractable lipid fraction for III A12 (Table 1). The differences in relative abundance of bacterial vs. archaeal GDGTs between the two lipid fractions of the well-developed podzolic horizon III A12 (Table 1) could be explained by the preferential transfer of archaeal GDGTs from the extractable to the non-extractable pool, as discussed above for the litter layer and surficial horizons of the temperate podzol (Table 1). The comparison of the GDGT distribution in the extractable and non-extractable fractions of the tropical podzol showed that archaeal GDGTs containing cyclopentyl rings (II–VI) were predominant among archaeal tetraethers, as observed in the temperate podzol (Table 1). In addition, GDGT IXa was observed to be by far the most abundant bacterial tetraether in the non-extractable and extractable fractions of all the Amazonian samples (e.g. Fig. 2), since it represented >74% of total non-extractable bacterial tetraethers and >80% of total extractable bacterial GDGTs (Table 1). The low degree of methylation of bacterial tetraethers in the tropical soil sequence is reflected in the high values of the MBT index (about 1.0) obtained for both extractable and non-extractable bacterial GDGTs (Table 2) and is consistent with the high MAT in Amazonia. In contrast, a higher degree of methylation is typically observed for soils from temperate and cold regions (Weijers et al., 2007), such as the temperate podzol investigated (Table 2). MAT was determined from MBT and soil pH using Eq. (4), because cyclopentyl-containing GDGT VIIIb was not detected in any of the Amazonian samples. MAT estimates derived from nonextractable and extractable lipid fractions were comparable, even though a slight difference between the two fractions was observed in the II A12 horizon. MAT values (between 23.7 and 28.3 °C; Table 2) were in rather close agreement with recorded temperature in the upper Amazon basin (average temperature 26 °C, maximum 31 °C and minimum 22 °C; Bardy et al., 2009). Nevertheless, values obtained from the well-developed podzol horizons were lower than those derived from the other samples, mainly because of the lower pH of II Bh and III A12 compared to the rest of the soil sequence (Table 2), as reported for the extractable lipid pool (Huguet et al., 2010a). Differences in the MAT values derived from the extractable lipid pool were observed between the three surficial samples (I AE, II A12 and III A12), I AE giving a higher MAT than the other samples (Table 2). These differences are difficult to explain, all the more so as I AE and II A12 are separated by only ca. 100 m and have similar pH values. Local calibrations could improve the accuracy of MAT reconstruction, as previously suggested (Sinninghe Damsté et al., 2008; Peterse et al., 2009a). Interestingly, MAT estimates derived from the non-extractable GDGT distribution are similar for I AE and II A12 (27.8 and 27.3 °C, respectively) and are consistent with the instrumental record of MAT.

As discussed above, GDGTs are likely to be transferred from the extractable to the non-extractable pool via a variety of mechanisms. They may be bound to or trapped in the organic macromolecular matrix. They may also become associated with soil mineral components, leading to the formation of organomineral complexes. Soil characteristics (e.g. mineralogy, OM composition and content) certainly directly affect the nature of the interaction between GDGTs and soil matrix. Mineral particles of different sizes are for example known to interact with SOM in different ways (Christensen, 1992), the sand fraction only exhibiting weak interaction with SOM, while clays provide a large negatively charged surface area for sorption of SOM. The mechanisms in play for the transfer of GDGTs from the extractable to the non-extractable pool are complex and are therefore likely to vary, depending on the nature of the soil, and even of the horizon, investigated. In particular, different mechanisms might be responsible for the transfer of bacterial GDGTs between the two lipid pools in the temperate and tropical podzols. This might explain why the MBT appeared to be lower from the non-extractable than from the extractable pool for the two AE horizons of the temperate podzol, whereas the opposite (i.e. higher MBT in the non-extractable pool) was observed for the II A12 horizon of the tropical podzol (Table 2). 4. Conclusions The abundance and distribution of bacterial and archaeal GDGTs were compared in the extractable and non-extractable lipid fractions of two podzols from temperate and tropical climatic environments. We observed that substantial amounts of GDGTs could be released after acid hydrolysis of solvent-extracted soils and that non-extractable and extractable GDGTs could show different distribution patterns. Thus, the average degree of methylation of bacterial GDGTs was found to be lower in the non-extractable than in the extractable lipid pool of the four mineral soil horizons of the temperate podzol, the largest difference having been observed in the two AE horizons. The differences in bacterial GDGT distribution between the extractable and non-extractable fractions might notably result from the preferential transfer of some bacterial GDGTs from the extractable to the non-extractable pool and/or from the preferential degradation of some bacterial GDGTs by soil microorganisms. Furthermore, the relative abundances of bacterial and archaeal GDGTs were compared along the temperate soil profile and the tropical soil sequence. The relative amount of bacterial over archaeal GDGTs was shown to be much higher in the extractable than in the non-extractable lipid fractions in the surficial horizons of the temperate podzol and in the well-expressed part of the tropical podzol. This difference seems to suggest that extractable archaeal GDGTs are preferentially transferred to the non-extractable lipid pool compared to extractable bacterial GDGTs in these surface horizons. This might be due to the fact that the types of polar head groups associated with bacterial GDGTs differ from, and are more labile than, those associated with archaeal GDGTs. Acknowledgments We thank the Regional Council of Ile de France for funding support. Two anonymous reviewers are thanked for constructive comments. Appendix A Glycerol dialkyl glycerol tetraether (GDGT) membrane lipids of archaeal and bacterial origin in French and Amazonian podzol samples and of the internal standard (IS) used for quantification.

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Archaeal GDGTs

m/z

HO O O

I

O O

HO

O O

OH O O

VIIb

O

OH

O

HO

1298

O

HO

O O

VIIIa

O O O

HO O O

OH

HO

HO

O

O

VI (+ VI’)

1294

O

O

O O

VIIIc IXa

OH

O O

HO O O

1292

1022

O O O

HO O

IXb

1020

O

O

O OH

OH

HO O O

OH O O

1032

OH

OH

O

O

1034

O

OH

O

1036 OH

VIIIb

O

HO

1046

OH

1296

O

O

O O

HO

OH

O

O O

VIIc

O

V

1048

O

OH

O

IV

1050

O O

HO

1300

O

HO

III

O O

1302 OH

O

II

m/z

Bacterial GDGTs

HO

VIIa

744

IXc

HO O O

O O OH

1018

Associate Editor – R.D. Pancost

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