Pergamon

Geochimica et Cosmochimica Acta. Vol. 58, NO. 13. pp. 2853-2863, 1994 Copyright Q I994 Elsevier Science Ltd Printed in the USA. All rights reserved

00 I6-7037/94 $6.00 t .OO

0016-7037(94)00083-2

Carbon isotopic fractionation in lipids from methsnotrophic bacteria: Relevance for interpretation of the geochemical record of biomarkers

ROGER E. SC’MMONS,’ LINDA L. JAHNKE,’ and ZARKO ROKSANDIC’

‘Australian Geological Survey Organisation, GPO Box 378, Canberra, ACT, 260 1, Australia *Planetary Biology Branch, NASA Ames Research Center, Moffett Field, CA 94035, USA

(Received December 2 1, 1993: accepted in revised.forrn Febrz1ar.v 10. 1994)

Abstract-Ex~~ments with cultured aerobic methane oxidising bacteria confirm that their biomarker lipids will be significantly depleted in “C compared to the substrate.

The methanotrophic bacteria Methylococcus capsulates and Methylomonus methanica, grown on methane and using the RUMP cycle for carbon assimilation, show maximum 13C fractionation of ap- proximately 30%0 in the resultant biomass. In M. cupsulatus, the maximum fractionation is observed in the earliest part of the exponential growth stage and decreases to approximately 16%0 as cells approach stationa~ phase. This change may be associated with a shift from the particulate form to the soluble form of the methane monooxygenase enzyme. Less than maximum fractionation is observed when cells are grown with reduced methane availability. Biomass of M. capsukztus grown on methanol was depleted by 9k compared to the substrate.

Additional strong 13C fractionation takes place during polyisoprenoid biosynthesis in methanotrophs. The 613C values of individual hopanoid and steroid biomarkers produced by these organisms were as much as 10% more negative than total biomass. In individual cultures, squalene was ‘3C-enriched by as much as 14%0 compared to the triterpane skeleton of bacte~ohopaneamino~ntol. Much of the isotopic dispersion in lipid metabolites could be attributed to shifts in their relative abundances, combined with an overall reduction in fractionation during the growth cycle. In cells grown on methanol, where there was no apparent effect of growth stage on overall fractionation there were still significant isotopic differences between closely related lipids including a 5.3%0 difference between the hopane and 3P-methylhopane skeletons. Hopane and sterane polyisoprenoids were also 13C-depleted compared to fatty acids.

These observations have significant impli~tions for the interpretation of specific compound isotopic signatures now being measured for hydrocarbons and other lipids present in sediments and petroleum. in particular, biomarker lipids produced by a single organism do not necessarily have the Same carbon isotopic composition.

INTRODUCI’ION

COMPOUND SPECIFIC ISOTOPE analysis using direct gas chro- matography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is a new tool now being applied in the field of organic geochemistry (e.g., HAYES et al., 1987; FREEMAN et al., 1990: FREEMAN, 199 1). In contrast to the abundance of information on carbon isotope fractionations associated with photoautotrophy, much less info~ation exists regarding fmctionations taking place during bacterial growth on reduced Cr compounds and any further fractionations which may take place during bacterial biosynthesis of the lipid biomarkers ultimately preserved in the sedimentary record.

Bacterial synthesis and consumption of methane is an im- portant process in the present-day biogeochemical carbon cycle (WHITICAR, 1990; CICERONE and OREMLAND, 1988) and likely played a key role in the global cycle since its genesis. One of the tangible clues for methane involvement in the early carbon cycle comes from the recognition of anomalous 13C depletion in kerogens of Archean age (SCHOELL and WELLMER, 198 1: HAYES et al., 1983; HAYES, 1993a). Organic carbon samples from the Superior Province (Canada: approx 2.7 Ga) and the Fortescue Group (Australia; approx 2.7 Ga) have isotopic compositions in the range -40 to -52%0 PDB and co-occur with carbonates near O%O. A model for the de- velopment of these 13C-depleted kerogens (HAYES, 1983;

HAYES, 1993a) was based on the extraordinarily low 13C con- tent of biogenic methanes in combination with the known fractionation associated with methane oxidation. A conse- quence of applying this model to late Archean sediments is the necessity for an appropriate oxidant to be identified and the likelihood that this was molecular oxygen (HAYES, 1983). These considerations are significant in terms of timing the transition from an anoxic world to one in which aerobiosis operated.

While there is strong evidence for the existence of anaerobic methane oxidation in nature (e.g., REEBURGH, 1976; PAN- CANIBAN et al., 1979), the identity of the organism(s), the nature of the electron acceptors and the biochemical pathways have so far eluded clarification (WHITICAR and FABER, 1985). The microbiology and biochemistry of aerobic methane ox- idation are, on the other hand, relatively well-known. Of par- ticular significance in the aerobic processes is the capacity for incorporation of methane-derived carbon into cellular biomass and an associated carbon isotope effect leading to ‘3C-enrichment in the residual methane (ZYAKUN et al,, 1980, 1984; BARKER and FRITZ, 198 I: COLEMAN et al., 198 1). Also impo~ant, in the context of the present study, is the absence of knowledge about the likely magnitude of “C-depletion in the biomass of aerobic methanotrophic bacteria. The recent observation of significant ‘3C-depletion (-32--80%0 PDB) in specific biomarker lipids which, on chemical structure

2853

2854 R. E. Summons, L. L. Jahnke, and Z. Roksandic

grounds, are traceable to methanotrophic sources (HAYES et al., 1987; FREEMAN et al., 1990; FREEMAN, 1991; COLLISTER et al., 1992) prompted us to begin this study linking, in a carefully controlled way, chemical structure in methanotroph lipids with specific compound isotopic data.

2 SERINE c 2 PEP

12c02 I-Y ti 2dAA CELL

d----l id- t-_-.-J

The pathway for carbon in methane oxidation is shown I I c in sequence. The initial 2 GLYCINE - GLYOXYLATE - MALYL- CoA

CH4 + CHSOH -+ HCHO + HCOOH + CO2 (1)

step of hydroxylation of methane to methanol, unique to methanotrophs, is catalysed by the enzyme methane mono- oxygenase (MMO). A variety of bacteria, collectively referred to as methylotrophs, are capable of growth on C, compounds including methane, methanol, or formaldehyde. Methano- trophs (i.e., bacteria which use methane in addition to other Cl compounds) are subdivided into Type-I, -II, or X ac- cording to their pathways of carbon assimilation and other characteristics ( WHITTENBURY and DALTON, 198 1). Type I methanotrophs use the ribulose monophosphate (RUMP) pathway to assimilate carbon from the stage of formaldehyde in Fig. 1. Type II methanotrophs use the serine pathway (Fig. 2) and differ from the RUMP types in that only 50-7096 of

cell carbon is derived from formaldehyde, with the rest de- rived from COz (ANTHONY, 1982). Type X methanotrophs, exemplified by M. capsulatus, have “less complex” RUMP type biochemistry combined with capacity for slight (2.5%) assimilation of COz via ribulose bisphosphate carboxylase oxygenase (RUBISCO) (TAYLOR et al,, 1981). We chose M. capsulatus for use in this study because of its obligate use of C, compounds for both energy and carbon source and because this organism has the unique capacity to concurrently syn- thesise sterols and hopanoids (BIRD et al., 197 1 a,b).

I ACETYL-CoA

GLYOXYLATE - ISOCITRATE - CITRATE

i SUCCINATE --)

FIG. 2. Assimilation pathway of carbon from methane oxidation for Type II methanotrophs such as Methylobacterium organophilum and Methylosinus trichosporium.

The ecology of methanotrophs is an important factor when drawing analogies between laboratory-controlled experiments and the biogeochemical fossil record. Type 1 methanotrophs are the ones most commonly isolated from natural popula- tions and these generally have methane oxidation capacity associated with a membrane-bound enzyme otherwise known as particulate MM0 (PRIOR and DALTON, 1985). This is also reflected in this study in the choice of Methvlococcus cap- sulatus and Methylomonas methanica, both of which use the Type-l RUMP pathway, and in the conditions of their growth.

Many factors may influence the ultimate carbon isotopic fractionations when methane is oxidised and incorporated into methanotroph biomass. In addition to the metabolic pathway variations described above, two distinct types (par- ticulate and soluble) of the MM0 enzyme have been recog- nised (ANTHONY, 1986). Methane and oxygen concentrations may also affect the isotopic outcome. Extreme methane lim- itation would result in minimal fractionation, while pOz may affect the rate of oxidation. Temperature, growth stage, cell density, and natural capacity for use of multiple substrates are other factors which must also be considered.

The biosynthetic pathways leading to pentacyclic triter- penoids and sterols in methanotrophs will also influence the isotopic compositions of their lipids. Obligate methanotrophic bacteria do not readily incorporate exogenous substrates with C-C bonds, thereby excluding study by conventional methods such as feeding isotopically labelled acetate or other biosyn- thetic intermediates. Information has been derived through detailed structural characterisation of intermediates and as- sumed analogy to biosynthesis in eukaryotes (e.g., BIRD et al., 1971b; BOUVIER et al., 1976; OURISSON et al., 1987). Cell-free systems have been utilised to study the specificity of squalene cyclases of M. capsulatus (ROHMER et al., 1980). Information has also been gained through study of related organisms such as the purple non-sulfur bacteria Rhodop-

seudomonas palustris and R. acidophila, the facultative methylotroph Methylobacterium organophilum and Aceto- batter sp. (ZUNDEL and ROHMER, 1985b; FLESCH and ROH- MER, 1988). Notable points of departure from triterpenoid biosynthesis in eukaryotes occur in the biosynthesis of the hydroxymethylglutaryI-CoA (HMGCoA) precursor for the isopentenyl units used to construct squalene and in the weak substrate specificity of the squalene cyclase(s) (ROHMER et al., 1980; OURISSON et al., 1987). In Rhodopseudomonas palustris and Methylobacterium organophilum, acetate does not appear to be incorporated directly into HMGCoA and there is evidence for separation of different acetate pools (FLESCH and ROHMER, 1988). Cyclisation of squalene into diploptene and/or diplopterol is a characteristic of various bacteria and in M. capsufatus this capacity coexists with one.

3 H-6-P 3 F-6-P

ILK/ jxl

3 R-5-P PYRUVATE

1 PEP

1 CELL 1

ICARBON ( - 3-PGA

FIG. 1. Assimilation pathway of carbon from methane oxidation for RUMP Type I and Type X methanotrophs such as Methyfococcus capsulatus.

Isotope fractionation of C in lipids 2855

whereby squalene (3S)-epoxide leads to lanosterol, 4,4-di-

methyl-, and 4a-methyl-&cholesta-8( 14)-en-3/3-ol. Further,

in M. capsulatus, biosynthesis of diplopterol and bacterio- hopanepolyol is accompanied by synthesis of 3&methyl an- alogues as depicted in Fig. 3. The exact mechanism by which this occurs has not been elucidated although it is known that L-methionine is a source of the intact methyl group when

3@methyl hopanoids are biosynthesised by other prokaryotes (ZUNDEL and ROHMER, 1985a).

Several different biomarker structures have been linked to methanotroph sources. The occurrence of ring-A methylated

hopanoids in sediments and petroleum has been known for

some time, although the exact structural relationships between

the methyl hopanoids of extant methanotrophs and their fossil

counterparts was only recently determined (SUMMONS and JAHNKE, 1990, 1992). 3@Methylbacteriohopanes are known

from M. capsulates and Acetobacter sp. and may well occur in other bacteria which have not yet been studied for their

hopanoid content. The potential for isotopic analysis to dis- tinguish between various possible sources and to identify fossil hopanoids derived from methanotrophs was subsequently demonstrated when it was found that 3/3-methyl- 17cu,2 ID(H)-

hopane, and 3fi-methyl- 17@,2 1/3(H)-norhopane in the lacus- trine Green River oil shale were isotopically depleted (ap-

HOE&m: Hobo..- 8(14),24dien-3p-01 10 8(14)-en-3p-d 11

FIG. 3. Possible biosynthetic pathways to hopanols and to methyl sterols in At. cupsulatus. Information is derived from BOUVIER et al. (1976) and ZUNDEL and ROHMER (1985a,b).

proximately -90 and -86%0 PDB, respectively) compared

to the 13C contents of total organic carbon (-27 to -33%0

PDB) and to other triterpenoids in the same sample (COL-

LISTER et al., 1992). The extreme 13C depletion of the Green River 3@-methylhopanes implicates a source from organisms utilising methane since it is the only natural carbon substrate

commonly found with such depletion.

The unusual nature of the aminopentahydroxy side-chain

of other bacteriohopanoids known to be abundant in certain

methanotrophs (ROHMER et al., 1984; ZUNDEL and ROHMER, 1985~) has also been noted. Cleavage of the oxygenated side-

chain during early diagenesis leads to facile formation of nor-

hopanes in sediments. This distinctive feature has also enabled

their recognition through a combination of structural and isotopic features~(FREEMAN et al., 1990; FREEMAN, 1991).

In this paper, we report the isotopic fractionation associated

with methane oxidation and assimilation into biomass for the RUMP pathway methanotrophs M. cupsdatus and Methylomonas methanica. We have measured the effects of substrate limitation and growth phase on the carbon isotopic composition of biomass and lipids. Intermolecular isotope

fractionations in individual lipids were determined so that

the range of isotopic signatures for methanotroph biomarkers isolated from sediments could be established.

MATERIALS AND METHODS

Growth of methanotrophs and control of methane and oxygen concentrations has been described previously by JAHNKE and NICHOLS ( 1986). Gas proportions were controlled using a rotometer with NZ as diluent. Dissolved methane concentrations [CH4 (as)] resulting from different gas proportions (47% (v/v) = 420 PM and 5% (v/v) = 50 PM) were measured by CC on a Poropak Q column before the inoculation of medium. An open system, where the growth medium was bubbled continuously with a calibrated gas mixture, was employed in order to minimise “closed system” effects and sim- plify measurement of isotope discriminations. Early exponentially grown cells (O.D. 0.22) contained a proportion of biomass (5-9%) from the starter culture (-57.9%0), although we have not corrected for this in our calculations of isotopic fractionation.

Bulk lipids were isolated from lyophilised cells by BLIGH and DYER (1959) extraction as modified by KATES (1975). Bacteriohopane- polyols were separated from neutral lipids by acetone precipitation (SUMMONS and JAHNKE, 1992) and the hopanol derivatives were prepared by periodate oxidation-NaBHe reduction (ROHMER et al., 1984: JAHNKE et al., 1992). Methylsterols, diplopterols. and the hy- drocarbons were isolated by preparative thin-layer chromatography (JAHNKE and NICHOLS, 1986: SUMMONS and JAHNKE, 1992).

Isotopic analysis of biomass, total lipid, Bligh-Dyer residue, and substrate methanol were determined in duplicate or triplicate by combustion in evacuated, sealed vycor tubes at 850°C for 3 h, using CuO as oxidant. In some early experiments, biomass and lipid samples were prepared with oxygen as oxidant, using a VG Isoprep apparatus directly coupled to the mass spectrometer. Isotopic measurements were made using VG SIRA 12 and Finnigan 252 mass spectrometers. Methane from two sources (6i3C = -34.4 and -30.2%0 PDB) was used as substrate and was combusted over CuO in evacuated quartz tubes at 850°C for 2.5 h. The CO2 from these combustions was dried and d”C analysis carried out at NASA using a Nuchde 6-60RMS mass spectrometer modified for small sample analysis (HAYES et al., 1977). Similar results for the substrate methane were obtained at AGSO. The sd for all these measurements varied between 1.0 and 0.1%0 and was generally 0.2%0 or less. In some cases we used the above sealed-tube combustion procedure to determine the isotopic compositions of the total sterol fraction and the total hopanols derived by oxidation of the bacteriohopanepolyol. In order to be sure that none of the material in the sterol fraction or the hopanol fraction

2856 R. E. Summons, L. L. Jahnke, and Z. Roksandic

TABLE I. Details oforganisms, growth conditions, substrates, and the carbon isotopic fmct~onation in biomass, buik lipid, and solvent-extracted residues.

CONDITIONS DENSilY SCURCE RESlWE

420nm mg.l-1 6 E E e

M. capsulatus 5’

6

7

8

9

IO

11

12’

13

14’

15

16

17

2

3

4

M. m&ha&a 18

M. trichosporium I9

M. organophilum 20

47% CH4 & 10% 02

*

5% CH4

5% CH4

1.2 mgli Cu 47% CH4 1.2 mgil Cu 47% CH4 0.5% CH30H

0.5% CHBOH

0.5% 02 47% CH4 1 .O% 02 47% CH4 2.7% 02 47% CH4 2.7% 02 47% CH4 10% 02 47% CH4 10% 02 47% CH4 0.5% CHJOH

0.22 23

0.35 35

0.4 37

0.58 72

0.95 126

1.11 177

1.46 244

0.23 34

0.97 727

0.26 37

1.3 160

0.79 102

1.69 131

0.7 a9

0.62 80

1.05 144

1.4 214

0.68 87

1.05 126

-34.2 31.3 29.1 nd

-34.2 28.9 23.2 nd

-34.2 24.4 24.0 nd

-34.2 19.5 21.5 23.2

-34.2 17.9 $7.8 19.4

-34.2 17.6 18.8 20.5

-30.4 15.8 nd 18.2

-34.2 14.5 17.2 nd

-34.2 3.8 4.2 72

-34.2 28.4 30.4 nd

-34.2 27,6 nd 27.8

-50.0 9.4 9.8 13.7

-50.0 9.7 9.2 t2.9

-30.4 19.6 nd 22.2

-34.2 19.3 nd 21.6

-34.2 18.6 nd 20 5

-30.4 18.2 nd 20.4

-34.2 29.3 nd 35.3

-34.2 9.9 nd 20.0

-50.0 3.9 5.5 nd

’ these data may be slightly affected by contribution from 5% innocutum at -57.9 per mil.

was being overlooked, the a13C values of these fractions were compared to the weighted average isotopic compositions of the individual sterols or hopanols as measured by the compound-specific isotopic tech- niques (see below). Where amounts of material were adequate to allow such comparisons, the results agree to within 0.5%0.

GC-C-IRMS analysis was conducted using a Finnigan MAT 252 mass spectrameter equipped with a Varian 1400 Gas Chromat~ph (GC) and CuO/Pt microvoiume combustion furnace interface op- emted at 850°C (FREEMAN, 199 1). Using a Septum Programmable Injector (SPI) programmed from 100-3OO’C at 20”C/min, the sam- ples were injected onto a 25 m X 0.25 mm J&W DB-5 or Chrompak CP Sil-SCB fused silica column using helium as carrier at 25 psi and programmed from SO-300°C at rates of 3,4, 10, or ZO”C/min, de- pending on the compound cfass being analysed. Fatty acids were anal@ as FAME derivatives prepared by mild alkaline methanolysis (GUCKERT et al., 1985) and their isotopic compositions were corrected for the effects of an additional carbon atom from the CH,OH (6 13C = -50,3460) derivatising reagent. Hydrocarbons, sterols, diplopterols, and the hopanols obtained by HjIOs oxidation and NaBN, reduction of bacte~ohopane~lyo~ (ROHMER et al., 1984) were underivatised. Primary ~n~di~tion was made using aiiquots of a standard COz gas introduced via one side of the dual inlet system at the start and finish of the ch~mato~phic run. In some cases, de&a vitlues were corrected by the deviation from the known 6°C values of coinjected

samples of C2dr C30, and C& perdeuterio n-alkanes. Standard deviation values for replicate injections varied from 0. I-0.6L and were gen- erally less than 0.3k. Identification of individual methanotroph lipids was made using a VG 70E mass spectrometer as described by SIJM- MONS and JAHNKE ( 1990, 1992).

RESULTS AND DISCUSSION

We have adopted the approach advocated by HAYES ( 1993b) and expressed the carbon isotopic fractionation for the process of conversion of methane (and methanols to bio- mass using the epsilon (6) notation

E = ((Y*,R ~- I) x 103 iZ)

where

a,&/a = RA/'RB = (loo0 + 6,)/(tooo -t &). (31

a is the f~ctionation factor, dA is the 613C of the substrate (methane or methanol), and & is that ofthe product in ques- tion. For comparison of the isotopic compositions of biomass

Isotope. fractionation of C in lipids 2857

Exponential phase ‘II,

‘0

Stationav phase

100 0 100

Cell density (mg 1-l ) 200

16-3,437

FIG. 4. Carbon isotopic fractionation in biomass from M. capsularus as a function of the change from exponential to stationary phase growth.

and various individual lipids we also present results as 6°C

PDB for the materials which were analysed. Table 1 summarises the calculated fractionation (6) in ex-

periments where Methylococcus capsulatus (Bath), Methy- lomonas methanica, Methylobacterium organophilum, and

Methylosinus trichosporium were grown on methane or methanol under a variety of conditions. The data include total biomass, the total lipid fraction and, in some cases, the

cell residue after removal of lipids. In M. capsulatus, growth on methanol resulted in considerably less fractionation than growth on methane. This suggests (but certainly does not prove) that it is the initial oxidation step of methane to meth-

anol, as opposed to the methanol to formaldehyde step, which determines the ultimate fractionation in methane oxidation.

Primary Controls on the Isotopic Composition of Methanotroph Biomass

Initially, it was intended that cells be harvested at specific times after innoculation. However, we found that there was sometimes a significant delay before active growth began. This made incubation time an uncertain parameter, especially in short-term experiments. We therefore recorded the optical density (O.D.) of the harvest medium as an index of cell density and hence, growth stage. Highest carbon isotopic fractionation of 3 1.3%0 was measured at the lowest cell den- sities, that is, during early exponential growth. As shown in Fig. 4, fractionation declined systematically with cell density and the age of the culture and approached 16%0 in cells grown for 5 days to densities of approximately 450 mg/L. Two pos- sible causes for the reduction in carbon isotopic fractionation are a diffusion-controlled limitation to the methane supply at high cell densities and/or growth rates or changes in the nature, proportion, and activities of the oxidation enzyme(s) as growth moves from exponential to stationary phase. Cor- relations between fractionation and observed MM0 type in this work suggest that the particulate and soluble forms likely have different isotope effects and that a switch from particulate to soluble MM0 results in the measured decrease in 13C frac- tionation. Details follow.

Cells grown on a reduced supply of methane (5% CH4 in

N2 vs. 47% CH4 in Nz) recorded the expected lowering of

isotopic fractionation at both low and high cell densities. In

any process involving isotopic discrimination, zero fraction- ation will be approached as all available substrate is con- sumed. During exponential growth, the respective isotopic

fractionations for 5% and 47% methane-grown cells were 14.5

and 3 1.3%0 (Table 1). In this respect, the data for cells grown on 5% methane show an isotopic effect consistent with meth-

ane-limitation. However, it should be noted that both meth- ane concentrations resulted in similar growth rates and, in

this regard, the 5% methane-grown cells were not “methane-

limited.”

We used two methods to determine if there were changes in the nature of MM0 enzymes during growth. Soluble MM0

may be detected calorimetrically via its ability to oxidise

naphthalene to naphthol (BRUSSEAU et al., 199 1) and by the

presence on SDS-polyacrylamide gel electrophoresis (SDS-

PAGE) of three major bands corresponding to fraction A (WOODLAND and DALTON, 1984). Gel electrophoresis was

unable to detect the soluble MM0 subunits in early expo- nential cells (prior to 0.5 O.D.), but these distinct protein

bands were present in cells harvested at 0.7 O.D. and throughout stationary phase (L. L. Jahnke and K. Zahiralis, unpubl. data). The calorimetric assay, however, did indicate the potential for some soluble MM0 type activity in earlier exponential cells.

Cells grown on a medium supplemented with copper sul- fate showed close to maximum fractionation even when har- vested at a high cell density ( 160 mg/L). Copper is known to be a potent inhibitor of the soluble form of MM0 (GREEN

et al., 1985; PRIOR and DALTON, 1985). Growth in this me- dium should reflect predominant or exclusive oxidation via

the particulate form of MMO. Both the naphthalene oxida- tion test and the SDS-PAGE analysis of the Cu-supplemented cells confirmed the absence of soluble MM0 in both expo- nential and stationary phase cultures and the carbon isotopic discriminations evident in their biomass were essentially identical at 28.4 and 27.6%0, respectively. Diffusion limitation

of methane supply was not evident in the carbon isotopic composition of stationary phase, Cu-supplemented cells. Thus, the combination of results for Cu-supplemented and cells grown on a low concentration of methane, suggests that reduction in the activity of membrane bound, particulate MM0 is accompanied by a decrease in the carbon isotopic fractionation in the oxidation of methane to methanol. Fur-

ther experiments are planned to pursue this question since the SDS-PAGE results only give information about the in- creasing content of soluble MM0 as cells approach stationary phase. We do not have independent information about the relative activities of the particulate and soluble MM0 forms during growth.

Methylomonas methanica is a RUMP cycle methanotroph in which the MM0 activity has been associated exclusively with the particulate form of the enzyme (COLBY et al., 1975). In an experiment where this organism was grown in a com- parable way to M. capsulatus the 13C discrimination was 29.3%0 and close to the maximum fractionation observed for M. capsulatus. This experiment provides some further evi-

2858 R. E. Summons, L. L. Jahnke, and Z. Roksandic

dence for larger fractionations approaching 300/M being as- sociated with the expression of particulate MMO.

Isotopic composition qf methanotroph biomass and the @ect qf oxygen levels

Celis grown at 10, 1, and 0.5% 02 showed little difference in ‘3C-fractionation when harvested at comparable ceil den- sities in the range 70-90 mg/L. This suggests that pOz per se is not a major factor in control ofthe fractionation associated with methane oxidation. The minor increase in fractionation that was observed at reduced oxygen could result from in- direct effects of& on cell growth rates.

Isotopic jiactionation associated wlith growth on methanol

In two experiments we measured fractionations near 9.5%0 when M. cupsulatus was grown on methanol. This was not significantly affected by the cell density at time of harvest suggesting that methanol supply was nonlimiting, an obser- vation also supported by the fact that growth on methanol, during the exponential phase, was 4.5 times as fast as growth on methane. We did not measure the final methanol con- centration in the growth medium but a calculation of carbon supplied vs. carbon assimilated into biomass indicated that. even at the highest cell density, cellular carbon accounted for only 7% of that available. While methanol is not likely to be as important as methane as a carbon source in natural en- vironments, the experiment did suggest that the first oxidation step, methane-to-methanol, was controlling carbon isotopic fractionation in methanotrophy. However, the situation here is potentially complex. It has been demonstrated that MM0 can oxidise a number of substrates including methanol (COLBY et ai., 1977). Particulate MM0 is also a constituitive enzyme being present in methanol-mown cells {PRIOR and DALTON, 1985). A complete unde~tanding of this fraction- ation step probably requires studies using isotated enzymes.

M. orgunophilum, a Type II methylotroph, grown on methanol, also showed a low fractionation (3.9%0).

Isotopic fractionation associated with lipid biosynthesis

In all experiments where the isotopic composition of a total lipid was determined, we observed a 13C-depletion rel- ative to the total biomass. This accords with the general frac- tionation associated with acetate formation from pyruvate (DENIRO and EPSTEW, 1977; MONSON and HAYES, 1982a) and the subsequent conversion of this acetate into lipid. The 2-4s depletion of lipid-C compared to biomass-C in :V. capszdatus and M. methanica is of the same order as found in other heterotrophic bacterial metabolism (e.g., BLAIR et al., 1985). The overall yield of extractable lipid from M. cap- sulatzts was 8% in exponential phase cells rising to 10% in stationary phase. In M. methanica and IV. organophilum, the lipid contents were 4.7 and 6.7%, respectively. The isotopic differences between biomass and the extracted residue were generally small and were not consistently either heavier or lighter. No attempt was made to isolate and analyse water soluble components of the biomass.

We measured the isotopic com~sitions of some fatty acids in the rM. cupsz~~aius experiments. Under our growth con-

ditions, the predominant fatty acids were (‘lh(,, a mixture ot four Clh:, isomers and lower amounts of CIA-C, and C, : (I (JAHNKE and NICHOLS, 1986; JA~IYKI:. 1992). It was not possible to measure all compounds individually due to in. adequate CC resolution or low relative compound abun- dances, However, we could determine C,,,,,, the composite of monounsaturates, and a composite delta value for all Ct,, fatty acids. In all cases which were tested, the delta value of the Ci6 composite (>90% of fatty acids) was within 0.5%~ of the delta value for the crude lipid fraction from which it was derived. The envelope of Ctb rnono-uIl~tLir~tes was I .S-2.0% heavier than Clb.o. We also observed that the composite delta value for Cl6 fatty acids was S-6%0 heavier than the composite delta value for sterols and the hopanoid skeleton of the bat- teriohopanepolyols. In Experiment #8, ti>r example, the composite ofC16 fatty acids, sterols, and hopanols were --S&S. -64.5, and --62.4%0 respectively. In Experiment k10, the respective values were -53.2, -58.5, and 58.6%,1. Our find- ing of significant 13C-enrichment in hl. tupsrdatus fktty acids as compared to polyisoprenoids is unusual with only one other example, the purple photosynthetic bacterium t’hw- ~~at~z~rn ~lin~~,~lfrn (WONG et al.. 1975) in the collection of empirical data brought to our notice by HA\ ES ( 1993b).

Independent studies of acetate isotopy (D~NJR~ and EP STEIN, 1977: MONSON and HAYES, 1982a.b) in II. t~jii and in yeast grown on various substrates suggested that the iso- topic “lightness” of lipid arose via a kinetic isotope eff‘ect in the action of the enzyme pyruvate dehydrogenase. This h! I pothesis for the “C-depletion in the carboxyl carbon ofacetyl- CoA, provided for a characteristic which could then be trans- ferred to acetate-derived products. By following and extending this logic, fatty acids, which have 1: 1 methyl-C:carboxvl-C’. compared to polyi~prenoids with 3:2 methyl-Ccarboxyl-C, should be pro~~ionately more “C-depleted (HAsfcs. 1993b).

Several uncertainties which are recognised and discussed by BLAIR et al. ( 1985) and HAYES ( 1993b) conspire to prevent an immediate resolution of this problem. in some experi- ments with E. coli grown on glucose, acetate carboxyl-C was observed to be “C-enriched compared to the carbon source (BLAIR et al., 1985). These authors noted the interpretational difficulties arising upon metabolism of different carbon sources and concluded that pyruvate dchydrogcnase wa\ probably not the sole control on fatty acid isotopic compo- sition. More recently, FLESCH and ROHMER (1988) have oh- served a probable compa~mentalisation in acetate metabo- lism during isotopic labelling experiments with the purpie non-suf fur bacteria Rhod~jp.setid(~mnnu.r i~uj~i,stri~s and Ellttt- d(?p,se~dorn~~nu.s u~~dop~i~a and in the methylotroph :\I. (Ji-

gan~)ph~~z{m. Their work revealed that acetate from two dis- tinct pools was incorporated into the isoprene moieties ot’ bacterial hopanoids. If multiple acetate pools arc a common feature in prokaryotes, they will have isotopic consequences and may explain the observations of MONWN and HAYE:S ( 1982b) and BLAIR et al. (1985), whereby the “C-enrichment pattern of acetate is reversed in different circumstances. It also seems that there may be presently unrecognised biosyn- thetic fractionations in bacterial lipid biosynthesis down- stream from acetate (BLAIR et al., 1985; HAYES, 1993b). Clearly, this is an issue where the CC-IRMS $001 can be ap- plied systematically to a range of organisms and trophic states,

Isotope fractionation of C in lipids 2859

TABLE 2. Summary of carbon isotopic compositions of biomass, lipid, and individual lipid biomarkers ofA4ethylococcus cupsztkzfus grown on methane and methanol.

EXPT# BIOMASS LIPID SqualeneDip'tene Dip'terol Hopanol3-MeHop Dimethyl sterol Methyl sterol 16:l FA 16:0 FA diene ene diene one corr. corr.

5 -63.5

6 -61.3

7

a

9

10

11

12

13

14

15

16

17

1

2

3

4

18

19

20

-57.2

-52.7

-51.2

-50.9

-45.5

-48.0

-37.9

-60.9

-60.1

-58.8

-59.1

-49.0

-52.5

-51.8

-47.7

-61.7

-43.7

-53.7

nd -62.6 -63.6 -68.8 -61.6 -68.3

nd -59.1 -61.6 -62.4 -68.2 -63.3 -65.0

nd -55.5 -56.9 -56.9 -66.5 -60.9 -59.0

-56.1 -54.1 -56.2 -58.4 -66.4 -58.3 -62.1

-52.6 -50.6 -54.3 -66.7 -56.0 -57.0

-53.6 -51.8 -53.9 -66.1 -56.1

-47.7

nd -52.1

-41.1 -45.3 -46.8 -45.0

nd -67.5 -68.7 -63.0 -68.5

-60.3 -57.1 -63.0 -66.0

-62.8 -62.0 -64.8

-62.1 -62.5 -64.0 -66.3 -61.0

-51.5

-54.6 -55.9

-53.6

-49.8

-67.1 -70.1

-53.1

nd -52.0 -54.5

-69.5 -69.1 -67.6 -62.8 -65.4

-67.8 -69.0 -69.9 -62.4 -63.7

-62.8 -65.2 -66.8

-66.5 -56.2 -57.6

-61.0 -51.7 -53.7

-55.6 -61.4 -52.6 -54.1

-47.5 -40.2 -42.1

-69.9 -70.5 -71.1

-67.3 -69.0

-64.9 -65.9 -61.0 -62.7

Biosynthesis of polycyclic triterpenoids

Since our primary purpose was to measure the isotopic compositions of lipids which were simultaneously character- istic of methanotrophs and which had potential for preser- vation in sediments, we made a detailed analysis of the iso- topic compositions of the triterpenoid squalene and its cy-

clisation products. In interpretations of 613C data for fossil hydrocarbons it has sometimes been assumed that metabolites along the same biosynthetic sequence in the same organism

will have similar carbon isotopic signatures. HAYES (1993b) has, however, drawn our attention to the potential for frac- tionations within lipid biosynthetic networks and the paucity of detailed study of this topic.

In our study of M. capsulatus, we were able to compare both oxygen-independent synthesis of hopanoids and oxygen- dependent synthesis of steroids. A common pattern emerged where each product was 13C-depleted with respect to its pre- sumed precursor. Complete data for the individual triterpen- oids we measured are shown in Table 2. As noted above,

Experiment #5 contained approximately 9% biomass (613C

-58.2%0) from the starter innoculum and hence, the isotopic compositions of individual triterpenoids in these experiments

may be anomalous. For example, sterol (1 1), and 3&meth- ylhopan-29-01, the preferred terminal products in their re- spective sequences are anomalously heavy at -67.6%0 and

-6 1.6%0 in this experiment. In observing the trends in Table 2, it can be seen that the biosynthetic sequence in order from squalene through diploptene, diplopterol, and hopanol-29- 01 is accompanied by progressive ‘3C-depletion. Readers should note that hopanamino-29-01 obtained by H,IO,/ NaBH, treatment of bacteriohopanepentol represents the in- tact thirty carbon atoms of the precursor squalene (ZUNDEL and ROHMER, 1985~). The isotopic consequences of carbon travelling through a reaction network have been elaborated (MONSON and HAYES, 1982a; BLAIR et al., 1985; HAYES, 1993b) and our finding of a progressive isotopic depletion is consistent with all-positive fractionation factors within the hopanoid biosynthetic sequence and is as expected. However it is the magnitude of the fractionations, that is up to 14%0

2860 R. E. Summons, L. L. Jahnke, and Z. Roksandic

difference between squalene and product hopan-29-01, which

is unexpected.

Isotopic fractionation associated with sterol biosynthesis

A pattern of progressive depletion also appeared for squa-

lene (1) 4,4-dimethyl-5a-cholest-8( 14)-en-3@-ol (9) and 4a- methyl-5a-cholest-8( 14)-en-3@-ol (11). In stationary phase cells there was an overall 9.6%0 difference between precursor squalene and the predominant endproduct (11) and a 6%0 difference between the two predominant sterols (9) and ( 1 1). If these differences were due solely to fractionations taking place during sterol biosynthesis it would require unprece- dented 13C discriminations for oxidative removal of methyl carbons at C-4 and C- I4 of lanosterol. If, however, the relative abundance of sterols shifted during cell growth, the isotopic separations could arise from the reduction in overall isotope discrimination which accompanies the change from expo- nential to stationary growth. Figure 5 shows the distribution of sterols and their isotopic compositions at different cell densities. In early exponential growth, the sterols are pre-

dominantly monomethyl and their 613C values range over only 2%0. In stationary phase, dimethyl sterol accounts for 456 and the predominant proportion being more recently

a. 00 0.22

!

L____-- LL--_-_

d. 00 1.11

'Ooo Scan number 1200

synthesised is approximately 6%0 more “C-enriched than monomethyl sterol. While a detailed mass balance has not been carried out, we observe that precursor sterol dienes are mostly heavier than their product monoenes.

In the experiments with Cu-enriched culture medium (#sl4 and 15) and for growth on methanol (#sl6 and 17) there was only a minor change in the overa fractionation between exponential and stationary phase cells (Table 1). The isotopic compositions of the sterols in these experiments (e.g.. Fig. 5g) were much closer to each other, spanning < 2.5%0 in #I4 and 1 %O in # 17, and showed progressive “C-depletion fol- lowing the biosynthetic sequence. 4,4-Dimethylcholest-8( 14)- en-3P-ol(9) was unusual in being the preferred sterol in cells grown to stationary phase in the copper-enrichment experi- ment. These data suggest that fractionations associated with interconversions of the intact carbon skeletons in sterol bio- synthesis are small compared to the reduction in overall car- bon isotopic fractionation which accompanies the growth cy- cle. The growth-related reduction combined with shifts in the balance of triterpenoid metabolites act in concert to give a wide dispersion of individual isotopic compositions under some circumstances. The combined effects of compound balance and overall fractionation are demonstrated in Figs. 5 and 6.

e. 00 0.23 50@l CH,

-57.6

f. OD 0.97 50/M CH,

-47 5

g. DO 0.26 I-71.0

1.2 mg 1-l CU++ /

d -69.9

-66.5

ji __- ~__.. ~~...'

-67.3

1000 Scan number '*O" 1400

,6-3143R

FIG. 5. Changes in the relative abundance and isotopic compositions of sterols of M. capsulatus during growth (a- d) and in response to different growth factors (e-h).

Isotope fractionation of C in lipids 2861

100

50

0

-50

-100

n methvlhooane% , I

q hopane%

0.2 0.3 0.4 0.6 0.9 1.1

Growth stage (O.D.)

FIG. 6. Changes in the proportions and isotopic compositions of the hopane skeletons of bacteriohopanepolyols during growth of M. capsulates. The data were obtained by CSIA analysis of the hopane- 29-01s prepared by cleavage of the polyhydroxy side-chain using per- iodate oxidation-NaBH4 reduction.

Isotopkfiactionation associated with hopanoid biosynthesis

Bacteriohopanepolyols with additional alkylation in

ring-A have restricted distributions in extant bacteria and cyanobacteria (ROHMER and OURISSON, 1976; ROHMER et

al., 1984; BISSERET et al., 1985). Presently known sources of 3@-methyl hopanols are M. capsulatus and Acetohacter pus- teurianus ssp pasteurianus, and details of their biosynthetic origins within the methanotroph have proven elusive (ZUN- DEL and ROHMER, 1985b). In our carbon isotopic fraction-

ation experiments, co-occurring hopan-29-01 and 3&meth- ylhopan-29-01 showed carbon isotopic differences as high as 10%0 in stationary phase cells. These compounds are derived by H5106 oxidation and removal of the ribose-derived side chain moiety from the corresponding bacteriohopaneami- nopentol. Except for 3@-methyl carbon from methionine, they are thought to contain equivalent C30 skeletons and originate from a common precursor squalene pool. Experiments to

elucidate details of their biosynthesis have been conducted using Acetohacter sp. (ZUNDEL and ROHMER, 198513).

Examination of the relative proportions of the two hopan-

29-01s during growth (Fig. 6) shows that bacteriohopane- aminopentol is more abundant in exponential phase and 3/3-methyl bacteriohopaneaminopentol accumulates prefer- entially in stationary phase cells. Because overall isotope fractionation decreases during the growth cycle, hopanoid biosynthesised in the later stages of growth would be from acetate which is ‘3C-enriched compared to acetate produced by exponential cells. Thus, reduction in ‘3C-discrimination

during growth, combined with a progressive shift from one metabolite to the other, could explain their 10%0 separation in cells harvested after long-term stationary phase growth. However, the large isotopic differences between hopanol and 3@-methylhopan-29-01 in Experiments #14 (with Cu-enrich- ment) and 17 (growth on methanol) suggests that this may not be the complete explanation. In Cu-supplemented cells

(except # 14) there was no reduction in fractionation and yet

there was still a 5.7%0 separation between the two metabolites.

Similarly, there is no significant difference between short- term and long-term fractionation in cells grown on methanol and yet the two hopanoids differ by 5.3%0 in the long-term

experiment (#17). Finally, in exponential phase cells grown on low-Cu medium, and exhibiting maximum fractionation

(Experiment #5), there was a 7.2%0 separation between the metabolites. It is unlikely that the single carbon atom which

distinguishes them could be the source of a difference of this

magnitude. However, the data could be explained if the tri- terpenoids were derived from spatially separated squalene

pools of different isotopic compositions. In the absence of

evidence to the contrary, it has generally been assumed that a homogeneous squalene pool provides the source for all the

C30polyisoprenoids in M. capsulatus.

Isotopic,fkctionation in Methylosinus trichosporium

M. trichosporium is a Type II methanotroph and, within this group, approximately 50-70s of cell carbon is derived

from methane oxidation with the remainder originating from COz. In one experiment with this organism grown on CH,

(-34.2%0) and CO2 (-40.6%0), the resultant biomass was -43.7%0 and had a lipid fraction with 613C -53.1%0. Frac-

tionations in both methane oxidation and COz assimilation are evidently much lower than other methane-oxidising or- ganisms. The large (10%0) separation between biomass and the lipid subfraction suggests that the lipid may be derived

exclusively from only one of the two possible sources of car- bon. If further experiments prove this to be the case, then lipid biomarkers from Type 1 and Type II methanotrophs may show different isotopic patterns.

SUMMARY

Carbon isotopic fractionation of M. capsulutus grown in

culture varies according to the stage of growth. This appears to be a consequence of changes in the nature and/or activity of different forms of the methane monooxygenase enzyme. Maximum isotopic fractionation of approximately 30%0 is observed in methanotrophs utilising exclusively membrane- bound MM0 such as Methylomonas methanica, Methylo- coccz~ cupsulutus grown on a Cu-supplemented medium and Methylococcus capsulatus in the very early stages of expo- nential growth. Growth on methanol was accompanied by much reduced fractionation. An additional isotopic fraction- ation of 2-4%0 is observed in the total lipid fraction of meth- anotrophs and individual metabolites were depleted by as much as IO%0 compared to whole biomass.

Large dispersions, as high as 14%0, were measured in the isotopic composition of intermediates in the biosynthetic pathways to bacteriohopaneaminopentol and methyl sterol under some circumstances. Some of this dispersion is linked to changes in the overall fractionation in methane oxidation and to the relative abundance of lipids during the growth cycle. In an experiment with Cu-supplemented medium, where fractionation did not change significantly during growth, isotopic compositions of metabolites along the sterol biosynthetic network still showed a progressive ‘3C-depletion leading to a 2.6%0 difference between methyl and dimethyl

2862 R. E. Summons, L. L. Jahnke. and Z. Roksandic

sterols. In this experiment there was also a 5%0 difference between the hopane and 3@-methylhopane skeletons.

In M. cupsufutus, the n-alkyl fatty acids are generally 13C- enriched compared to polyisoprenoids. This is contrary to the theoretical “C-depletion resulting from biosynthesis from different proportions of acetate methyl-C and acetate car- boxy1C when the carboxyl-C is depleted compared to methyl- C. The observation indicates that there must be fundamental distinctions between the way organisms such as A4 cupsuiatus

and more conventional counterparts construct lipids and that there may be unrecognised sources of discrimination against 13C in bacterial biosynthesis of polyisoprenoids.

The availability of the GC-IRMS tool for compound-spe- cific isotope analysis (CSIA) will give information about bio- synthetic pathways which are difficult to study by other methods.

Our results indicate that care must be exercised in the in- terpretation of compound-specific isotope data for sedimen- tary lipids. Biomarker lipids from the same organism may show significantly different isotopic compositions if the iso- topic composition of substrate, isotopic fractionation during assimilation, and the relative abundances of lipids changes during the growth cycle. Conversely, significantly different isotopic compositions in biomarker lipids may not necessarily

rule out a common source organism.

No& Added in Prouj ROHMER et al. (1993) have recently reported that Eubacteria biosynthesise the C5 framework of polyisoprenoids via a pathway which is fundamentally different than the Bloch-Lynen route. The isotopic consequences of this new finding are not known but will certainly impinge on the interpretation of the results reported here. Reference: ROHMER M., KNANI M., SIMONIN P., SUTTER B.. and SAHM H. (1993) lsoprenoid biosynthesis in bacteria: A novel pathway for the early steps leading to isopentenyl diphosphate. Biochem. J. 295, 5 17-524.

.3~~knoMfledgm~~r.s-John Hayes, Dawn Merritt, Margaret Ricci. and Martin Schoell provided invaluable advice and assistance concerning the operation and standardisation of our GC-combustion-isotope ratio mass spectrometer. Neil Blair, John Hayes, Andrew Murray, Chris Boreham, Peter Nichols, Brian Popp, and an anonymous GCA re- viewer assisted greatly with comments on earlier versions of this manuscript. We are indebted to David Des Marais, Lesley Dowling, Zoltan Horvath, Algis Juodvalkis, Eleanor Laing, Julian Olley, Anne Tharpe, and Karen Zahiralis for help with isotopic measurements and other technical assistance during the course of the project. This work was partially supported by a grant to L.L.J. from the NASA Exobiology program. R.E.S. and Z.R. publish with permission from the Director. AGSO.

Ediroriul handling: G. Fame

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