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Animal Feed Science and Technology 166–167 (2011) 87–92

Contents lists available at ScienceDirect

Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Analysis of archaeal ether lipids in bovine faeces

a b a b b a

F.L. Gill , R.J. Dewhurst , R.P. Evershed , E. McGeough , P. O’Kiely , R.D. Pancost , a,∗

I.D. Bull

Organic Geochemistry Unit, Bristol Biogeochemistry Research Centre, University of Bristol, School of Chemistry, Cantock’s Close, Bristol BS8 1TS, UK

Teagasc, Animal & Grassland Research and Innovation Centre, Grange, Dunsany, Co. Meath, Ireland

a r t i c l e i n f o a b s t r a c t

This study evaluated concentrations of archaeol, a lipid biomarker for Archaea and, presum-

ably, methanogens in cattle faeces. Twelve continental cross-bred steers were allocated at

random to receive either a concentrate based diet or a grass silage based diet. Lipids were

Keywords: extracted from dried faeces, separated into speciﬁc compound classes and analysed by gas

Archaea chromatography/mass spectrometry, and CH4 production was estimated using the sulphur

Archaeol

hexaﬂuoride (SF6) technique. Steers fed the grass silage based diet consumed less feed (9.15

Dialkyl glycerol ether lipids

versus 11.43 kg dry matter (DM)/d; SED = 0.824; P<0.05), emitted more CH4 (341 versus

Cattle

174 g/d; SED = 60.5; P<0.05) and produced faeces with higher concentrations of archaeol

Rumen

Methane (30.6 versus 5.1 mg/kg DM; SED = 5.42; P<0.001) than those fed the concentrate based diet.

Other dialkyl glycerol lipids, such as hydroxyarchaeol, macrocyclic archaeol and unsatu-

rated archaeol analogues, were not detected. Since the feeds contained no archaeol, it must

have been produced in the ruminant digestive tract, most likely by Archaea in the rumen. The

relationship between CH4 emission and faecal archaeol concentration for individual ani-

mals was weak. Possible explanations include the inherent limitations of the SF6 technique,

the sampling regime, selective retention of Archaea in the rumen and possible degradation

of the archaeol during gut transit and post excretion. Despite these limitations, faecal lipid

biomarker analysis shows potential as a new method to study ruminant methanogens.

This paper is part of the special issue entitled: Greenhouse Gases in Animal Agriculture –

Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest

Editors: K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology,

1. Introduction

Methanogens belong to the domain Archaea, and comprise less than 0.05 of rumen prokaryotes (Janssen and Kirs, 2008),

but can lead to losses of up to 0.12 of gross energy intake by ruminants as CH4, which is a potent greenhouse gas. Bacte-

rial membrane lipids are usually comprised of a phosphate head group attached to a glycerol backbone esteriﬁed to acyl

chains in the sn-1,2 positions. In contrast, archaeal membrane lipids are comprised of a phosphate or phosphor-sugar head

group attached to a glycerol backbone to which isoprenoid chains are ether-linked at the sn-2,3 positions forming dialkyl

glycerol ether (DAGE) lipids. Archaeol (2,3-diphytanyl-O-sn-glycerol) is the simplest DAGE with two phytanyl chains and is

Abbreviations: CON, concentrate rich diet; DAGE, dialkyl glycerol ether; DM, dry matter; GS, grass silage rich diet; LW, live weight.

∗

Corresponding author. Tel.: +44 117 3317192; fax: +44 117 9251295.

E-mail address: [email protected] (I.D. Bull).

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88 F.L. Gill et al. / Animal Feed Science and Technology 166–167 (2011) 87–92

Table 1

Ingredient (g/kg) and chemical composition (g/kg dry matter) of the concentrates used for the concentrate and grass silage diets.

Diet

Concentrate Grass silage

Barley grain 820 460

Soybean meal 100 460

Molasses 50 50

Mineral/vitamin premix 20 20 b

Vegetable oil 10 10

Dry matter, g/kg 866 860

Ash 56 73

Crude protein 159 297

aNDFom 157 138

Starch 485 235

Supplying (per kg of concentrate) 10,000 IU vitamin A, 2000 IU vitamin D3, 50 IU vitamin E, 0.50 mg Se and 10 mg Cu.

Goldstar Lakeland Winter Blend 70; Goldstar Oils Ltd., Kilkenny, Ireland.

widespread among Archaea. Other DAGE lipids, including hydroxyarchaeol, macrocyclic archaeol and unsaturated analogues

of archaeol are found in some Archaea (Koga and Morii, 2005).

DAGE lipids have been used as biomarkers in a wide range of matrices. Bai et al. (2000) determined the DAGE content of

paddy rice soils, as well as phospholipid fatty acids and hydroxyl fatty acids of lipopolysaccharides, and used the results to

estimate the microbial biomass and community structure. Pancost et al. (2001) investigated the occurrence of DAGE lipids

in two Eastern Mediterranean mud dome ﬁelds where archaeol was found to be the most abundant diether lipid. Moreover

␦13 13

the C results obtained for archaeol in all but two of the samples were depleted in C (−41.3 to −95.8 ‰), suggesting that

archaeal progenitors were either directly or indirectly involved in CH4 production. Recent studies have demonstrated the

occurrence of archaeol in faeces of herbivores with foregut (rumen) fermentation, but not in the faeces of other herbivores

(Gill et al., 2010).

Our aim was to develop archaeol as a proxy for CH4 production in ruminants and perhaps overcome some of the limitations

of current techniques of measuring CH4 production in ruminants. For example, respiration chambers (Blaxter and Clapperton,

1965) are laborious and cannot be used with grazing animals. The SF6 technique (Johnson et al., 1994) has been shown to be

highly variable and may not be suitable for comparison of CH4 emissions among individuals (Pinares-Patino˜ and Clark, 2008).

The ﬁrst step in this research was to quantify archaeol in bovine faeces and compare variations in its concentration among

animals fed the same, and different, diets. The objective was to determine if the concentration of archaeol was positively

correlated with CH4 production.

2. Materials and methods

2.1. Animals and diets

Twelve continental cross-bred steers (initial live weight (LW) 541 ± 41.8 kg) were blocked according to LW and randomly

allocated to one of two dietary treatments based of either grass silage or concentrate being: (1) a ﬁxed allocation (1.25 kg

DM/head daily in a single meal) of grass silage with ad libitum concentrates (diet CON); and (2) a ﬁxed allocation (2.58 kg

DM/head daily in a single meal) of concentrates with ad libitum grass silage (diet GS). The ingredient composition of the

concentrates used for each diet is in Table 1. Samples of feeds were collected daily during the collection weeks and composited

by collection week. Chemical analysis methods for feeds are described by McGeough et al. (2010).

2.2. Management of animals

Steers were housed and trained to receive their diet through Calan gates (American Calan Inc., Northwood, NH, USA) so

that individual feed intake could be recorded. Steers were adapted to receive their allocated diets over the ﬁrst two weeks of

the study. Feed offered and refused was recorded daily. Steers were fed the same diet throughout the study. Measurements

of exhaled/eructated CH4 were collected from three steers per treatment after 5 and 16 weeks of provision of their respective

diets.

2.3. Animal measurements

Steers remained on the same dietary treatments, but were housed in individual stalls in a separate building for 10 d prior

to CH4 measurements. The SF6 technique (Johnson et al., 1994; Hart et al., 2009) was used for individual measurements of

CH4 production. Boluses containing SF6, of known release rate (3.1 ± 0.88 mg/day), were put into the rumen 10 d prior to

collection of respiratory gases. Evacuated canisters were attached to the head halter and used to collect expired air over 24 h

from each steer. A minimum of 3, and up to 5, separate daily samples were collected from each steer for estimation of daily CH4 emissions.

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F.L. Gill et al. / Animal Feed Science and Technology 166–167 (2011) 87–92 89

␤ ␤

Fig. 1. Partial gas chromatogram of the sterol fraction isolated from bovine faeces; (A) 5 -cholestan-3 -ol (coprostanol); (B) 5␤-cholestan-3␣-ol

(epicoprostanol); (C) cholest-5en-3␤-ol (cholesterol); (D) 5␣-cholestan-3␤-ol (5␣-coprostanol); (E) 24-methyl-5␤-cholestan-3␤-ol (5␤ campestanol);

␤ ␣

(F) 24-methyl-5 -cholestan-3 -ol (5␤-epicampestanol); (G) 24-ethyl-5␤-cholest-22-en-3␤-ol; (H) 24-ethyl-5␤-cholestan-3␤-ol (5␤-stigmastanol); (I)

␤ ␣

24-ethyl-5 -cholestan-3 -ol (5␤-epistigmastanol); (J) 24-ethyl-cholest-5en-3␤-ol (sitosterol); (K) 24-ethyl-5␣-cholestan-3␤-ol (5␣-stigmastanol); (L)

2,3-diphytanyl-O-sn-glycerol (archaeol).

Faecal samples were collected once daily over 5 d during the week after CH4 measurement. Rectal grab samples of faeces

∼ ◦ ◦

( 200 g) were obtained, stored at −20 C, thawed, composited and dried at 60 C for 48 h. Faecal samples were not collected

at exactly the same time as the CH4 emissions were made because of experimental constraints, and this may mask subtle

relationships between CH4 emitted and archaeol concentration, especially differences within individual steers fed the same

diet.

2.4. Laboratory analysis

An extract of total lipids was obtained from samples (0.5–1 g) of dried, ground (1 mm screen) faeces and feeds through

solvent extraction in 200 ml of dichloromethane:acetone (9:1 v/v) for 24 h using a Soxhlet apparatus. Internal standards

␮

(22.5 g each of preg-5-en-3␤-ol and hyocholic acid) were added to the extract. An aliquot of the total lipid extract was

saponiﬁed and initially separated into two fractions as described by Bull et al. (2003). The second of these fractions was

further separated by column chromatography using a slightly modiﬁed version of the procedures of Bull et al. (1999). The

ﬂash column chromatography step was shortened by reducing extraction to a three solvent system being: dichloromethane,

dichloromethane:methanol 1:1 and methanol, which were applied in order of polarity to give three fractions identiﬁed as:

“apolar”, “alcohol” and “polar”.

Analytes in the alcohol fraction were derivatised to their respective trimethylsilyl ethers by adding 50 ␮l of N,O-

bis(trimethylsilyl)triﬂuoroacetamide containing 0.01 trimethylchlorosilane, and 50 ␮l pyridine to the sample and heating

◦

at 70 C for 1 h. The samples were dissolved in ethyl acetate prior to analysis by gas chromatography (Carlo Erba HRGC 5300

equipped with a non-polar fused silica capillary column, CPSil-5CB, 50 m × 0.32 mm × 0.12 ␮m, Varian Chrompack, Oxford,

UK). Gas chromatography mass spectrometry was conducted using a ThermoQuest TraceMS GC-MS equipped with a column

◦

identical to that used for GC analyses. In both cases, the temperature program used was: initial temperature 70 C, rising to

◦ ◦ ◦ ◦ ◦

130 C at 20 C/min, then to 300 C at 4 C/min, with a ﬁnal hold at 300 C for 25 min. Archaeol was identiﬁed on the basis of

its characteristic mass spectrum (Fig. 1), featuring prominent fragment ions with a mass to charge ratio (m/z) of 130, 278,

412 and 426 (Teixidor and Grimalt, 1992) and quantiﬁed by comparison to the internal standard.

Methane and SF6 were measured in gas samples using gas chromatography (Hart et al., 2009) using a gas chromatograph

equipped with both ﬂame ionization and electrochemical detectors.

2.5. Statistical analysis

Effects of dietary treatments were analysed using analysis of variance (Genstat, 2007) with ‘diet’ as a treatment factor

and blocking according to measurement week.

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90 F.L. Gill et al. / Animal Feed Science and Technology 166–167 (2011) 87–92

Table 2

Effects of dietary treatment on feed intake, methane production and faecal archaeol concentration.

Ad libitum concentrates Ad libitum grass silage SED P

Dry matter intake, kg/day 11.43 9.15 0.824

Methane production, g/day 174 341 60.5

Methane production, g/kg DM intake 15.0 37.4 6.09

***

Faecal archaeol, mg/kg DM 5.1 30.6 5.42

* P<0.05. ** P<0.01. *** P<0.001.

3. Results

The chemical composition of the concentrates used for diets CON and GS is in Table 1. The grass silage had a pH of 3.81

and contained 252 g DM/kg and, on a DM basis, 100 g ash/kg, 138 g CP/kg, 511 g aNDFom/kg, 106 g lactic acid/kg, 26.6 g acetic

acid/kg, 11.9 g butyric acid/kg and 18.6 g ethanol/kg.

Feed intake, CH4 emissions and faecal archaeol concentrations are in Table 2. Feed intake was higher (P<0.05) for the

ad libitum concentrate diet (11.43 kg/d) compared to the ad libitum grass silage diet (9.15 kg/d). Methane production was

higher for the grass silage diet than the concentrate diet, whether expressed on a daily basis (341 g/d versus 174 g/d) or

relative to DM intake (37.4 g/kg DM intake versus 15.0 g/kg DM intake). The concentrate diet also resulted in lower (P<0.001)

faecal archaeol concentrations compared to the grass silage diet (5.1 mg/kg DM versus 30.6 mg/kg DM; Fig. 2). The relative

difference in faecal archaeol concentration (x6) between the silage and concentrate diet was much bigger than the difference

in CH4 production (x2), indicating a possible positive non-linear relationship between them. There was no difference in the

relationship between faecal archaeol concentration (mg/kg DM) and CH4 production (g/kg DM intake) for measurements

made in week 5 and 16 of the experiment (Fig. 2). We did not detect any of the other DAGE lipids (i.e., hydroxyarchaeol,

macrocyclic archaeol, unsaturated analogues of archaeol), commonly observed in unrelated samples analysed routinely by

this laboratory, in feeds or faeces.

4. Discussion

4.1. Methane production

Treatment means for CH4 production (g/kg DM intake) corresponded closely to the lowest and highest treatment means

in the studies reviewed by Beauchemin et al. (2008). Animals fed a predominantly grass silage based diet produced more CH4

than those fed a predominantly concentrate based diet. The relationship between forage:concentrate ratio and CH4 produc-

tion is well recognized in cattle (e.g., Johnson and Johnson, 1995). Methane emissions are lower on high concentrate diets

due to several factors, including higher ruminal passage rates, an increase in the ﬂow of reducing equivalents to propionate

Fig. 2. Archaeol concentrations in bovine faeces from steers fed concentrate or grass silage diets ad libitum versus methane production as estimated by the

SF6 technique.

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F.L. Gill et al. / Animal Feed Science and Technology 166–167 (2011) 87–92 91

(Johnson and Johnson, 1995) and direct inhibition of methanogens at low pH (Van Kessel and Russell, 1996). Recent studies

suggest that variation in CH4 production may not be correlated with the population size of rumen methanogens (Morgavi

et al., 2010), although this is equivocal since estimates of archaeal populations are likely to have large experimental errors.

Another possible source of variation in the relationship between archeaol and CH4 production is that not all Archaea in the

rumen are methanogens. Whilst most rumen Archaea are known to be methanogens, this has not yet been conﬁrmed for

Archaea belonging to Rumen Cluster C which are abundant under some dietary conditions (Janssen and Kirs, 2008).

4.2. Faecal dialkyl glycerol ether lipids

This study conﬁrmed that archaeol is generated during passage through the digestive tract, since none was detected in

the diets. Several observations indicate that the rumen is the most probable source of archaeol. For example, the population

of prokaryotes in the rumen is at least an order of magnitude higher than populations in the hind gut or faeces (Mould et al.,

2005), whilst analysis of the 16S RNA gene provided no evidence for a higher proportion of Archaea in the hind gut or faeces

compared with the rumen (Lin et al., 1997). The evidence for a rumen origin is implicit in the absence of detectable levels

of archaeol derived from the archaeal populations in faeces in non-ruminant herbivores such as horses, zebra, elephant and

rhinoceros.

Non-ruminant gastric systems produce CH4 and there will almost certainly be archaeol that is derived from methanogenic

Archaea in these systems, albeit below the limit of detection using the methods employed by this and previous research (Gill

et al., 2010). Nonetheless, further studies are required to elucidate the relationships between concentrations of archaeol in

faeces, the size of the methanogen population in the rumen, and CH4 output. The unknown elements of this relationship

include selective retention of Archaea in the rumen, differences in the species composition of the methanogen community,

which might differ in archaeol concentration per cell, differences in the post-rumen digestibility of DAGE, and the production

of archaeol by non-methanogenic Archaea.

There was only a weak relationship between faecal archaeol concentration and CH4 production/kg DM intake (r = 0.55;

P<0.05), despite the large effect of dietary treatment. This may be related to uncertainties about faecal archaeol outlined

above, as well as experimental error with the SF6 technique (Pinares-Patino˜ and Clark, 2008).

The absence of hydroxyarchaeol, macrocyclic archaeol and unsaturated analogues of archaeol in bovine faeces is con-

sistent with other information about the family level composition of ruminant Archaea (Janssen and Kirs, 2008) and their

membrane lipids (Koga and Morii, 2005). The review by Janssen and Kirs (2008) suggested that most rumen Archaea belong to

the families Methanobacteriaceae and Methanomicrobiaceae, which do not contain hydroxyarchaeol, macrocyclic archaeol or

unsaturated archaeol. Some members of the family Methanosarcinaceae contain hydroxyarchaeol and unsaturated archaeol,

but are rare in the rumen (0.02–0.03 of the archaeal population). There is some uncertainty about the presence of members

of the family Methanococcaceae, which contain hydroxyarchaeol, in the rumen, but these results suggest that they were not

present in substantial numbers.

5. Conclusions

Archaeol is produced in the ruminant digestive tract, presumably by methanogenic Archaea within the rumen (Gill et al.,

2010). Large differences in faecal archaeol concentration between cattle fed diets with different forage/concentrate ratio

mirrored effects on CH4 production, but further research is needed to before this approach can be used as a direct indicator

or CH4 emissions in ruminants.

Conﬂict of interest

None.

Acknowledgements

Funding to carry out the research reported herein was provided by the Leverhulme Trust (grant F/00 182/BS awarded to

IDB). NERC provided funding for the mass spectrometry facilities at Bristol (Contract no. R8/H12/15; www.lsmsf.co.uk).

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