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Organic Geochemistry 39 (2008) 167–177

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OrganicGeochemistry

Occurrence of unusual steroids and hopanoids derived fromaerobic methanotrophs at an active marine mud volcano

Marcus Elvert a,*, Helge Niemann b,c

a Organic Geochemistry Group, Department of Geosciences, University of Bremen, Leobener Strasse, D-28359 Bremen, Germanyb Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany

c Alfred Wegener Institute for Polar and Marine Research, D-27515 Bremerhaven, Germany

Received 26 June 2007; received in revised form 5 November 2007; accepted 15 November 2007Available online 22 November 2007

Abstract

Surface sediment samples from two microbial habitats (centre and adjacent Beggiatoa mats) at an active CH4-emittingmud volcano on the Norwegian margin of the Barents Sea (Haakon Mosby Mud Volcano – HMMV) were analyzed fortheir steroid and hopanoid biomarker distributions and associated stable carbon isotopic composition. Substantial abun-dances of a suite of six steroids (four 4a-methyl sterols and two 4a-methyl steroid diols) and three hopanoids (diploptene,diplopterol and D2-diplopterol) were found, together with biomarkers from anaerobic methanotrophic consortia (ANME-3/Desulfobulbus aggregates) at the surface sediment of the mats. All of the steroid and hopanoid biomarkers, some notbeen described before, were substantially depleted in 13C (d13C between �77‰ and �68‰), proving biosynthesis by anorganism involved in methane consumption. Molecular analysis (16S rRNA sequencing and fluorescence in situ hybrid-isation) performed in a parallel study showed that, besides ANME-3/Desulfobulbus consortia, aerobic methanotrophsof the order Methylococcales are also abundant with high cell numbers. These independent findings suggest a spatiallyclose vicinity of active aerobic and anaerobic methanotrophic communities on the order of millimetres, not reported beforefor the marine environment. At the centre location, where methane oxidation is performed solely by Methylococcales spe-cies, predominantly 4a-methyl-3b-sterols, together with high abundances of specific fatty acids (i.e. C16:1x9c and C16:1x8c),were detected. The additional presence of new and unusual biomarkers for aerobic methanotrophy at the Beggiatoa matsseems to indicate either varying strains of Methylococcales types or an energy stress adaptation of the dominant species atthe latter sampling site. Moreover, consideration is given to the new biomarkers being intermediates in the biosyntheticformation of steroids and hopanoids in methanotrophic bacteria.� 2007 Elsevier Ltd. All rights reserved.

1. Introduction

Steroids and hopanoids are important buildingblocks of living cells, influencing membrane rigidity

0146-6380/$ - see front matter � 2007 Elsevier Ltd. All rights reserveddoi:10.1016/j.orggeochem.2007.11.006

* Corresponding author. Tel.: +49 421 21865706; fax: +49 42121865715.

E-mail address: melvert@uni-bremen.de (M. Elvert).

and permeability. Whereas steroids occur ubiqui-tously in eukaryotic organisms (Volkman, 2003),hopanoids are dominantly attributed to the bacte-rial kingdom (Rohmer et al., 1984). Therefore, thepresence of these biomarkers in environmentalsamples reveals ecological and chemotaxonomicinformation. Some aerobic methanotrophic bacteriaof the order Methylococcales have, however, the

.

168 M. Elvert, H. Niemann / Organic Geochemistry 39 (2008) 167–177

unique capability of biosynthesising large quantitiesof both groups of cellular membrane components(Rohmer et al., 1980). Diagnostic steroids (4,4-dimethyl and 4a-methyl sterols) and hopanoids(diploptene, diplopterol, 3b-methyl diplopterol)have been reported from Methylococcus capsulatus

(Bird et al., 1971; Bouvier et al., 1976; Zundel andRohmer, 1985a), whereas the psychrophilic methan-otroph Methylosphaera hansonii showed the pres-ence of a diverse suite of characteristic steroids,but no hopanoids (Schouten et al., 2000).

Aerobic methanotrophy has rarely been detectedin marine sediments. Exceptions have been docu-mented for surface sediments of active methane-emit-ting mud volcanoes, namely Haakon Mosby MudVolcano (HMMV) in the Barents Sea (Niemannet al., 2006a), and Kazan and Chefren mud volcanoesin the eastern Mediterranean Sea (Werne et al., 2002;Stadnitskaia et al., 2007). However, other studies forwhich indications of aerobic methanotrophy in mar-ine sediments were found linked the process to pastactivity at the oxycline of an upper water column(Schouten et al., 2001; Hinrichs et al., 2003). The glo-bal relevance of aerobic methanotrophy in marinesediments is not much understood because its pres-ence is generally suppressed due to the anaerobic oxi-dation of methane (AOM) using sulfate as terminalelectron acceptor. Sulfate constantly diffuses intothe sediment from the water column, such thatAOM most often prevents the discharge of methaneinto the upper sediment and ocean water (Niemannet al., 2006b; Reeburgh, 2007).

Biological research on surface sediments ofHMMV has shown that the system is dominatedby novel microbial communities functioning in bothmethanotrophic modes (Niemann et al., 2006a): (1)aerobic methanotrophic c-proteobacteria of theorder Methylococcales at the centre habitat, and(2) anaerobic methanotrophic consortia consistingof ANME-3 archaea and sulfate reducing bacteria(Desulfobulbus spp. – DBB) at thiotrophic Beggia-

toa mats. However, 16S rRNA gene sequence anal-ysis and cell counting via fluorescence in situhybridisation (FISH) performed on the Beggiatoamats sample showed that aerobic methanotrophsof the order Methylococcales are also present in con-siderable amounts (3.9 ± 2.5 � 108 cells cm�3 at 0–1 cm sediment depth; Losekann et al., 2007).Because of the high abundance of Beggiatoa-derivedfatty acids (i.e. C16:1x7c) at the site, signatures of aer-obic methanotrophy via diagnostic fatty acids (e.g.C16:1x9c and C16:1x8c) were obscured. Consequently,

we re-investigated sediments from the two surfacehabitats for indications of aerobic methanotrophs,specifically targeting characteristic steroid andhopanoid biomarkers and their associated stablecarbon isotopic compositions.

2. Materials and methods

2.1. Study area

HMMV is located at 72�00.250N, 14�43.500E in1250 m water depth in the SW Barents Sea (Vogtet al., 1997). It has a circular shape 1 km in diameter(0.8 km2), a relief of up to 10 m above sea floor, andis surrounded by a circular ca. 200 m wide depres-sion (Vogt et al., 1997; Hjelstuen et al., 1999). Theseafloor is roughly divided into three major concen-tric habitats (Niemann et al., 2006a): (1) the thermalcentre, (2) adjacent Beggiatoa mats covered sedi-ments and (3) an outer rim colonized by siboglinidtubeworms. Thermal gradients at the centre, wheremethane-rich sediments and fluids are expelledthrough a central conduit, are high, with values ofup to 3 �C m�1, and decrease towards the outerrim (Kaul et al., 2006). Methane in gas hydrates atHMMV is dominantly of microbial origin, with ad13C value of �60.6‰ (Lein et al., 1999) and is>99.8% pure (Ginsburg et al., 1999). Relative meth-ane consumption (as % total methane flux into thewater column) by aerobic methanotrophs andANME-3/DBB consortia is up to 2% at the centreand 14% at the sulfide-oxidizing Beggiatoa mats,respectively (Niemann et al., 2006a). Much greatermethane removal of up to 24% is, however,observed at siboglinid tubeworm fields surroundingthe Beggiatoa mats and the centre habitat.

2.2. Sample collection and storage

Surface sediment samples were recovered fromtwo of the three specific habitats (Beggiatoa mats,centre) during a cruise with RV L’Atalanteequipped with ROV VICTOR 6000 in 2001. Theywere collected with ROV push cores and sliced into2 cm sections for lipid analysis. Sections were trans-ferred to cleaned glass vials and stored at �20 �Cuntil extraction.

2.3. Lipid biomarker analysis

Surface sediment samples (0–2 cm depth hori-zons) were extracted, separated into compound clas-

M. Elvert, H. Niemann / Organic Geochemistry 39 (2008) 167–177 169

ses (hydrocarbons, alcohols, fatty acids) and ana-lyzed using gas chromatography-flame ionizationdetection (GC-FID), gas chromatography-massspectrometry (GC–MS) and gas chromatography–isotope ratio mass spectrometry (GC–IRMS)according to reported methods (Elvert et al., 2003,2005; Niemann et al., 2005). Briefly, a total lipidextract (TLE) was obtained for each sample fromca. 5 g of wet sediment by subsequent ultrasonica-tion using solvents of decreasing polarity: (I) dichlo-romethane (DCM)/MeOH (1:2, v/v), (II) DCM/MeOH, (2:1 (v/v) and (III) DCM. Internal standardsof known concentration and carbon isotopic compo-sition were added prior to extraction. Fatty acid moi-eties present in glyco- and phospholipids werecleaved off by saponification with methanolic KOH(6%). After extraction of the neutral lipid fraction,fatty acids (FAs) were extracted at pH 1 and methyl-ated with BF3 in MeOH, affording methyl esters(FAMEs). Neutral lipids were further separated intohydrocarbons, ketones and alcohols with a SPE sil-ica glass cartridge (0.5 g packing) with solvents ofincreasing polarity: (I) n-hexane/DCM (95:5, v/v),(II) n-hexane/DCM (2:1, v/v) and (III) DCM/ace-tone (9:1, v/v). Alcohols were derivatized withN,O-bis(trimethylsilyl)triflouracetamide (BSTFA)in pyridine.

GC-FID was performed using a HP 5890 Series IIchromatograph equipped with a split/splitless injec-tor and a 50 m apolar HP-5 fused silica column(0.32 mm i.d., film thickness 0.17 lm; Hewlett Pack-ard). Fractions were injected in splitless mode at300 �C using He as carrier gas. Initial oven tempera-ture was 60 �C (hold 1 min), increased to 150 �C at10 �C/min and raised to 310 �C at 4 �C/min (25 minhold). Structural assignment was based on relativeretention time and comparison with published massspectra. Relative retention of 3a- and 3b-sterols oncapillary columns with apolar stationary phaseswas tested via the reduction of a mixture of 4-chole-sten-3-one and cholestan-3-one using NaBH4 inMeOH, affording a mixture of 3a- (10%) and 3b-(90%) sterols. Double bond positions in FAMEs, aswell as trimethylsilyl (TMS) derivatives of shortchain n-alcohols and sn-1-mono-O-alkyl glycerolethers (MAGEs) were determined by preparation ofdimethyl disulfide (DMDS) adducts. Stable carbonisotope values are reported in the d-notation as permil (‰) deviation from the Vienna Pee Dee Belemnitestandard (VPDB). The analytical error is less than1.0‰ and values were corrected for the introductionof additional carbon atoms during derivatization.

3. Results

Fig. 1 shows a gas chromatogram of the alcoholfraction from the surface sediment at the Beggiatoamats. Biomarkers for anaerobic methanotrophicarchaea (ANME-3; Niemann et al., 2006a) such asphytanol, sn-2-phytanyl-mono alkyl glycerol ether(MAGE), archaeol, and sn-2-hydroxyarchaeol weredetected in significant amounts (up to 3.7 lg/g dryweight (dwt.) for sn-2-hydroxyarchaeol) and withspecific 13C-depletion (d13C between �106‰ and�91‰). Moreover, a set of unknown isoprenoidalglycerol diethers, which contain molecular struc-tures similar to dihydroxyarchaeols (A. Bradley,personal communication), were found with 13C-depletions of ca. �108‰. Indications of sulfatereducing bacteria were also present (n-alcoholC16:1x5c and MAGE). In addition, an abundantsuite of 13C-depleted steroids and hopanoids withconcentration values of up to 1.9 lg/g dwt. weredetected (d13C from �77‰ to �68‰; Table 1). Allthe steroid (pairs Ia/Ib, IIa/IIb and IIIa/IIIb; seeFig. 3 for numbering) were assigned as 4a-methylsterols, indicated by the significant presence of anm/z 227 ion in the spectra of the TMS derivatives(Fig. 1, enlarged insert); 4,4-dimethyl sterols (m/z

241 chromatogram) were only detected in minorconcentration. Based on interpretation of publishedspectra of the corresponding acetates (Bouvier et al.,1976), Ib and IIb were assigned as 4a-methylcholest-8(14)-en-3b-ol and 4a-methylcholesta-8(14),24-dien-3b-ol, respectively. The differences in the mass spec-tra of sterol acetates vs. opposed to TMS derivativesused in this study are minor and essentially resultsfrom the mass difference of both derivatizing agents.

The second eluting 4a-methyl sterol (compoundIIa) showed a virtually identical mass spectrum tothat of IIb, except for an increased m/z 210 ion,which is an unusual fragment for sterols in generaland obviously not prominent in the case of 4a-methyl-3b-sterols (Bouvier et al., 1976). A literaturesurvey did not reveal that IIa could be a doublebond isomer, i.e. containing a double bond at C-7,8, or 9(11) (e.g. lathosterol, lanosterol, or parkeol,respectively). Therefore, we presume that it couldbe a stereoisomer of IIb, possibly related to one orother of the chiral centres at C-3 and C-4. The lattercan be excluded because it has been shown that 4b-methyl sterols elute later on apolar stationaryphases than their 4a-methyl sterol counterparts(Knapp et al., 1975). In contrast, the opposite hasbeen observed for cholestanols and cholestenols

Fig. 1. Gas chromatogram of alcohols (TMS derivatives) from the Beggiatoa mats surface sediment. Abundant compounds not shown inTable 1 have been annotated with their carbon isotopic signature in brackets. Enlarged insert shows m/z 227 and 131 chromatograms,indicating 4a-methyl sterols (Ia–IIIb) and sterols and hopanols containing a terminal tertiary alcohol in the side chain (IIIa, IIIb, IV, anddiplopterol), respectively. MAGE: mono alkyl glycerol ether.

Table 1Concentration (lg/g dwt.) and d13C value (‰ VPDB, parentheses) of lipid biomarkers indicative of aerobic methanotrophs in surface sediment samples(0–2 cm depth) from the Beggiatoa mats site and the centre habitat

Hydrocarbons Sterols/hopanolsa Fatty acids

Squalene Diploptene IIa Ib IIb IIIa IIIb IV Diplopterol C16:1x9c C16:1x8c

Beggiatoa mats 2.1 (�49) 0.3 (�75) 0.5 (�40)b 0.6 (�73) 1.8 (�77) 1.3 (�75) 1.5 (�76) 1.9 (�75) 1.4 (�68) 1.2 (n.m.)c 1.5 (n.m.)c

Centre 0.4 (�52) 0.2 (�70) n.d.d 0.3 (�71) 1.1 (�72) 0.1 (n.det.)e n.d.d 0.1 (n.det.)e 0.1 (n.det.)e 5.2 (�69) 11.6 (�80)

aCompound Ia (4a-methylcholest-8(14)-en-3a-ol) not included due to prescence chromatographic background.

b d13C value determined on target compound co-eluting with ergost-22-en-3b-ol.c Not measured due to co-elution with highly-abundant C16:1x7c fatty acid (74.7 lg/g dwt.) dominantly derived from Beggiatoa species.d Not detected.e

Not determined due to low concentration.

170 M. Elvert, H. Niemann / Organic Geochemistry 39 (2008) 167–177

containing the OH group in the 3a-position (Arin-ger, 1978; Serizawa et al., 1981). This is corrobo-rated by the observation of a shorter retentiontime of the 3a-sterols than the 3b-sterols formedduring the chemical reduction of a mixture of 4-cholesten-3-one and cholestan-3-one. Therefore,

we tentatively assigned IIa to 4a-methylcholesta-8(14),24-dien-3a-ol. Based on relative retentiontimes and mass spectral characteristics, we analo-gously assigned Ia as 4a-methylcholest-8(14)-en-3a-ol (tentative assignment), which was onlydetected in the chromatographic background.

M. Elvert, H. Niemann / Organic Geochemistry 39 (2008) 167–177 171

The later eluting 4a-methyl sterols IIIa and IIIbadditionally showed a base peak at m/z 131(Fig. 1, enlarged insert), as observed in the spectrumof diplopterol (hopan-22-ol), a triterpenoid oftenencountered in environmental samples (ten Havenet al., 1989) and abundant at the Beggiatoa matssite. This specific fragment ion also indicates thepresence of a terminal tertiary alcohol carbon inthe side chain of 25-hydroxysterols (Park andAddis, 1985; Pizzoferrato et al., 1993), so IIIa andIIIb were assigned as 4a-methyl-3,25-steroid diols.The mass spectra of these newly detected biomark-ers are shown in Fig. 2A and B and the fragmenta-tion pattern, showing the loss of two TMSOHmoieties, is in good agreement with a diol steroidalstructure. Like the pairs Ia/Ib and IIa/IIb, the two

Fig. 2. Mass spectra of tentatively assigned 4a-methyl-3,25-steroid diol(ten Haven et al., 1989; D). Specific mass fragments are indicated.

4a-methyl-3,25-steroid diols IIIa and IIIb showalmost identical spectra, with the former revealingthe presence of a more abundant m/z 210 ion.Although the exact nature and formation of thisspecific fragment are unknown, we accordinglyassigned IIIa and IIIb as 4a-methylcholest-8(14)-en-3a,25-diol and 4a-methylcholest-8(14)-en-3b,25-diol, respectively.

An m/z 131 ion was also observed for anunknown, but very abundant, unsaturated hopanoidalcohol (compound IV; Fig. 2C) with a spectrumshowing analogies to that of diplopterol (Fig. 2D).The presence of unsaturation is revealed by theM+. at m/z 498 and a fragment at m/z 365, the latterindicating the presence of a double bond in thepentacyclic ring structure. Furthermore, the sole

s (IIIa and IIIb; A and B), D2-diplopterol (IV; C) and diplopterol

172 M. Elvert, H. Niemann / Organic Geochemistry 39 (2008) 167–177

presence of an m/z 189 ion, characteristic of C-ringcleavage vs. the m/z 189/191 doublet in the spectrumof diplopterol suggests that the double bond islocated in ring A or B. During synthesis of 2-methyldiplopterol from 22-hydroxyhopan-3-one, Bisseretet al. (1985) isolated D2-diplopterol as an intermedi-ate having MS characteristics identical to those ofour unsaturated hopanol. Hence, we assigned IV asD2-diplopterol (hop-2(3)-en-22-ol). Characteristicmethyl hopanoids, indicated by the presence of dou-blet fragments at m/z 205 and 383, were not detected.

4. Discussion

4.1. Aerobic methanotrophy in surface sediments of

Beggiatoa mats

Two independent lines of evidence indicate thepresence of aerobic methanotrophy in surface sedi-ments at the Beggiatoa mats of HMMV: (1) abun-dant diagnostic 13C-depleted steroid and hopanoidbiomarkers (this study) and (2) 16S rRNA genesequences and FISH analyses showing live Methylo-

coccales strains (Losekann et al., 2007), which areclosely related to known triterpenoid-producingmethanotrophic bacteria such as M. capsulatus (Birdet al., 1971; Bouvier et al., 1976) and M. hansonii

(Schouten et al., 2000). The presence of microorgan-isms oxidizing methane under aerobic conditions atthe Beggiatoa mats site is quite remarkable consider-ing the close vicinity to abundant and strictly anaer-obic ANME-3/DBB consortia near the sedimentsurface (Niemann et al., 2006a). Nevertheless,in situ oxygen measurements have provided evidencethat, even at the Beggiatoa mats site, oxygen pene-trates ca. 1 mm into the sediment (bottom water oxy-gen concentration of �0.3 mmol L�1), where it isclearly separated from the underlying sulfide frontby ca. 6 mm (de Beer et al., 2006). The general poten-tial of aerobic methanotrophs to grow at very lowoxygen levels has been demonstrated (Jahnke,1986; Jahnke and Nichols, 1986) and so mightexplain the simultaneous presence of both modesof methane oxidation separated by only <1 cm.Some studies have revealed the presence of 13C-depleted hopanoids in AOM environments, suggest-ing that they are produced anaerobically (Elvertet al., 2000; Pancost et al., 2000; Thiel et al., 2003).However, the simultaneous occurrence of steroidshere points to the availability of oxygen to the Meth-

yloccocales species, which is crucial for the first stepin sterol biosynthesis, the epoxidation of squalene by

the enzyme squalene monooxygenase (Summonset al., 2006; Fischer and Pearson, 2007).

In surface sediments of the centre habitat, whereaerobic methanotrophy is dominant, no ANME-3/DBB aggregates could be detected (Niemann et al.,2006a). The biomarker inventory of this habitatis dominated by 13C-depleted C16:1x9c and C16:1x8c

FAs (R = 16.8 lg/g dwt.; Table 1). Such a fingerprintof FAs indicates type I methanotrophs (Makula,1978; Nichols et al., 1985), which are significantlyreduced in concentration at the Beggiatoa mats(R = 2.7 lg/g dwt.). Here, biomarkers characteristicof aerobic methanotrophy are composed mainly ofpolycyclic triterpenoids dominated by the unusual4a-methyl steroid diols and diplopterol-related hopa-noids, as well as their irregular precursor isoprenoid,squalene (R = 11.4 lg/g dwt.), all of which werefound in much smaller amounts at the centre habitat(R = 2.3 lg/g dwt.). This finding of varying bio-marker patterns might point to the presence of differ-ent strains of Methylococcales at the two HMMVsampling sites. The dominant Methylococcales typeshave been shown to consist of two separated clustersof c-proteobacteria (HMMV-MetI and -MetII);however, both clusters have been detected in an evendistribution at the centre habitat as well as theBeggiatoa mats (Losekann et al., 2007). Hence, apotential difference between the Methylococcalescommunities at the centre and Beggiatoa mats habi-tat of HMMV must be, if at all, at the sub-strain level.

Alternatively, it is possible that Methylococcales

types at HMMV adapt their biomarker inventorydepending on specific environmental factors suchas temperature difference, oxygen availability, orpH. Whereas oxygen availability and pH werealmost identical at both sampling sites (de Beeret al., 2006), temperatures as high as 25.8 �C havebeen measured in deeper parts of the central mudconduit (Kaul et al., 2006). However, because ofthe very high heat loss, temperatures of theupward-migrating mud are uniformly �0.8 �C (i.e.bottom water temperature) at the sediment surfaceof both sampling sites. Consequently, the biomarkerinventory in aerobic methanotrophs at HMMVseems not to be regulated by environmental factors.

As a third option, it should be considered thatthe functional role of sterols in living cells has beensuggested to influence their membrane properties.For example, the reduction in leakage of protonsas well as Na+ and K+ has been proposed (Haines,2001). More specifically, 25-hydroxysterols (i.e., ste-roid diols), have been shown to increase the uptake

M. Elvert, H. Niemann / Organic Geochemistry 39 (2008) 167–177 173

of cations such as Ca2+, Mg2+, or Mn2+ of lipo-somes (Holmes and Yoss, 1984), whereas oxysterolsin general have been documented to affect thephospholipid packing in cell membranes (Szosteket al., 1991). The specific presence of 4a-methyl ste-roid diols and diplopterol-related hopanoids in theMethylococcales species at the Beggiatoa mats sitemight point to such effects, which seem to be relatedto the conservation of metabolic ATP energy withinthe cell (Haines, 2001). Energy conservation ispotentially essential during the competition fornutrients and substrates of aerobic methanotrophsas opposed to sulfide-oxidizing Beggiatoa andANME-3/DBB consortia at the location (i.e. oxy-gen/nitrate and methane, respectively). At the centrehabitat, in contrast, sulfidic conditions cannot beestablished due to high upflow velocity of sulfate-free fluid, which prevents the diffusion of water col-umn sulfate into the sediment and so, environmen-tal conditions are unfavourable for AOM (de Beeret al., 2006; Niemann et al., 2006a). Such a situationprobably reduces the energy stress for the Methylo-

coccales species, leading to the observed biomarkerpattern dominated by FAs.

4.2. Biosynthesis of steroids and hopanoids by

psychrophilic methanotrophs at HMMV

The abundance of new and additional biomark-ers for aerobic methanotrophs such as 4a-methyl-3,25-steroid diols, diplopterol, and D2-diplopterolat the Beggiatoa mats sample site implies the pres-ence of an undetected biosynthetic pathway in aero-bic methanotrophs. Therefore, we tentativelypropose a pathway of steroid and hopanoid biosyn-thesis in Methylococcales species of HMMV that isactive at the Beggiatoa dominated microbial mats(Fig. 3). However, such a putative pathway has tobe validated with pure culture studies. The precur-sor squalene (d13C �52 and �49‰), as well as itssimple hydrocarbon cyclisation product diploptene(d13C �75 and �70‰), have been detected at bothsampling sites (Table 1). Moreover, the presenceof D2-diplopterol as an intermediate in the produc-tion of methyl hopanoids has been predicted (Zun-del and Rohmer, 1985b). The latter authors usedtheoretical stereochemical considerations of the cyc-lisation of squalene via the squalene-hopene cyclasein bacteria that indicated a D2-hopanoid as a plausi-ble candidate. The biosynthesis might occur eitherdirectly from squalene or by desaturation betweenC-2 and C-3 of prior-formed diplopterol. There-

upon, the formation of 3b-methyl diplopterol, asobserved in M. capsulatus (Zundel and Rohmer,1985a; Summons et al., 1994), would be finally per-formed by the transfer of a methyl group from S-adenosylmethionine (S-AdoMet), a general processfunctioning in various bacteria and leading to meth-ylated compounds as well as products with a cyclo-propane ring (Grogan and Cronan, 1997; Elvertet al., 2003). However, a cyclopropane intermediatein the methyl hopanoid biosynthesis has beenexcluded (Zundel and Rohmer, 1985b).

4a-Methyl sterols have been identified in methan-otrophic bacteria (Bouvier et al., 1976; Schoutenet al., 2000), but not as steroid diols bearing a ter-tiary hydroxyl group at C-25 in the side chain; 25-hydroxysterols and other so-called oxysterols havebeen often documented from mammalian cells tosuppress enzyme activity, resulting in marked reduc-tion of sterol biosynthesis (Schroepfer, 2000). InMethylococcales species at HMMV, the formationof these might occur during the oxidative removalof methyl carbons at C-14 and C-4 of lanosterolor of another, yet undescribed, protosterol, e.g.parkeol (Pearson et al., 2003). It has also been sug-gested that M. capsulatus uses an alternative enzy-matic pathway for the C-4 demethylase reaction(Summons et al., 2006).

The presence of two series of closely identical ste-roidal compounds (Ia–IIIa and Ib–IIIb) seems toindicate that the enzymatic rearrangement of thesterol molecule from lanosterol takes place in a spe-cific but different fashion relative to the knownmodifications in eukaryotes (Brown, 1998; Piironenet al., 2000). Hence, there might be the possibility ofthe specific formation of methyl sterols containingthe OH group in the tentatively assigned 3a-positionof ring A, leading to the observed biomarker patternof 3a- and 3b-hydroxy methyl sterols in Methylo-

coccales species at HMMV. According to earlierstudies by Bouvier et al. (1976), the common endproducts of this proposed metabolic pathway wouldbe unsaturated 4a-methyl sterols (Fig. 3). However,the unusual and novel compounds comprise animportant part of the steroid inventory at HMMV,but are especially present at the Beggiatoa mats hab-itat, whereas the centre site is dominantly character-ized by 4a-methyl-3b-sterols.

5. Conclusions

A suite of known and novel steroids and hopa-noids has been detected in surface sediments of the

Fig. 3. Tentative biosynthetic pathway of steroid and hopanoid formation in psychrophilic Methylococcales species at the Beggiatoa matssite of HMMV. Basic information on the general pathway is provided by Bouvier et al. (1976) and Summons et al. (1994).

174 M. Elvert, H. Niemann / Organic Geochemistry 39 (2008) 167–177

centre and Beggiatoa mats habitats of Haakon Mos-by Mud Volcano. Characteristic 13C-depletion pro-vides evidence that these lipid biomarkers are

derived from aerobic methanotrophic bacteria.Members of the Methylococcales cluster are the dom-inant aerobic methanotrophs in these sediments,

M. Elvert, H. Niemann / Organic Geochemistry 39 (2008) 167–177 175

which prevail in the oxygenated upper few millime-tres. The spatially close vicinity of aerobic and anaer-obic methanotrophic communities of the order ofmillimeters at the Beggiatoa mats is remarkable andhas not been reported for the marine environment.This situation is likely an important component ofall marine methane seeps and mud volcanoes over-lain by an oxygenated water column. The additionalpresence of new and unusual biomarkers for aerobicmethanotrophy (4a-methyl steroid diols, D2-diplop-terol) at the Beggiatoa mats compared to the centrehabitat seems to indicate either different strains ofMethylococcales types or an energy stress adaptationof the dominant species. Furthermore, the novel bio-markers point to an undetected biosynthetic pathwayin aerobic methanotrophs.

Acknowledgements

We thank the officers and crew, the ROV-team ofVICTOR 6000, and the shipboard scientific partyfor excellent support during RV L’Atalante cruiseto the HMMV in 2001. Antje Boetius (MPI, Bre-men) is specifically thanked for cruise organizationand sediment handling. Daniel Birgel (RCOM, Bre-men) provided fruitful comments on an earlier ver-sion of the manuscript. Alex Bradley (MIT,Cambridge) provided helpful thoughts during thediscussion about unknown isoprenoidal archaealdiethers. Constructive criticism by Philippe Schaef-fer, Martin Blumenberg and an anonymous re-viewer is highly appreciated. The study was partof the programs MUMM I and II (MikrobielleUMsatzraten von Methan in gashydrathaltigen Sed-imenten, grants 03G0554A and 03G0608C) sup-ported by the Bundesministerium fur Bildung undForschung (BMBF, Germany) and the DeutscheForschungsgemeinschaft (DFG, Germany). Furthersupport was provided from the Max-Planck-Gesell-schaft (Germany) and the Research Centre OceanMargins (RCOM) at the University of Bremenfunded by the DFG. This is publication GEO-TECH-292 of the R&D program GEOTECHNOL-OGIEN, and RCOM publication No. 0533.

Associate Editor—P. Schaeffer

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