Recognition of the Sources of Isoprenoid
Alkanes in Recent Environments
M. M. QUIRK*, R. L. PATIENCE*, J. R. MAXWELL*
and R. E. WHEATLEY**
* Organic Geochemistry Unit, School of Chemistry, University of
Bristol, Bristol BS8 ITS, England
**Department of Microbiology, Macaulay Institute for Soil
Research, Aberdeen AB9 2 Qj, Scotland

ABSTRACT
Alkanes are ubiquitous components of contemporary aquatic and t e r r e s t r i a l environments and derive from natural and/or p o l l u t a n t sources. In many cases, a
detailed consideration of the structure and stereochemistry of individual components is necessary to distinguish the sources of these components. High
resolution gas chromatography and computerised gas chromatography-mass spectrometry allow determination of the s t r u c t u r e s , including stereochemistry, of
certain w i d e l y - d i s t r i b u t e d alkanes in recent sediments. Three examples of such
compounds have been chosen to i l l u s t r a t e the p r i n c i p l e s by which these s t r u c t u r a l determinations permit assignment to p a r t i c u l a r sources: ( i ) pristane
(2,6,10,14-tetramethylpentadecane) from a lacustrine sediment is shown to
derive from a mixed source ( b i o l o g i c a l and p o l l u t a n t ) ; ( i i ) 17ßH-hopane in
the same sediment appears to derive from reduction of hop-22(29)-ene, an abundant alkene of Microcystis aeruginosa, present in the lake; ( i i i ) 17a(H)homohopane in a sphagnum peat arises mainly as a r e s u l t of bacterial decay of
Sphagnum cuspidatum.
Keywords: p r i s t a n e , 173(H)-hopane, 17a(H)-homohopane, Microcystis aeruginosa,
Sphagnum cuspidatum, peat, sediments.
INTRODUCTION
Alkanes occur widely in recent sediments as complex mixtures readily amenable
to analysis by c a p i l l a r y gas chromatography (GLC) and computerised gas chromatography-mass spectrometry (C-GC-MS) ( e . g . Giger and Schaffner, 1978;
Thompson and E g l i n t o n , 1978). These compounds can derive from a number of
sources, which include a contribution from l i v i n g organisms ( e i t h e r d i r e c t l y ,
or i n d i r e c t l y by way of a precursor compound converted to the alkane in the
sediment). Alkanes may derive from a v a r i e t y of p o l l u t a n t sources. In some
cases the gross structure of an i n d i v i d u a l alkane, or the d i s t r i b u t i o n of a
series of alkanes, r e f l e c t s a p a r t i c u l a r i n p u t . For example, the presence of
7- and 8-methylheptadecanes is c h a r a c t e r i s t i c of a d i r e c t contribution from

23


24

M. M. Quirk et

al.

blue-green algae ( e . g . Han, McCarthy and Calvin, 1968; E g l i n t o n , Maxwell and
P h i l p , 1974). Since steroidal alkanes (steranes) ( e . g . Mülheim and Ryback,
1975) are geological maturation products of s t e r o l s , t h e i r presence in contemporary environments is good evidence of a f o s s i l fuel-derived input
(Cardoso, 1976; Brassell and others, 1978).
The origins of certain other alkanes which are ubiquitous in recent sediments
can only be determined from elucidation of t h e i r stereochemistry. Three
examples have been chosen to i l l u s t r a t e t h i s approach: 2,6,10,14-tetramethylpentadecane (pristane) ( I ) , 17ß(H)-hopane ( I I ) and 17ot(H)-homohopane ( I I I ,
R=CH3). Pristane occurs widely in marine organisms, and in Zooplankton and a
higher marine organism comprises solely the 6(R),10(S) isomer ( l a ) (Cox and
others, 1972; Patience, Rowland and Maxwell, 1979). In mature geological
samples, including petroleum, the alkane comprises a mixture of la : lb + Ic in
the r a t i o 1:1 (Patience, Rowland and Maxwell, 1979). Thus, determination of
the configuration of pristane in a recent sediment should allow d i s t i n c t i o n
between a biological and a p o l l u t a n t source.

^χ-s. ^ t S v L x * - . . ^ ^ s s v
Compound I I also occurs widely in
|
| | ]
T22
R

recent sediments ( e . g . Brooks and
I I I 1^ J
I
others, 1977), but possible sources
y
//
s>
( ^^^ ^
\^\^^
are more d i f f i c u l t to assign. I t
:
j
I
i n
does not occur in petroleum, so i s
X^^As^^X
unlikely to derive from a p o l l u t a n t
y^^
source; there is only one report
of i t s occurrence in an a c i d o p h i l i c bacterium (De Rosa and others, 1973).
Petroleum triterpanes are mainly of the 17a(H)-hopane type and are characterised (> C3-1) by the presence of ca. 1:1 mixtures of C-22 diastereoisomers
(e.g. Ensminger and others, 1974J7 The occurrence of such a d i s t r i b u t i o n in
recent sediments has been taken as evidence for petroleum-derived p o l l u t i o n
(Dastillung and Albrecht, 1976). However, in many recent sediments the C31
member ( I I I ) occurs as an unequal mixture, with the l a t e r - e l u t i n g isomer
(GLC) more abundant; this points to an additional ( b i o l o g i c a l ) o r i g i n for t h i s
isomer although i t has never been found in organisms (Brooks and others, 1977).
In the present study the o r i g i n of each of these compounds in a p a r t i c u l a r sediment has been determined by application of gas chromatography and computerised
gas chromatography-mass spectrometry techniques: ( i ) pristane in a lacustrine
sediment, ( i i ) 17$(H)-hopane in the same sediment, ( i i i ) 17a(H)-homohopane in

a sphagnum peat.


Recognition of Sources of Isoprenoid Alkanes

EXPERIMENTAL
Sample Collection
Rostherne sediment. Cores were collected using a Gilson mud sampler, and
stored frozen before sectioning and analysis (Gaskell, 1974).
Microcystis aeruginosa. A sample of the alga was collected from the surface
of the lake. An a l i q u o t was kept f o r analyses. A second portion was suspended (18 wk) j u s t below the lake surface (0.5m) in a conical flask with the
neck packed with s t e r i l e glass wool; subsequently, i t was allowed to stand
(53 wk) on the lake bed (30m). The laboratory culture was grown by Mr.B.
Capel at Porton Microbiological Research Establishment. Growth occurred (7d)
at ambient in an aerated polycarbonate vessel (40£) containing a mineral salts
solution with aqueous garden s o i l e x t r a c t . The c e l l s were harvested by centrifugation.
Sphagnum cuspidatum. A fresh sample was collected from the Lyne of Skene peat
bog. Part was kept for analysis w h i l s t a second portion was allowed to decay
(ca. 13 mnth) in a culture room at 30OC under aerobic conditions in the dark.
In a d d i t i o n , a sample of the moss base was collected f o r analysis.
Extraction and Separation
Samples were extracted using e i t h e r a Dawes soniprobe (sediment samples; i PrOH/hexane, 4 : 1 ) , a Soxhlet apparatus (algal samples; (CH3)2C0 followed by
CH2Cl2/MeOH, 2 : 1 ) , a d i r e c t r e f l u x (moss base and decayed moss, O^CWhexane;
l i v i n g moss, (CH3J2CO followed by C^C^/MeOH, 2 : 1 ) . In each case, the
' n e u t r a l ' f r a c t i o n of the t o t a l organic e x t r a c t was separated from the ' a c i d '
f r a c t i o n by shaking with aqueous (or methanolic) KOH (4 10% w/v; ca_. 50ml)
followed by extraction with hexane or Ch^C^. The neutral f r a c t i o n was separated by t h i n - l a y e r chromatography (TLC) on SiO? (CH2CI2 developer) to y i e l d
a hydrocarbon band (Rf * 0 . 8 ) . Urea adduction (3X) separated the hydrocarbons
i n t o an adduct and non-adduct (branched and c y c l i c ) components; the l a t t e r
was f u r t h e r fractionated by TLC (10% AgN03/SiÛ2; hexane developer) to y i e l d

the branched and c y c l i c alkanes (Rf * 0 . 7 ) .
Gas Chromatography
Diastereoisomers of pristane were separated using a modified Perkin Elmer F-17
gas Chromatograph (flame i o n i s a t i o n d e t e c t o r ) . This was f i t t e d with a DEGS
glass c a p i l l a r y column (100m) using helium (30 psig) as c a r r i e r gas. The oven
temperature was programmed from 40°C to 80°C at 2°/min.
Gas Chromatography-Mass Spectrometry
A l l samples were analysed using a Finnigan 9610 gas Chromatograph coupled
d i r e c t l y to a Finnigan 4000 mass spectrometer. The Chromatograph was f i t t e d
with an 0V-1 glass c a p i l l a r y column (20m) using helium (ca. 10 psig) c a r r i e r
gas. Temperature programme conditions were 50°C to 260°IT~at 6°/min. Typical
mass spectrometer conditions were: ion source temperature 200°C, electron
energy 70 eV, filament current 430 yA. Data were collected (ca. 1.5 sec per
scan) on a DEC PDP 8/e (32K core) laboratory computer. Mass Tragmentograms
and mass spectra were p l o t t e d using a Calcomp 565 p r i n t e r .

25


M. M. Quirk et

26

al.

RESULTS AND DISCUSSION
Rostherne Mere, Cheshire (U.K.) is a small (0.5 km 2 ), eutrophic lake with permanent oxygen depletion at the deepest part (c_a. 30m). The dominant algal
species is the blue-green Microcystis aeruginosa, which is p a r t i c u l a r l y abundant as intense blooms during the summer months. I t is probably the major
contributor of organic matter to the bottom sediment (Belcher and Storey,
1968; Reynolds and Rogers, 1976). Skene Moss (0.5 km2) is situated near the

Lyne of Skene, Aberdeenshire (U.K.) and is an o l i g o t r o p h i c , raised moss. The
upper 2.5m of peat i s composed mainly of Sphagnum cuspidatum remains; the
peat has been used as a fuel so the present surface dates from the sub-Boreal
period (2000-3000 yr B.P.) except for the upper 1cm deposited w i t h i n the l a s t
100 years (Wheatley, Greaves and Inkson, 1976).
A l l the hydrocarbons were i d e n t i f i e d from comparison of mass spectra with
standards, except for those in the moss which were assigned by mass fragmentography (m/e 191, 205) and retention data.
Pristane in Rostherne Sediment
Pristane was analysed by GLC on diethyleneglycol succinate (DEGS). The d i a stereoisomer separation f o r three sediment sections (0-7cm, 7-18cm, 18-30cm),
a sample of the alga collected from the lake surface, and a standard (1:1 mixture of the 6(R),10(R) and 6(R),10(S) isomers, lb,a) is shown in F i g . l . The

Fig. 1.

GLC traces of pristane (100m
DEGS): i ) 1:1 mixture 6(R),10(R)
and 6(R),10(S) isomers; i i ) - i v )
from d i f f e r e n t depths of Rostherne
Mere sediment; v) from M.aeruginosa in Rostherne.

r e l a t i v e contribution from the 6(R),10(S) isomer ( l a ) varied among the samples
and was at a maximum in the alga. Since pristane of b i o l o g i c a l o r i g i n should
comprise solely the 6(R),10(S) isomer (Cox and others, 1972; Patience, Rowland and Maxwell, 1979) each sample therefore contains a contribution from a
p o l l u t a n t source. A p l o t of the proportion of pollutant-derived pristane f o r
each sample shows a r e l a t i v e decrease for t h i s component with increasing depth
of sediment, as would be expected ( F i g . 2 ) . The alga, although containing
mainly the phytol-derived 6(R),10(S) isomer ( l a ) , s t i l l has a s i g n i f i c a n t cont r i b u t i o n from a contamination o r i g i n . The most l i k e l y source of the p o l l u t i o n
is petroleum-derived hydrocarbon material entering the lake via a small sewage


Recognition of Sources of Isoprenoid Alkanes


effluent and/or run-off from a nearby trunk road.

o C3|ocß
x PRISTA

7o

POLLUTANT -DERIVED

Fig.

2.

Plot of percent contribution of pollutant-derived
pristane and 17a(H)-homohopane in Rostherne alga
and d i f f e r e n t sediment depths.

17ß(H)-Hopane in Rostherne Sediment
Pentacyclic triterpanes of the hopane type show an abundant ion at m/e 191 in
t h e i r mass spectra and can be recognised readily in complex mixtures by mass
fragmentography, using t h i s i o n . F i g . 3D shows the m/e 191 fragmentogram of
the alkane f r a c t i o n from a sediment core (0-20cm). The extended (> C31)
17a(H)-hopanes ( I I I , R=C2H5,n-C3H7,n-C4Hg,n-C5H]-|) occur as ca_. 1:1 doublets
for the two C-22 diastereoisomers ( I I I ) . The d i s t r i b u t i o n is t h a t t y p i c a l l y
observed in mature geological samples and indicates that these components
arise from a petroleum-derived source (Dastillung and Albrecht, 1976). The
C30 alkane, 173(H)-hopane is also a s i g n i f i c a n t component ( F i g . 3D) but cannot
derive from a p o l l u t a n t source (see above). Formally, i t can only arise from
a contemporary b i o l o g i c a l source or from an erosion of an immature ancient

sediment where the 173(H)-hopanes have not been replaced by the more stable
17a(H) series t y p i c a l of mature samples ( e . g . Ensminger and others, 1974; Van
Dorsselaer, Albrecht and Ourisson, 1977). In Rostherne Mere, the geology of
the area is such that the l a t t e r p o s s i b i l i t y is not l i k e l y and a b i o l o g i c a l
o r i g i n is more f e a s i b l e . Mass fragmentography reveals, however, the absence
of 173(H)-hopane in the cultured sample of M.aeruginosa ( F i g . 3A). The two
most abundant hopane derivatives recognised (2 and 390 ppm dry weight respect i v e l y ) were hop-13(18)-ene ( I V ; Fig. 3A) in the alkane f r a c t i o n (hindered
double bond) and hop-22(29)-ene (V) in the alkenes. In the sample of the alga
from the lake surface and the sample suspended in the lake p r i o r to lowering

27


M. M. Quirk et

28

al.

to the lake bed, the concentrations (ppm dry weight) were: hop-13(18)-ene, 5
and 6 respectively (Fig. 3B,C); hop-22(29)-ene, 820 and 450; 17B(H)-hopane,
0.3 and 2 (Fig. 3B,C). This is strong circumstantial evidence that the hopane
originates from reduction of the abundant hop-22(29)-ene present. The culture
sample, although not axenic, had no detectable iso- or anteiso-acids (Ci 5 and
Ci7) of bacterial origin, whereas the lake and TäFe bed samples had both isoand anteiso-acids present (70 and 1290 ppm t o t a l , respectively) (Quirk anci~
Maxwell, unpublished results). I t is possible, therefore, that the hopane
arises from bacterial reduction of the alkene. This may represent a general
pathway for the origin of the widespread 17ß(H)-hopane in recent sediments.

ΗΟΡ-13(1β)-ΕΝΕ

(X)

^Ju^J^J

B

D

C 3 1 «ßHOPANE

C

AA~JLJ'H^y^-A—^
Fig. 3.

^-«ßHOPANES^
C
32 C 33 C34
35

O Λ

Mass fragmentograms (m/e 191) of branched/cyclic
alkanes from: A. Culture of M. aeruginosa; B.
Rostherne surface alga; C. Rostherne alga (lake
bed experiment); D. Rostherne sediment (0-20cm).

Of the two C-22 diastereoisomers of l7a(H)-homohopane ( I I I , R=CH3) in the sediment (Fig. 3D), the second isomer (by GLC) is in much higher relative abundance,
unlike the C32 - C35 members of pollutant origin alone. The second isomer i s ,
therefore, mainly of biological origin, and the pollutant contribution (both

isomers in ca. 1:1 ratio) decreases (Fig. 2) with increasing depth of sediment
(cf. pristane).
17a(H)-Homohopane in Lyne of Skene Peat
More detailed information about the origin of the second eluting isomer in the
sedimentary environment was obtained by examination of the pentacyclic t r i t e r panes in this peat and in a major contributor of organic matter, Sphagnum


Recognition of Sources of Isoprenoid Alkanes

cuspidatum. GC-MS analysis of the alkanes from various depths of a peat core
showed the major t r i t e r p a n e by f a r to be 17a(H)-homohopane ( I I I , R=CH3) (Quirk
and Maxwell, unpublished r e s u l t s ) . A s i m i l a r s i t u a t i o n has been observed i n
another peat (Gaskell, 1974) and in a l i g n i t e (Van Dorsselaer, Albrecht and
Connan, 1977) and i t is possible that t h i s remarkable predominance in the
branched and c y c l i c alkanes may be c h a r a c t e r i s t i c of certain peat types and
t h e i r ancient counterparts. The branched and c y c l i c alkanes of the l i v i n g
moss showed only a trace amount of 17a(H)-homohopane, which was only revealed
by mass fragmentography of m/e 191 ( F i g . 4A) and 205 (not shown). The alkane
was present as a ca. 1:1 r a t i o of the two C-22 isomers, i n d i c a t i n g a p o l l u tant o r i g i n . In "tiïe moss base ( c o l l e c t e d immediately below the l i v i n g moss,
age ca. 1 yr) a dramatic increase in the r e l a t i v e abundance of the second
e l u t i n g isomer was observed ( F i g . 4B). This s i t u a t i o n was paralleled in the
branched and c y c l i c alkane f r a c t i o n from the moss sample allowed to decay
under dark, aerobic conditions ( F i g . 4C). The o r i g i n of the abundant second
isomer of 17a(H)-homohopane ( I I I , R=CH3) in the peat, t h e r e f o r e , appears to be
associated with b a c t e r i a l decay. I t is not known i f i t arises from a l t e r a t i o n
of a precursor in the moss or i f i t is solely a bacterial product. The l a t t e r
appears more l i k e l y since extended (> C30) hopane derivatives have only been
found in bacteria and blue-green algae ( e . g . Rohmer and Ourisson, 1976).

Fig. 4. Mass fragmentograms (m/e 191)

of branched/cyclic alkanes from
S. cuspidatum: A. Living moss;
B. Moss base; C. Decayed moss
(laboratory).

^^^jjl

^KJVA^JU^.

29


M. M. Quirk et

30

al.

ACKNOWLEDGEMENTS
We thank Mrs. A.P. Gowar and Mr. A. Turrington f o r technical assistance. Two
of us (MMQ and RLP) thank the Science Research Council and the Natural Environment Research Council (NERC) respectively, for Research Studentships. Acknowledgement is made to the Donors of The Petroleum Research Fund, administered
by the American Chemical Society, f o r p a r t i a l support. We are also grateful
to NERC f o r f i n a n c i a l support (GR3/2951).
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Patience, R.L., S.J. Rowland and J.R. Maxwell (1979). The e f f e c t of maturat i o n on the configuration of pristane in sediments and petroleum. Geochim. Cosmochim. Acta, 43, In press.
Reynolds, C.S. and D.A. Rogers (1976). Seasonal variations in the v e r t i c a l d i s t r i b u t i o n and buoyancy of Microcystis aeruginosa Kutz. emend. Elenkin in


Recognition of Sources of Isoprenoid Alkanes

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31