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C25 and C30 highly branched isoprenoid alkenes in laboratory cultures of two marine diatoms
Org. Geochem. Vol. 21, No. 3/4, pp. 407-413, 1994
Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0146-6380/94 $7.00+ 0.00
Pergamon
C~ and C30 highly branched isoprenoid alkenes in laboratory cultures of two marine diatoms JOHN K. VOLKMAN,STEPHANIEM. BARRETT and GRAEMEA. DUNSTAN CSIRO Division of Oceanography, G.P.O. Box 1538, Hobart, Tasmania 7001, Australia Al~ract---C25 and C30 highly branched isoprenoid (HBI) alkenes have been isolated from laboratory cultures of the marine diatoms Haslea ostrearia and Rhizosolenia setigera. At least seven C25 isoprenoid alkenes, with carbon skeleton II, were found in the culture of H. ostrearia. A C25:4HBI alkene was the major hydrocarbon present. Other hydrocarbons included a C2s:3alkene, a different C25:4alkene, two C25:s aikenes and two unidentified minor components that exhibited properties consistent with alkenes. Three C30:5 HBI alkenes and smaller amounts of two C3o:6alkenes, all with carbon skeleton Ill, were identified in R. setigera. These isoprenoid hydrocarbons were not present in thirteen other species of diatom examined. All species possessed the alkene n-heneicosa-3,6,9,12,15,18-hexaene (n-C21:6), and eight also had n-heneicosa-3,5,9,12,15-pentaene (n-C21:5). Highly branched isoprenoid alkenes are commonly reported in marine sediments and waters, but this is their first identification in marine microalgae. These data support previous suggestions that these alkenes might be derived from diatoms in many contemporary marine environments. Key words--alkenes, biomarkers, diatoms, isoprenoid, Haslea, Rhizosolenia
INTRODUCTION C20, C25, and to a lesser extent C3o, highly branched isoprenoid (HBI) alkenes are commonly the most abundant natural hydrocarbons found in marine sediments and waters. They were first reported in 1976 in coastal sediments off Florida, U.S.A. (Gearing et aL, 1976), and subsequently they were found in marine regions as diverse as Puget Sound (Barrick et ai., 1980), Peru (Volkman et al., 1983), Spain (Albaig6s, 1984a,b) and Antarctica (Nichols et al., 1988). Rowland and Robson (1990) have reviewed the sedimentary occurrence of these compounds and listed 15 marine environments where these isoprenoid alkenes had been found. It seems probable that they also exist in most present-day marine environments. Yon et al. (1982) established that the saturated C20 hydrocarbon carbon skeleton is formed by an unusual linkage of C5 isoprene units (I; Fig. 1). The C2s and C30 alkane structures were later shown to involve further addition of C5 units to this basic skeleton (II, m ; Fig. 1) (Bayona et al., 1983; Robson, 1987). Attempts to identify the position and geometry of the double bonds using standard derivatization methods have not been successful. In view of the geochemical importance of these compounds, there has been considerable speculation about their likely sources. Some authors noted a link to branched hydrocarbons with similar structures produced by Actinomycetes, which led to the suggestion that marine bacteria were a possible source
(Farrington et al., 1977; Boehm and Quinn, 1978; Requejo and Qulnn, 1983). However, the occurrence of these alkenes in seawater particulate matter and sediment trap samples (Prahl et al., 1980; Volkman et al., 1983) implied that a more likely origin was from certain species of phytoplankton. Support for this view came from the work of Nichols et al. (1988) who identified a di-unsaturated C25 HBI alkene in Antarctic sea-ice communities shown by microscopy to consist solely of diatoms. More recently, a new C25 HBI alkene has been isolated from benthic microbial communities dominated by diatoms (Summons et al., 1993). Here we report the first identification of C25 and C30 HBI alkenes in laboratory cultures of two marine diatoms, thus confirming that diatoms are the likely source of these unusual hydrocarbons in many present-day marine environments.
I II III Fig. 1. Structures of the parent C20, C25 and C30 carbon skeletons. The C2o alkane is a major constituent of the hydrocarbons isolated from Rozel Point crude oil. Its structure was elucidated by Yon et al. (1982). In marine sediments and seawater, the C25 and C3o hydrocarbons mainly occur as highly unsaturated alkenes.
407
408
JOHN K . VOLKMAN el al. Table I. Culture conditions and hydrocarbon contents of diatoms studied
Diatom species
White light (IrE m ~ s i ) Axenic
Culture medium
Culture age at harvest (days)
Cell counts × 105 cells/ml
Temp. (C)
f, t~ f2 f: f, f, 1, f? l, f. (3 G2 f,
10 6 13 6 13 10 7 7 7 12 6 I2 12
10.5 ~3 0.02 l 3.3 II 8.4 17.3 14.2 66 4 7 !. 2 1.2 0.87
20 20 20 20 25 20 20 20 25 28 2(I 20 20
70.80 * t00 [00 10(t 100 70 80* iotJ 10t) lot) 7tL 80* I 0(t 7080* 7080*
t, f,
13 10
1.2 0.33
20 20
100 7t~-80"
Amphiphora hyalina CS-28 Amphora sp. CS-10c Coscinodiscus sp. CS-151 Cylindrotheca fusiformis CS- 13c Fragilaria pinnata CS- 121 Navicula sp. CS-46c Nitzsehia closterium CS-5c Skeletonema costatum CS-181 Skeletonema sp. CS-252 Thalassionema nitzschioides CS- 146 Thalassiosira stellaris CS- 16 Thalassiosira rotula CS-77 Thalassiothrix heteromorpha CS-132/8 Species containing HBI alkenes: Haslea ostrearia CS-250 Rhizosolenia setigera CS-62
~ ~ + ....
..... ~
n-C:~
n-C2~ 5 fig/cell)
fig/cell)
41 81 1550"i" 63 9 23 13 ND ND 15 ND ND ND
117 80 2400";" 129 3! 13 3(I 103 28 20 190 265 323
ND ND
396 52
*Cultures transferred after 3-5 days to blue light at 40/aE m - Z s ~.
+Coscinodiscus sp. cell size is quite large; approx. 70/~m in diameter. ND, not detected.
EXPERIMENTAL
Algal cultures Fifteen diatom species were obtained from the CSIRO Algal Culture Collection from genera known to be present in marine environments where HBI alkenes had been identified. These were cultured in Erlenmeyer flasks under a 12 : 12 light: dark cycle. Cultures were grown under white light for 6-13 days at 100/~Em-2s ~ except for those marked • (Table 1) which were grown initially in white light for 3-5 days at 70-80/~ E m 2 s- ~and then transferred to blue light at 40/~Em-2s -1. Light levels were measured with a QSL-100 (Biospherical Instruments) light meter. All cultures were grown in 1 litre Erlenmeyer flasks containing 700-800ml of culture medium t"2(Guillard and Ryther, 1962), or medium G (Jeffrey, 1980) at full strength (CS-16) or half strength (CS-77). Most were aerated with 1% CO2 in air with a 12:12 light:dark cycle; the exceptions were CS-146 and CS-132/8 which were aerated with air alone. The initial inoculum was 75 ml of log-phase culture. Cell counts were measured with a Neubauer haernocytometer. Lipid extraction and analysis After 6--13 days (depending on growth rate), the cells were filtered onto glass fibre filters and extracted with chloroform-methanol-water (Volkman et al., 1992). The total hydrocarbons were isolated by open column chromatography on silica gel and eluted with hexane and hexane:toluene (1:1). The hydrocarbon fractions were analysed by capillary gas chromatog• raphy on a non-polar HPI methyl silicone fused silica column , (50 m x 0.32 mm i.d., Hewlett-Packard) using a Shimadzu 9A gas chromatograph equipped with a F I D and cooled on-column injector (SGE, Australia) (Volkman et al., 1992). Peak areas were quantified using DAPA software (DAPA Scientific,
Kalamunda, Western Australia). Electron-impact mass spectra were obtained using an HP 5890 GC
and 5790 MSD fitted with a direct capillary inlet (Volkman et al., 19921. Hydrogenation A portion of the hydrocarbon fractions from Haslea ostrearia and Rhizosolenia setigera dissolved in 200ill iso-octane (2,2,4-trimethylpentane) was added to a test tube containing 2 ml of iso-octane and 30 mg of Adam's catalyst (PtO2) flushed with hydrogen for 3 h. Following the addition of purified water (Milli-Q"- system), the hydrogenation products were removed in the upper iso-octane layer (4 extractions) and analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). RESULTS
All but two of the diatom species had very simple hydrocarbon distributions. Each of the 15 species studied contained n-heneicosa-3,6,9,12,15,18hexaene ( n - C 2 1 : 6 ; Table 1) which is believed to be formed by decarboxylation of the fatty acid 22:6 (n-3) (docosahexa-4,7,10,13,16,19-enoic acid) which is a minor constituent in all of the diatoms studied (Dunstan et al., 1994). This hydrocarbon is common in diatoms and many other classes of microalgae (Lee and Loeblich, 1971). Eight of the species also contained n-heneicosa-3,6,9,12,15-pentaene (n-C2~:5; Table 1), which we assume is synthesized in like manner by decarboxylation of the 22:5 (n-6) fatty acid. This alkene is rarely reported in microalgae. Trace amounts of squalene were also found in some of the diatoms. Two of the diatoms contained unusual highly branched isoprenoid (HBI) alkenes. Haslea ostrearia contained a suite of at least seven C25 HBI alkenes having 3, 4 and 5 double bonds [Fig. 2(a,b) and Table 2]. These eluted well before n-C25 (in fact just after n-C2j ) on the methyl silicone column indicating that they were highly branched. The major constituent was a C25:4 alkene which comprised 78.5% of the
Highly branched isoprenoid alkenes
409
Ha 25:4
H7
R2
n-C22 IS
25:5
30:5
H1
IS (17.'0 FAME)
n-C21:e H2"
i
Pe
n-C21:6
3O:5
H6
,/~:5(7)
" 3O:6
/Hs
R5
H4. I
i
24
28
~:6 32
I
I
~
~
"lime (minutes)
I
I
I
I
24
28
32
36
I
I
~
Time (minutes)
Fig. 2. Partial capillary gas c h r o m a t o g r a m s of the total h y d r o c a r b o n s in (a) H. ostrearia and (b) R. setigera. Peak identifications are given in Table 2. Alkenes are denoted Cx:y where x is the n u m b e r of c a r b o n a t o m s and y is the n u m b e r of double bonds.
total C25 alkenes. Other constituents were two C25:5 alkenes (2.6 and 8.2%), another C25:4 alkene (3.2%), at least one C25:3 alkene (6.4%) and two other minor components which could not be identified due to weak mass spectra. Major ions in the mass spectra of the alkenes are listed in Table 2. The mass spectrum of the major C25:4 alkene is shown in Fig. 3(b). The presence of
ions at m/z 233, 259, 287 is consistent with a highly branched structure and base peak at m/z 69 indicates the presence of multiple double bonds. Molecular ions were present in the mass spectra of most of the alkenes [Fig. 3(a-d)] allowing the number of double bonds to be assigned (Table 2). Hydrogenation of the total hydrocarbons produced two alkanes: n-eicosane (n-C2~) from n-C21:6
Table 2. C2s and C3o isoprenoid alkenes in Haslea ostrearia and Rhizosolenia setigera GC peak number
ECL*
Conc. fig/cell)
Conc. (/~g/g)
% of total HBI alkencs
Haslea ostrearia H~ C25:3
21.06
189
242
6.4
H2
C25:4 ?
21.34
96
123
3.2
Ha
C2s:4
21.44
2340
2990
78.5
H4
?
21.58
11
15
0.4
H5
?
21.73
22
28
0.7
AIkene
H6
C25:5~
21.91
77
99
2.6
H7
C25:5
22.00
244
313
8.2
Rhizosolenia setigera RI C30:s
25.07
232
88
14.3
R2
C3o:5
25.50
624
237
38.6
R3
C3o:5
25.60
473
179
29.3
R4
C30:6
25.81
212
80
13.1
R5
C3o:6
26.00
76
29
4.7
*ECL, equivalent chain length o n H P I capillary column. tPercentage abundance in parenthesis.
Major ions in electron impact mass spectrumt 41 (63), 55(98), 69(100), 83(85), 95(67), 261 (16), 289 (8), 346 (3) 41 (63), 55 (42), 69 (100), 81 (81), 95(85), 259 (1), 275 (5), 344 (3) 41 (58), 55 (31), 69 (1O0), 81 (62), 95 (35), 177 (4), 259 (5), 275 (3), 329 (I), 344 (0.5) 41 (73), 55 (64), 69 (100), 81 (55), 95(41), 189 (5), 231 (2), 259 (12) 41 (69), 55 (29), 69 (100), 81 (49), 95 (27), 203 (5), 259 (5), 273 (5), 287 (I), 299 (2) 41 (66), 55 (26), 69 (100), 81 (45), 95 (26), 259 (3), 273 (5), 299 (2) 41 (66), 55(21), 69(100), 81 (49), 95 (26), 257 (6), 273 (6), 299 (2), 327 (I)
163(11), 233(21),
41 (60), 55 (37), 69 (100), 81 (42), 95 (29), 299(8), 357(6), 412(1) 41 (56), 55 (100), 69 (71), 81 (57), 95 (58), 259 (10), 315 (22), 327 (3), 412 (14) 41 (59), 55 (37), 69 (100), 81 (62), 95 (31), 231 (2), 299(3), 357(3), 412(3) 41 (72), 55 (54), 69(100), 81 (56), 95 (56), 259(8), 273 (8), 313 (12), 341 (2), 410 (5) 41 (62), 55 (34), 69(100), 81 (41), 95 (28), 246 (2), 273 (8), 341 (0.5), 410 (2)
135 (10), 231 (6),
123 (23), 165(17), 123 (10), 163 (3), 123(15), 163(3), 109 (20), 189 (12), 109(18), 175(5), 121 (12), 189(41),
135 (18), 191 (4), 137(12), 191 (15), 135(22), 189(5), 135 (11), 191 (6),
410
JOHN K. VOLKMANet al.
1-1 (a)
Ic) Rhizosolenia $etigera
Haslea ostrearia PeakH1 (C2s:3)
PeekR= (C~.d
\
55
8OOO
233
4oooi
~ 400o"1
/
289
1/77
I J]
20OO
20(10,
i
O' 50
100
(b)
\
L,
150
I[ ,/
200 250 300 Mass/Charge
350
,i
400
107
259 315
, , , , l o , , , | , , , , l l ,
, ,i
, , , , i
41~
i,
, ,i
,
100 150 200 250 300 350 400
50
M~Charge
Haslea ostrearia
(d) Rhizosolenia se~gera
PeakHs (C-~:4)
~oooo
69
PeakFI4 (C~:e)
\ 69
8000
60001
60O0
X5
0
, 50
259
,1 ,,
/
121
/
.LL
i0b"isb"~"asb"so6"~6"46o Mass/Charge
11!11
/I
s6' i 0 6 " i 5 6 " ~ " ~
300 ~
\
!
400
Mass/Charge
Fig. 3. Electron impact mass spectra of (a) the C25:~(peak number Hi) and (b) the C2s:4 (peak number H2) alkenes in H. ostrearia, (c) the C30:5(peak number R2) and (d) the C30:6(peak number R4) alkenes in R. setigera. Mass spectra were obtained using a Hewlett-Packard Mass Selective Detector (Volkman et al., 1992).
and a single C25 HBI alkane from the C25 alkenes thus confirming that all of the alkenes had the same carbon skeleton (If; Fig. 1). The electron impact mass spectrum of the C25 alkane showed a major ion at m / z 238 and minor ions at m / z 210 and 266 in addition to the usual alkyl ion series. This spectrum is identical with that previously published for the HBI alkane 2,6,10,14-pentamethyl-7-(3-methylpentyl)-pentadecane (Robson and Rowland, 1986; Rowland and Robson, 1990), and its ECL (equivalent chain length) index of 21.10 confirms this assignment. The mass spectrum did not give a molecular ion under electron impact conditions which is common with this class of alkanes (Rowland et al., 1990). The hydrocarbon distribution in R. setigera showed, in addition to n-C21:6, a suite of hydrocarbons eluting between n-C:5 and n-C26. The mass
spectra of these alkenes [Fig. 3(c,d)] confirmed the presence of two C30 highly branched alkenes with 6 double bonds and three with 5 double bonds (Table 2). Two of the C30:5 alkenes (peak numbers RI and R3) had similar mass spectra to that of a C30 penta-unsaturated alkene referred to as "HC412" by Barrick and Hedges (1981), and as the monocyclic tetraene "C30:4:~" by Prahl et al. (1980). Hydrogenation produced a single C30 alkane plus small amounts of two C30 monounsaturated alkenes. The alkane had an identical mass spectrum and retention index to that previously reported for 2,6,10,14,18pentamethyl- 7- (3- methylpentyl)- nonadecane (III; Robson and Rowland, 1988; Rowland and Robson, 1990). The presence of the two monoenes indicates that one or more of the double bonds is in a hindered position and resistant to hydrogenation (as also
Highly branched isoprenoid alkenes concluded by Barrick and Hedges, 1981). Some C25 alkenes also contain a double bond that is difficult to hydrogenate (Rowland et al., 1990). DISCUSSION The isolation of both C25 (II, Fig. 1) and C30 (IlI; Fig. 1) HBI aikenes from the two diatoms H. ostrearia and R. setigera, plus the earlier isolation of a C25 diene from a natural sea-ice diatom population, strongly suggests that diatoms are a major source of C25 and C30 HBI alkenes in present-day marine sediments and seawater. This result explains why these alkenes are abundant in marine environments where diatoms are major constituents of the phytoplankton (e.g. upwelling areas off Peru, productive coastal regions, some algal mats and sea-ice diatom communities). Furthermore, carbon isotope data obtained for compounds isolated from a Messinian evaporite showed that the C25 HBI alkanes had the same ratio of ~2C to 13C as algal-derived sterols (Kohnen et al., 1992). The isotopic compositions of a C20 HBI alkane and two C25 HBI alkenes isolated from diatomaceous microbial communities also suggested they were of microalgal origin (Summons et al., 1993). Several of the cultures, including H. ostrearia and R. setigera, were not axenic (Table 1), but the level of bacteria present was extremely low. Branched-chain fatty acids typical of bacteria represented less than 1 and 3.3% of the total fatty acids in H. ostrearia and R. setigera respectively (unpublished data), indicating a very minor contribution of bacteria to the extractable lipids. Similar amounts of bacteria were present in other cultures which did not contain HBI alkenes. Moreover, the HBI alkenes were much more abundant than the n-C2~:6 alkene, which is of undisputed algal origin, so a contamination source for the HBI alkenes in the cultures can be safely ruled out. It should be noted, however, that the distribution of C25 HBI isomers found in Haslea is different from those previously reported in marine sediments and seawater even though the alkenes possess the same unusual carbon skeleton. For example, in coastal sediments off Peru, a suite of tri- and tetraunsaturated C25 HBI alkenes dominate the hydrocarbon distributions (Volkman et al., 1983). These elute on the non-polar capillary column in the same region as the Haslea HBI alkenes, but did not co-elute with them. However, the triunsaturated C25 HBI alkenes found in the particulate matter and sediments delta of the Erbo River, Spain (Albaigrs et al., 1984a,b), referred to as i-25:3 (Rloa_ 5 2119 and RisE.30 2140) have similar mass spectra to that of the C25:3 HBI (peak Hi) found in H. ostrearia. C30 HBI alkenes appear to be common in marine sediments, but are rarely reported as major constituents. It is apparent from the limited amount of retention data reported to date that at least some isomers are different from those found here in
411
R. setigera. These compounds should not be confused with the complex mixture of C30 alkenes, containing two to five double bonds, identified as hydrosqualenes which have been found in some sediments (AIbaigrs et al., 1984a). These elute after n-C27 (i.e. much later than the HBI alkenes found in R. setigera), and have significantly different mass spectra. However, a suite of C3o HBI alkenes have been found in mussels and seawater particulate matter in Port Phillip Bay, southeastern Australia (A. P. Murray and J. K. Volkman, unpublished data), which show a similar distribution to those found in R. setigera. Two C30:5 alkenes and one C30:6 alkene co-elute with those found in R. setigera (peaks Rl, R3 and Rs) while the other major C30:6 alkene has a different retention index. This study of mussels and seawater particles was performed at the time of a bloom of R. chunii in Port Phillip Bay which rendered mussels and shellfish in the area bitter-tasting and hence unpalatable for many months (Parry et al., 1989). This strongly suggests that the source of the alkenes found was R. chunii; the slightly different hydrocarbon distribution to that found in R. setigera is probably due to interspecies variations within the genus Rhizosolenia. Cultures of R. chunii are not yet available to test this hypothesis. It is clear from our data that only a small proportion of diatoms synthesize these unusual hydrocarbons. The two species found here belong to different Orders (Haslea is in Pennales while Rhizosolenia is in Centrales), but many other species from both of these Orders do not contain HBI alkenes (Table 1). Both algal genera are common in marine environments and thus could be significant sources of alkenes in particular cases, but it seems that other species are likely to be major sources. The genus Haslea contains at least 9 species, some formerly of the genus Navicula, which have been isolated from both temperate and tropical marine environments (Ricard, 1987a). Haslea ostrearia is common in coastal areas and it produces an unusual pigment called marennine which is responsible for the bluegreen colouration of the gills found in oysters grown in sea basins along the French coast (Neuville and Daste, 1978; Robert, 1986). Rhizosolenia is a common genus in coastal and oceanic marine waters world-wide, with several species found in Antarctica and some in the Arctic regions (Priddle et al., 1990; Priddle and Fryxell, 1985). There are about sixty marine and only five freshwater species known (Ricard, 1987b). In some cases, the taxonomy, even to genus level, is uncertain (Priddle and Fryxell, 1985). Sundstr6m (1986) has proposed a revision of the genus Rhizosolenia, and recommended that some species, including R. setigera, should be transferred to other genera. Large mats and rafts of diatoms from the genus Rhizosolenia are found over wide ranges in the Pacific, Indian and Atlantic oceans (e.g. Carpenter et al., 1977; Alldredge and Silver, 1983; Sancetta
412
JOlly K. VOLKMANet al.
et at., 1991 ). These mats can be up to 4.4 m 3 in volume (Martinez et al., 1983) and consist of intertwining chains of either single or two or more different species o f Rhizosolenia (Carpenter et al., 1977; Alldredge and Silver, 1982). They are not always visible from ships, but are very obvious when sampled using scuba techniques to prevent mat disintegration (Alldredge and Silver, 1982; Martinez et al., 1983). The mats sink quickly to the sea floor when the rhizosolenid cells lose buoyancy, carrying large amounts of organic material to the sediments (Sancetta et al.. 1991). Rising mats of Rhizosolenia may also be an important means of bringing nitrate from depth to surface waters in the North Pacific (Villareal et al., 1993). It is believed that mass sinking of rhizosolenid mats may be a c o m m o n occurrence (Sancetta et aL, 1991). The impact of sinking diatoms, present and past, on the benthos and formation of sediments could thus be much greater than currently realized. Vast regions of the worlds seas are believed to have been covered by floating mats of diatoms of the genus Thalassiothrix from 4.5 to 15 million years ago, with the total area covered being about the size of Australia (Kemp and Baldauf, 1993; Sancetta, 1993). These mats formed laminated sediments under reducing conditions so that the organic matter was well preserved (Kemp and Baldauf, 1993). However, the delicacy and size of rhizosolenid diatoms makes their preservation in the sediments very unlikely (Sancetta et al., 1991). Floating mats can be formed by several genera of" diatoms characterized by elongate cells (Sancetta, 1993), so the sinking of mats provides a route to the sediments of HBI alkenes produced by rhizosolenid or other, as yet undiscovered, source genera. This may be one reason why HBI alkanes and alkene diagenetic products have been found in ancient sedimentary rocks and crude oils (Yon et al., 1982; Sinninghe Damst6 et al., 1989) It is possible that the previous isolation of a C20 HBI alkane and monoene and a C25 diene in the macroscopic green alga E n t e r o m o r p h a proliJera (Rowland et al., 1985), may have been due to presence of epiphytic diatoms, particularly since n-C:~ 6 was also observed in the hydrocarbon fraction and this alkene is more commonly associated with a microalgal source (Lee and Loeblich, 1971). These HBI alkenes are not present in the closely related green alga E. intestinalis (Volkman, unpublished data). However, the possibility that these compounds might be synthesized by other algal classes cannot be excluded, especially since unsaturated isoprenoid alkenes of the botryococcene and lycopadiene type are known in microalgae from the green algal genus Botryococcus (Metzger and Casadevall, 1987; Metzger et al., 1985, 1991). It may be significant that a C20 HBI alkene has yet to be isolated from diatoms in laboratory culture. Further studies of other classes of microalgae need to be carried out, but the results presented here clearly establish diatoms as a major
source of these unusual hydrocarbons in marine sediments. CONCLUSIONS
Two species of marine diatoms, Haslea ostrearia and Rhizosolenia setigera, have been shown to produce C25 and C30 highly branched isoprenoid alkenes. This represents the first isolation and identification of these unusual hydrocarbons from cultures of marine microalgae. Without excluding the possibility that other classes of algae may produce these isoprenoid hydrocarbons, our data show clearly that diatoms are likely to be a major source in present-day marine sediments and waters. Acknowledgements---We thank Dr S. W. Jeffrey for providing the algal cultures, Ms Jeannie-Marie LeRoi for culturing the algae and cell counts, Dr Roger Summons for many helpful discussions and Dr Andrew Revill for commenting on the manuscript. This work was funded in part by the Chevron Oil Field Research Company and by the Fishing Industry Research and Development Corporation.
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Highly branched isoprenoid alkenes Gearing P., Gearing J. N., Lytle T. F. and Lytle J. S. (1976) Hydrocarbons in 60 northeast Gulf of Mexico shelf sediments: a preliminary survey. Geochim. Cosmochim. Acta 40, 1005-1017. Guillard R. R. L. and Ryther J. H. (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Can. J. Microbiol. g, 229-239. Jeffrey S. W. (1980) Cultivating unicellular marine plants. In CSIRO Division o f Fisheries and Oceanography, Annual Report (1977-1979), pp. 22-43. Kemp A. E. S. and Baldauf J. G. (1993) Vast Neogene laminated diatom mat deposits from the eastern equatorial Pacific ocean. Nature 362, 141-144. Kohnen M. E. L., Schouten S., Sinninghe Damst6 J. S., de Leeuw J. W., Merritt D. A. and Hayes J. M. (1992) Recognition of paleobiochemicals by a combined molecular sulfur and isotope geochemical approach. Science 256, 358-362. Lee R. F. and Loeblich A. R. III (1971) Distribution of 21:6 hydrocarbon and its relationship to 22:6 fatty acid in algae. Phytochemistry 10, 593-602. Martinez L., King J. M. and Alldredge A. L. (1983) Nitrogen fixation by floating diatom mats: a source of new nitrogen to oligotrophic ocean waters. Science 221, 152-154. Metzger P. and Casadevall E. (1987) Lycopadiene, a tetraterpenoid hydrocarbon from new strains of the green alga Botryococcus braunii. Tetrahedron Lett. 34, 3931-3934. Metzger P., Largeau C. and Casadevall E. (1991) Lipids and macromolecular lipids of the hydrocarbon-rich microalga Botryococcus braunii. Chemical structure and biosynthesis. Geochemical and biotechnological importance. In Progress in the Chemistry of Organic Natural Products (Edited by Herz W., Kirby G. W., Steglich W. and Tamm C.), pp. 1 70. Springer, Vienna. Metzger P., Casadevall E., Pouet M. J. and Pouet Y. (1985) Structures of some botryococcenes: branched hydrocarbons from the B-race of the green alga Botryococcus braunii. Phytochemistry 24, 2995-3002. Neuville D. and Daste P. (1978) Recherches sur la d&erminisme de la production de marennine par la Diatom6e marine Navicula ostrearia (Gaillon) Bory en culture in vitro. Rev. Gen. Bot. 85, 255-303. Nichols P. D., Volkman J. K., Palmisano A. C., Smith G. A. and White D. C. (1988) Occurrence of an isoprenoid C25 diunsaturated alkene and high neutral lipid content in Antarctic sea-ice diatom communities. J. Phycol. 24, 90-96. Parry G. D., Langdon J. S. and Huisman J. M. (1989) Toxic effects of a bloom of the diatom Rhisozolenia chunii on shellfish in Port Phillip Bay, Southeastern Australia. Mar. Biol. 102, 25-41. Prahl F. G., Bennett J. T. and Carpenter R. (1980) The early diagenesis of aliphatic hydrocarbons and organic matter in sedimentary particulates from Dabob Bay, Washington. Geochim. Cosmochim. Acta 44, 1967-1976. Priddle J. and Fryxell G. (1985) Handbook of the Common Plankton Diatoms o f the Southern Ocean: Centrales Except the Genus Thalassiosira, pp. 73-95. British Antarctic Survey, Cambridge. Priddle J., Jordan R. W. and Medlin L. K. (1990) Family Rhizosoleniaceae. In Polar Marine Diatoms (Edited by Medlin L. K. and Priddle J.), Chap. 15, pp. 115-127. British Antarctic Survey, Cambridge. Requejo A. G. and Quinn J. G. (1983) Geochemistry of C25 and C30 biogenic alkenes in sediments of
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Narragansett Bay estuary. Geochim. Cosmochim. Acta 47, 1075-1090. Ricard M. (1987a) Atlas du Phyto~olankton Marin. Vol. H: Diatomophycbes, pp. 102-103. Editions du Centre Nationale de la Recherche Scientifique, Paris. Ricard M. (1987b) Atlas du Phytoplankton Marin. Vol. H: Diatomophycbes, pp. 55-56. l~ditions du Centre Nationale de la Recherche Scientifique, Paris. Robert J.-M. (1986) Greening of oyster-ponds by the diatom Haslea ostrearia Bory: a model of the phenomenon. In Proceedings o f the 8th Diatom Symposium (Edited by Ricard M.), pp. 517-523. Koeltz Scientific Books, Koenigstein. Robson J. N. (1987) Synthetic and biodegradation studies of some sedimentary isoprenoid hydrocarbons Ph.D. thesis, Plymouth Polytechnic, England. Robson J. N. and Rowland S. J. (1986) Identification of novel widely distributed sedimentary acyclic sesterpenoids. Nature 324, 561-563. Robson J. N. and Rowland S. J. (1988) Synthesis of a highly branched C30 sedimentary hydrocarbon. Tetrahedron Lett. 29, 3837-3840. Rowland S. J. and Robson J. N. (1990) The widespread occurrence of highly branched acyclic C20, C25 and C30 hydrocarbons in recent sediments and biota--a review. Mar. Environ. Res. 30, 191-216. Rowland S. J., Hird S. J., Robson J. N. and Venkatesan M. I. (1990) Hydrogenation behaviour of two highly branched C25 dienes from Antarctic marine sediments. Org. Geoehem. 15, 215-218. Rowland S. J., Yon D. A., Lewis C. A. and Maxwell J. R. (1985) Occurrence of 2,6,10-trimethyl-7-(3-methylbutyl)dodecane and related hydrocarbons in the green alga Enteromorpha prolifera and sediments. Org. Geochem. 8, 207-213. Sancetta C. (1993) Green sea, black mud. Nature 362 108-109. Sancetta C., Villareal T. and Falkowski P. (1991) Massive fluxes of rhizosolenid diatoms: a common occurrence? Limnol. Oceanogr. 36, 1452-1457. Sinninghe Damst6 J. S., Van Koert E. R., Kock-van Dalen A. C., de Leeuw J. W. and Schenck P. A. (1989) Characterization of highly branched isoprenoid thiophenes occurring in sediments and immature crude oils. Org. Geochem. 14, 555-567. Summons R. E., Barrow R. A., Capon R. J., Hope J. M. and Stranger C. (1993) The structure of a new CE5 isoprenoid alkene biomarker from diatomaceous microbial communities. Aust. J. Chem. 46, 907-915. Sundstr6m B. G. (1986) The marine genus Rhizosolenia. A new approach to taxonomy. Ph.D. dissertation. Lund University, as cited in Priddle et al. (1990). Villareal T. A., Altabet M. A. and Culver-Rymsza K. (1993) Nitrogen transport by vertically migrating diatom mats in the North Pacific Ocean. Nature 363, 709-712. Volkman J. K., Barrett S. M., Dunstan G. A. and Jeffrey S. W. (1992) C30~C32alkyl diols and unsaturated alcohols in microalgae of the class Eustigrnatophyceae. Org. Geochem. 18, 131-138. Volkman J. K., Farrington J. W., Gagosian R. B. and Wakeham S. G. (1983) Lipid composition of coastal marine sediments from the Peru upwelling region. In Advances in Organic Geochemistry 1981 (Edited by Bjor~y M. et al.), pp. 228-240. Wiley, Chichester. Yon D. A., Maxwell J. R. and Ryback G. (1982) 2,6,10trimethyl-7-(3-methylbutyl)-dodecane, a novel sedimentary biological marker compound. Tetrahedron Lett. 23, 2143-2146.
Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0146-6380/94 $7.00+ 0.00
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C~ and C30 highly branched isoprenoid alkenes in laboratory cultures of two marine diatoms JOHN K. VOLKMAN,STEPHANIEM. BARRETT and GRAEMEA. DUNSTAN CSIRO Division of Oceanography, G.P.O. Box 1538, Hobart, Tasmania 7001, Australia Al~ract---C25 and C30 highly branched isoprenoid (HBI) alkenes have been isolated from laboratory cultures of the marine diatoms Haslea ostrearia and Rhizosolenia setigera. At least seven C25 isoprenoid alkenes, with carbon skeleton II, were found in the culture of H. ostrearia. A C25:4HBI alkene was the major hydrocarbon present. Other hydrocarbons included a C2s:3alkene, a different C25:4alkene, two C25:s aikenes and two unidentified minor components that exhibited properties consistent with alkenes. Three C30:5 HBI alkenes and smaller amounts of two C3o:6alkenes, all with carbon skeleton Ill, were identified in R. setigera. These isoprenoid hydrocarbons were not present in thirteen other species of diatom examined. All species possessed the alkene n-heneicosa-3,6,9,12,15,18-hexaene (n-C21:6), and eight also had n-heneicosa-3,5,9,12,15-pentaene (n-C21:5). Highly branched isoprenoid alkenes are commonly reported in marine sediments and waters, but this is their first identification in marine microalgae. These data support previous suggestions that these alkenes might be derived from diatoms in many contemporary marine environments. Key words--alkenes, biomarkers, diatoms, isoprenoid, Haslea, Rhizosolenia
INTRODUCTION C20, C25, and to a lesser extent C3o, highly branched isoprenoid (HBI) alkenes are commonly the most abundant natural hydrocarbons found in marine sediments and waters. They were first reported in 1976 in coastal sediments off Florida, U.S.A. (Gearing et aL, 1976), and subsequently they were found in marine regions as diverse as Puget Sound (Barrick et ai., 1980), Peru (Volkman et al., 1983), Spain (Albaig6s, 1984a,b) and Antarctica (Nichols et al., 1988). Rowland and Robson (1990) have reviewed the sedimentary occurrence of these compounds and listed 15 marine environments where these isoprenoid alkenes had been found. It seems probable that they also exist in most present-day marine environments. Yon et al. (1982) established that the saturated C20 hydrocarbon carbon skeleton is formed by an unusual linkage of C5 isoprene units (I; Fig. 1). The C2s and C30 alkane structures were later shown to involve further addition of C5 units to this basic skeleton (II, m ; Fig. 1) (Bayona et al., 1983; Robson, 1987). Attempts to identify the position and geometry of the double bonds using standard derivatization methods have not been successful. In view of the geochemical importance of these compounds, there has been considerable speculation about their likely sources. Some authors noted a link to branched hydrocarbons with similar structures produced by Actinomycetes, which led to the suggestion that marine bacteria were a possible source
(Farrington et al., 1977; Boehm and Quinn, 1978; Requejo and Qulnn, 1983). However, the occurrence of these alkenes in seawater particulate matter and sediment trap samples (Prahl et al., 1980; Volkman et al., 1983) implied that a more likely origin was from certain species of phytoplankton. Support for this view came from the work of Nichols et al. (1988) who identified a di-unsaturated C25 HBI alkene in Antarctic sea-ice communities shown by microscopy to consist solely of diatoms. More recently, a new C25 HBI alkene has been isolated from benthic microbial communities dominated by diatoms (Summons et al., 1993). Here we report the first identification of C25 and C30 HBI alkenes in laboratory cultures of two marine diatoms, thus confirming that diatoms are the likely source of these unusual hydrocarbons in many present-day marine environments.
I II III Fig. 1. Structures of the parent C20, C25 and C30 carbon skeletons. The C2o alkane is a major constituent of the hydrocarbons isolated from Rozel Point crude oil. Its structure was elucidated by Yon et al. (1982). In marine sediments and seawater, the C25 and C3o hydrocarbons mainly occur as highly unsaturated alkenes.
407
408
JOHN K . VOLKMAN el al. Table I. Culture conditions and hydrocarbon contents of diatoms studied
Diatom species
White light (IrE m ~ s i ) Axenic
Culture medium
Culture age at harvest (days)
Cell counts × 105 cells/ml
Temp. (C)
f, t~ f2 f: f, f, 1, f? l, f. (3 G2 f,
10 6 13 6 13 10 7 7 7 12 6 I2 12
10.5 ~3 0.02 l 3.3 II 8.4 17.3 14.2 66 4 7 !. 2 1.2 0.87
20 20 20 20 25 20 20 20 25 28 2(I 20 20
70.80 * t00 [00 10(t 100 70 80* iotJ 10t) lot) 7tL 80* I 0(t 7080* 7080*
t, f,
13 10
1.2 0.33
20 20
100 7t~-80"
Amphiphora hyalina CS-28 Amphora sp. CS-10c Coscinodiscus sp. CS-151 Cylindrotheca fusiformis CS- 13c Fragilaria pinnata CS- 121 Navicula sp. CS-46c Nitzsehia closterium CS-5c Skeletonema costatum CS-181 Skeletonema sp. CS-252 Thalassionema nitzschioides CS- 146 Thalassiosira stellaris CS- 16 Thalassiosira rotula CS-77 Thalassiothrix heteromorpha CS-132/8 Species containing HBI alkenes: Haslea ostrearia CS-250 Rhizosolenia setigera CS-62
~ ~ + ....
..... ~
n-C:~
n-C2~ 5 fig/cell)
fig/cell)
41 81 1550"i" 63 9 23 13 ND ND 15 ND ND ND
117 80 2400";" 129 3! 13 3(I 103 28 20 190 265 323
ND ND
396 52
*Cultures transferred after 3-5 days to blue light at 40/aE m - Z s ~.
+Coscinodiscus sp. cell size is quite large; approx. 70/~m in diameter. ND, not detected.
EXPERIMENTAL
Algal cultures Fifteen diatom species were obtained from the CSIRO Algal Culture Collection from genera known to be present in marine environments where HBI alkenes had been identified. These were cultured in Erlenmeyer flasks under a 12 : 12 light: dark cycle. Cultures were grown under white light for 6-13 days at 100/~Em-2s ~ except for those marked • (Table 1) which were grown initially in white light for 3-5 days at 70-80/~ E m 2 s- ~and then transferred to blue light at 40/~Em-2s -1. Light levels were measured with a QSL-100 (Biospherical Instruments) light meter. All cultures were grown in 1 litre Erlenmeyer flasks containing 700-800ml of culture medium t"2(Guillard and Ryther, 1962), or medium G (Jeffrey, 1980) at full strength (CS-16) or half strength (CS-77). Most were aerated with 1% CO2 in air with a 12:12 light:dark cycle; the exceptions were CS-146 and CS-132/8 which were aerated with air alone. The initial inoculum was 75 ml of log-phase culture. Cell counts were measured with a Neubauer haernocytometer. Lipid extraction and analysis After 6--13 days (depending on growth rate), the cells were filtered onto glass fibre filters and extracted with chloroform-methanol-water (Volkman et al., 1992). The total hydrocarbons were isolated by open column chromatography on silica gel and eluted with hexane and hexane:toluene (1:1). The hydrocarbon fractions were analysed by capillary gas chromatog• raphy on a non-polar HPI methyl silicone fused silica column , (50 m x 0.32 mm i.d., Hewlett-Packard) using a Shimadzu 9A gas chromatograph equipped with a F I D and cooled on-column injector (SGE, Australia) (Volkman et al., 1992). Peak areas were quantified using DAPA software (DAPA Scientific,
Kalamunda, Western Australia). Electron-impact mass spectra were obtained using an HP 5890 GC
and 5790 MSD fitted with a direct capillary inlet (Volkman et al., 19921. Hydrogenation A portion of the hydrocarbon fractions from Haslea ostrearia and Rhizosolenia setigera dissolved in 200ill iso-octane (2,2,4-trimethylpentane) was added to a test tube containing 2 ml of iso-octane and 30 mg of Adam's catalyst (PtO2) flushed with hydrogen for 3 h. Following the addition of purified water (Milli-Q"- system), the hydrogenation products were removed in the upper iso-octane layer (4 extractions) and analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). RESULTS
All but two of the diatom species had very simple hydrocarbon distributions. Each of the 15 species studied contained n-heneicosa-3,6,9,12,15,18hexaene ( n - C 2 1 : 6 ; Table 1) which is believed to be formed by decarboxylation of the fatty acid 22:6 (n-3) (docosahexa-4,7,10,13,16,19-enoic acid) which is a minor constituent in all of the diatoms studied (Dunstan et al., 1994). This hydrocarbon is common in diatoms and many other classes of microalgae (Lee and Loeblich, 1971). Eight of the species also contained n-heneicosa-3,6,9,12,15-pentaene (n-C2~:5; Table 1), which we assume is synthesized in like manner by decarboxylation of the 22:5 (n-6) fatty acid. This alkene is rarely reported in microalgae. Trace amounts of squalene were also found in some of the diatoms. Two of the diatoms contained unusual highly branched isoprenoid (HBI) alkenes. Haslea ostrearia contained a suite of at least seven C25 HBI alkenes having 3, 4 and 5 double bonds [Fig. 2(a,b) and Table 2]. These eluted well before n-C25 (in fact just after n-C2j ) on the methyl silicone column indicating that they were highly branched. The major constituent was a C25:4 alkene which comprised 78.5% of the
Highly branched isoprenoid alkenes
409
Ha 25:4
H7
R2
n-C22 IS
25:5
30:5
H1
IS (17.'0 FAME)
n-C21:e H2"
i
Pe
n-C21:6
3O:5
H6
,/~:5(7)
" 3O:6
/Hs
R5
H4. I
i
24
28
~:6 32
I
I
~
~
"lime (minutes)
I
I
I
I
24
28
32
36
I
I
~
Time (minutes)
Fig. 2. Partial capillary gas c h r o m a t o g r a m s of the total h y d r o c a r b o n s in (a) H. ostrearia and (b) R. setigera. Peak identifications are given in Table 2. Alkenes are denoted Cx:y where x is the n u m b e r of c a r b o n a t o m s and y is the n u m b e r of double bonds.
total C25 alkenes. Other constituents were two C25:5 alkenes (2.6 and 8.2%), another C25:4 alkene (3.2%), at least one C25:3 alkene (6.4%) and two other minor components which could not be identified due to weak mass spectra. Major ions in the mass spectra of the alkenes are listed in Table 2. The mass spectrum of the major C25:4 alkene is shown in Fig. 3(b). The presence of
ions at m/z 233, 259, 287 is consistent with a highly branched structure and base peak at m/z 69 indicates the presence of multiple double bonds. Molecular ions were present in the mass spectra of most of the alkenes [Fig. 3(a-d)] allowing the number of double bonds to be assigned (Table 2). Hydrogenation of the total hydrocarbons produced two alkanes: n-eicosane (n-C2~) from n-C21:6
Table 2. C2s and C3o isoprenoid alkenes in Haslea ostrearia and Rhizosolenia setigera GC peak number
ECL*
Conc. fig/cell)
Conc. (/~g/g)
% of total HBI alkencs
Haslea ostrearia H~ C25:3
21.06
189
242
6.4
H2
C25:4 ?
21.34
96
123
3.2
Ha
C2s:4
21.44
2340
2990
78.5
H4
?
21.58
11
15
0.4
H5
?
21.73
22
28
0.7
AIkene
H6
C25:5~
21.91
77
99
2.6
H7
C25:5
22.00
244
313
8.2
Rhizosolenia setigera RI C30:s
25.07
232
88
14.3
R2
C3o:5
25.50
624
237
38.6
R3
C3o:5
25.60
473
179
29.3
R4
C30:6
25.81
212
80
13.1
R5
C3o:6
26.00
76
29
4.7
*ECL, equivalent chain length o n H P I capillary column. tPercentage abundance in parenthesis.
Major ions in electron impact mass spectrumt 41 (63), 55(98), 69(100), 83(85), 95(67), 261 (16), 289 (8), 346 (3) 41 (63), 55 (42), 69 (100), 81 (81), 95(85), 259 (1), 275 (5), 344 (3) 41 (58), 55 (31), 69 (1O0), 81 (62), 95 (35), 177 (4), 259 (5), 275 (3), 329 (I), 344 (0.5) 41 (73), 55 (64), 69 (100), 81 (55), 95(41), 189 (5), 231 (2), 259 (12) 41 (69), 55 (29), 69 (100), 81 (49), 95 (27), 203 (5), 259 (5), 273 (5), 287 (I), 299 (2) 41 (66), 55 (26), 69 (100), 81 (45), 95 (26), 259 (3), 273 (5), 299 (2) 41 (66), 55(21), 69(100), 81 (49), 95 (26), 257 (6), 273 (6), 299 (2), 327 (I)
163(11), 233(21),
41 (60), 55 (37), 69 (100), 81 (42), 95 (29), 299(8), 357(6), 412(1) 41 (56), 55 (100), 69 (71), 81 (57), 95 (58), 259 (10), 315 (22), 327 (3), 412 (14) 41 (59), 55 (37), 69 (100), 81 (62), 95 (31), 231 (2), 299(3), 357(3), 412(3) 41 (72), 55 (54), 69(100), 81 (56), 95 (56), 259(8), 273 (8), 313 (12), 341 (2), 410 (5) 41 (62), 55 (34), 69(100), 81 (41), 95 (28), 246 (2), 273 (8), 341 (0.5), 410 (2)
135 (10), 231 (6),
123 (23), 165(17), 123 (10), 163 (3), 123(15), 163(3), 109 (20), 189 (12), 109(18), 175(5), 121 (12), 189(41),
135 (18), 191 (4), 137(12), 191 (15), 135(22), 189(5), 135 (11), 191 (6),
410
JOHN K. VOLKMANet al.
1-1 (a)
Ic) Rhizosolenia $etigera
Haslea ostrearia PeakH1 (C2s:3)
PeekR= (C~.d
\
55
8OOO
233
4oooi
~ 400o"1
/
289
1/77
I J]
20OO
20(10,
i
O' 50
100
(b)
\
L,
150
I[ ,/
200 250 300 Mass/Charge
350
,i
400
107
259 315
, , , , l o , , , | , , , , l l ,
, ,i
, , , , i
41~
i,
, ,i
,
100 150 200 250 300 350 400
50
M~Charge
Haslea ostrearia
(d) Rhizosolenia se~gera
PeakHs (C-~:4)
~oooo
69
PeakFI4 (C~:e)
\ 69
8000
60001
60O0
X5
0
, 50
259
,1 ,,
/
121
/
.LL
i0b"isb"~"asb"so6"~6"46o Mass/Charge
11!11
/I
s6' i 0 6 " i 5 6 " ~ " ~
300 ~
\
!
400
Mass/Charge
Fig. 3. Electron impact mass spectra of (a) the C25:~(peak number Hi) and (b) the C2s:4 (peak number H2) alkenes in H. ostrearia, (c) the C30:5(peak number R2) and (d) the C30:6(peak number R4) alkenes in R. setigera. Mass spectra were obtained using a Hewlett-Packard Mass Selective Detector (Volkman et al., 1992).
and a single C25 HBI alkane from the C25 alkenes thus confirming that all of the alkenes had the same carbon skeleton (If; Fig. 1). The electron impact mass spectrum of the C25 alkane showed a major ion at m / z 238 and minor ions at m / z 210 and 266 in addition to the usual alkyl ion series. This spectrum is identical with that previously published for the HBI alkane 2,6,10,14-pentamethyl-7-(3-methylpentyl)-pentadecane (Robson and Rowland, 1986; Rowland and Robson, 1990), and its ECL (equivalent chain length) index of 21.10 confirms this assignment. The mass spectrum did not give a molecular ion under electron impact conditions which is common with this class of alkanes (Rowland et al., 1990). The hydrocarbon distribution in R. setigera showed, in addition to n-C21:6, a suite of hydrocarbons eluting between n-C:5 and n-C26. The mass
spectra of these alkenes [Fig. 3(c,d)] confirmed the presence of two C30 highly branched alkenes with 6 double bonds and three with 5 double bonds (Table 2). Two of the C30:5 alkenes (peak numbers RI and R3) had similar mass spectra to that of a C30 penta-unsaturated alkene referred to as "HC412" by Barrick and Hedges (1981), and as the monocyclic tetraene "C30:4:~" by Prahl et al. (1980). Hydrogenation produced a single C30 alkane plus small amounts of two C30 monounsaturated alkenes. The alkane had an identical mass spectrum and retention index to that previously reported for 2,6,10,14,18pentamethyl- 7- (3- methylpentyl)- nonadecane (III; Robson and Rowland, 1988; Rowland and Robson, 1990). The presence of the two monoenes indicates that one or more of the double bonds is in a hindered position and resistant to hydrogenation (as also
Highly branched isoprenoid alkenes concluded by Barrick and Hedges, 1981). Some C25 alkenes also contain a double bond that is difficult to hydrogenate (Rowland et al., 1990). DISCUSSION The isolation of both C25 (II, Fig. 1) and C30 (IlI; Fig. 1) HBI aikenes from the two diatoms H. ostrearia and R. setigera, plus the earlier isolation of a C25 diene from a natural sea-ice diatom population, strongly suggests that diatoms are a major source of C25 and C30 HBI alkenes in present-day marine sediments and seawater. This result explains why these alkenes are abundant in marine environments where diatoms are major constituents of the phytoplankton (e.g. upwelling areas off Peru, productive coastal regions, some algal mats and sea-ice diatom communities). Furthermore, carbon isotope data obtained for compounds isolated from a Messinian evaporite showed that the C25 HBI alkanes had the same ratio of ~2C to 13C as algal-derived sterols (Kohnen et al., 1992). The isotopic compositions of a C20 HBI alkane and two C25 HBI alkenes isolated from diatomaceous microbial communities also suggested they were of microalgal origin (Summons et al., 1993). Several of the cultures, including H. ostrearia and R. setigera, were not axenic (Table 1), but the level of bacteria present was extremely low. Branched-chain fatty acids typical of bacteria represented less than 1 and 3.3% of the total fatty acids in H. ostrearia and R. setigera respectively (unpublished data), indicating a very minor contribution of bacteria to the extractable lipids. Similar amounts of bacteria were present in other cultures which did not contain HBI alkenes. Moreover, the HBI alkenes were much more abundant than the n-C2~:6 alkene, which is of undisputed algal origin, so a contamination source for the HBI alkenes in the cultures can be safely ruled out. It should be noted, however, that the distribution of C25 HBI isomers found in Haslea is different from those previously reported in marine sediments and seawater even though the alkenes possess the same unusual carbon skeleton. For example, in coastal sediments off Peru, a suite of tri- and tetraunsaturated C25 HBI alkenes dominate the hydrocarbon distributions (Volkman et al., 1983). These elute on the non-polar capillary column in the same region as the Haslea HBI alkenes, but did not co-elute with them. However, the triunsaturated C25 HBI alkenes found in the particulate matter and sediments delta of the Erbo River, Spain (Albaigrs et al., 1984a,b), referred to as i-25:3 (Rloa_ 5 2119 and RisE.30 2140) have similar mass spectra to that of the C25:3 HBI (peak Hi) found in H. ostrearia. C30 HBI alkenes appear to be common in marine sediments, but are rarely reported as major constituents. It is apparent from the limited amount of retention data reported to date that at least some isomers are different from those found here in
411
R. setigera. These compounds should not be confused with the complex mixture of C30 alkenes, containing two to five double bonds, identified as hydrosqualenes which have been found in some sediments (AIbaigrs et al., 1984a). These elute after n-C27 (i.e. much later than the HBI alkenes found in R. setigera), and have significantly different mass spectra. However, a suite of C3o HBI alkenes have been found in mussels and seawater particulate matter in Port Phillip Bay, southeastern Australia (A. P. Murray and J. K. Volkman, unpublished data), which show a similar distribution to those found in R. setigera. Two C30:5 alkenes and one C30:6 alkene co-elute with those found in R. setigera (peaks Rl, R3 and Rs) while the other major C30:6 alkene has a different retention index. This study of mussels and seawater particles was performed at the time of a bloom of R. chunii in Port Phillip Bay which rendered mussels and shellfish in the area bitter-tasting and hence unpalatable for many months (Parry et al., 1989). This strongly suggests that the source of the alkenes found was R. chunii; the slightly different hydrocarbon distribution to that found in R. setigera is probably due to interspecies variations within the genus Rhizosolenia. Cultures of R. chunii are not yet available to test this hypothesis. It is clear from our data that only a small proportion of diatoms synthesize these unusual hydrocarbons. The two species found here belong to different Orders (Haslea is in Pennales while Rhizosolenia is in Centrales), but many other species from both of these Orders do not contain HBI alkenes (Table 1). Both algal genera are common in marine environments and thus could be significant sources of alkenes in particular cases, but it seems that other species are likely to be major sources. The genus Haslea contains at least 9 species, some formerly of the genus Navicula, which have been isolated from both temperate and tropical marine environments (Ricard, 1987a). Haslea ostrearia is common in coastal areas and it produces an unusual pigment called marennine which is responsible for the bluegreen colouration of the gills found in oysters grown in sea basins along the French coast (Neuville and Daste, 1978; Robert, 1986). Rhizosolenia is a common genus in coastal and oceanic marine waters world-wide, with several species found in Antarctica and some in the Arctic regions (Priddle et al., 1990; Priddle and Fryxell, 1985). There are about sixty marine and only five freshwater species known (Ricard, 1987b). In some cases, the taxonomy, even to genus level, is uncertain (Priddle and Fryxell, 1985). Sundstr6m (1986) has proposed a revision of the genus Rhizosolenia, and recommended that some species, including R. setigera, should be transferred to other genera. Large mats and rafts of diatoms from the genus Rhizosolenia are found over wide ranges in the Pacific, Indian and Atlantic oceans (e.g. Carpenter et al., 1977; Alldredge and Silver, 1983; Sancetta
412
JOlly K. VOLKMANet al.
et at., 1991 ). These mats can be up to 4.4 m 3 in volume (Martinez et al., 1983) and consist of intertwining chains of either single or two or more different species o f Rhizosolenia (Carpenter et al., 1977; Alldredge and Silver, 1982). They are not always visible from ships, but are very obvious when sampled using scuba techniques to prevent mat disintegration (Alldredge and Silver, 1982; Martinez et al., 1983). The mats sink quickly to the sea floor when the rhizosolenid cells lose buoyancy, carrying large amounts of organic material to the sediments (Sancetta et al.. 1991). Rising mats of Rhizosolenia may also be an important means of bringing nitrate from depth to surface waters in the North Pacific (Villareal et al., 1993). It is believed that mass sinking of rhizosolenid mats may be a c o m m o n occurrence (Sancetta et aL, 1991). The impact of sinking diatoms, present and past, on the benthos and formation of sediments could thus be much greater than currently realized. Vast regions of the worlds seas are believed to have been covered by floating mats of diatoms of the genus Thalassiothrix from 4.5 to 15 million years ago, with the total area covered being about the size of Australia (Kemp and Baldauf, 1993; Sancetta, 1993). These mats formed laminated sediments under reducing conditions so that the organic matter was well preserved (Kemp and Baldauf, 1993). However, the delicacy and size of rhizosolenid diatoms makes their preservation in the sediments very unlikely (Sancetta et al., 1991). Floating mats can be formed by several genera of" diatoms characterized by elongate cells (Sancetta, 1993), so the sinking of mats provides a route to the sediments of HBI alkenes produced by rhizosolenid or other, as yet undiscovered, source genera. This may be one reason why HBI alkanes and alkene diagenetic products have been found in ancient sedimentary rocks and crude oils (Yon et al., 1982; Sinninghe Damst6 et al., 1989) It is possible that the previous isolation of a C20 HBI alkane and monoene and a C25 diene in the macroscopic green alga E n t e r o m o r p h a proliJera (Rowland et al., 1985), may have been due to presence of epiphytic diatoms, particularly since n-C:~ 6 was also observed in the hydrocarbon fraction and this alkene is more commonly associated with a microalgal source (Lee and Loeblich, 1971). These HBI alkenes are not present in the closely related green alga E. intestinalis (Volkman, unpublished data). However, the possibility that these compounds might be synthesized by other algal classes cannot be excluded, especially since unsaturated isoprenoid alkenes of the botryococcene and lycopadiene type are known in microalgae from the green algal genus Botryococcus (Metzger and Casadevall, 1987; Metzger et al., 1985, 1991). It may be significant that a C20 HBI alkene has yet to be isolated from diatoms in laboratory culture. Further studies of other classes of microalgae need to be carried out, but the results presented here clearly establish diatoms as a major
source of these unusual hydrocarbons in marine sediments. CONCLUSIONS
Two species of marine diatoms, Haslea ostrearia and Rhizosolenia setigera, have been shown to produce C25 and C30 highly branched isoprenoid alkenes. This represents the first isolation and identification of these unusual hydrocarbons from cultures of marine microalgae. Without excluding the possibility that other classes of algae may produce these isoprenoid hydrocarbons, our data show clearly that diatoms are likely to be a major source in present-day marine sediments and waters. Acknowledgements---We thank Dr S. W. Jeffrey for providing the algal cultures, Ms Jeannie-Marie LeRoi for culturing the algae and cell counts, Dr Roger Summons for many helpful discussions and Dr Andrew Revill for commenting on the manuscript. This work was funded in part by the Chevron Oil Field Research Company and by the Fishing Industry Research and Development Corporation.
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