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Geochimica et Cosmochimica Acta 72 (2008) 1396–1414 www.elsevier.com/locate/gca
Okenane, a biomarker for purple sulfur bacteria (Chromatiaceae), and other new carotenoid derivatives from the 1640 Ma Barney Creek Formation
Jochen J. Brocks a,*, Philippe Schaeffer b
a Research School of Earth Sciences and Centre for Macroevolution and Macroecology, The Australian National University, Canberra, ACT 0200, Australia b Laboratoire de Ge´ochimie Bio-organique, CNRS UMR 7177, Ecole Europe´enne de Chimie, Polyme`res et Mate´riaux, 25 rue Becquerel, 67200 Strasbourg, France
Received 20 June 2007; accepted in revised form 12 December 2007; available online 23 December 2007
Abstract
Carbonates of the 1640 million years (Ma) old Barney Creek Formation (BCF), McArthur Basin, Australia, contain more than 22 different C40 carotenoid derivatives including lycopane, c-carotane, b-carotane, chlorobactane, isorenieratane, b-iso- renieratane, renieratane, b-renierapurpurane, renierapurpurane and the monoaromatic carotenoid okenane. These biomark- ers extend the geological record of carotenoid derivatives by more than 1000 million years. Okenane is potentially derived from the red-colored aromatic carotenoid okenone. Based on a detailed review of the ecology and physiology of all extant species that are known to contain okenone, we interpret fossil okenane as a biomarker for planktonic purple sulfur bacteria of the family Chromatiaceae. Okenane is strictly a biomarker for anoxic and sulfidic conditions in the presence of light (photic zone euxinia) and indicates an anoxic/oxic transition (temporarily) located at less than 25 m depth and, with a high proba- bility, less than 12 m depth. For the BCF, we also interpret renierapurpurane, renieratane and b-renierapurpurane as biomarkers for Chromatiaceae with a possible contribution of cyanobacterial synechoxanthin to the renierapurpurane pool. Although isorenieratane may, in principle, be derived from actinobacteria, in the BCF these biomarkers almost certainly derive from sulfide-oxidizing phototrophic green sulfur bacteria (Chlorobiaceae). Biological precursors of c-carotane, b-carotane and lycopane are found among numerous autotrophic and almost all phototrophic organisms in the three domains of life. In the BCF, a paucity of diagnostic eukaryotic steroids suggests that algae were rare and, therefore, that cyanobacterial carotenoids such as b-carotene, echinenone, canthaxanthin and zeaxanthin are the most likely source of observed b-carotane. c-Carotane may be derived from cyanobacteria, Chlorobiaceae and green non-sulfur bacteria (Chloroflexi), while the most likely biological sources for lycopane in the BCF are carotenoids of the lycopene, rhodopin and spirilloxanthin series abundant in purple sulfur bacteria. 2007 Elsevier Ltd. All rights reserved.
1. INTRODUCTION that are distinguished by a large variety of functional groups and different cyclic and linear end-groups. All photosyn- Carotenoids preserved in sediments or detected in living thetic eukaryotes, bacteria, and halophilic archaea, and a organisms have been used to study environmental condi- large variety of non-photosynthetic organisms, can biosyn- tions, evolutionary processes and biological origins (e.g. thesize carotenoids de novo. The usually yellow to red col- Britton et al., 1995; Frank et al., 1999; Xiong et al., 2000). ored carotenoids function as accessory pigments in the More than 600 different carotenoid structures are known light harvesting complex of phototrophic organisms, and are important for photoprotection, phototropism and the * Corresponding author. coloration of plants and animals. Although the plethora of E-mail address: [email protected] (J.J. Brocks). functionalized carotenoids is based on a very limited
0016-7037/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.gca.2007.12.006 Author's personal copy
Okenane and other carotenoid derivatives 1397 number of different carbon skeletons, some hydrocarbon tions of a shallow lacustrine or sabkha depositional setting carotenoids retain a taxonomically diagnostic structure in (Jackson et al., 1988) have been abandoned, and the BCF ancient sedimentary rocks and function as important bio- is now recognized as a marine succession that accumulated markers for paleoenvironmental reconstructions. Particu- in a quiet, sub-wave base environment (Bull, 1998; Jackson larly the aromatic carotenoid derivatives isorenieratane I et al., 2000; Shen et al., 2002). The basin was most likely con- and chlorobactane II and their breakdown products have be- nected to the ocean to the north-east or east. However, the come important paleoenvironmental proxies. They are com- syndepositionally active Emu fault on the eastern perimeter monly interpreted as biomarkers for green sulfur bacteria probably formed a sill and may have restricted exchange of (Chlorobiaceae), indicating anoxic and sulfidic (euxinic) con- basin waters with the ocean. Just how extensively the basin ditions in the photic zone of the water column (Schaefle´ et al., was connected to the ocean remains unknown. 1977; Summons and Powell, 1986; Kohnen et al., 1992). Thirty-eight samples were collected from the upper BCF In modern ecosystems, planktonic Chlorobiaceae are in the Glyde River sub-basin at the southern end of the Batten commonly restricted to stratified lakes and silled fjords. Trough (drill cores GR3, 5, 7, 8 and 10). The sample depths Open marine habitat in the modern, mostly oxygenated for all samples, and presence or absence of carotenoid deriv- oceans does not offer suitable habitat (with the notable atives are given in Brocks et al. (2005). The BCF in the Glyde exception of the Black Sea; Repeta et al., 1989). However, River region arguably contains the best preserved organic during Oceanic Anoxic Events in the Phanerozoic, photo- matter in the world older than one billion years (Jackson trophic sulfur bacteria (PSB) existed in the open ocean (Sin- et al., 1986). The dominant lithologies are thinly bedded or ninghe Damste´ and Ko¨ster, 1998), and they may have planar laminated, dolomitic, carbonaceous and pyritic silt- played a particularly important ecological role in the oceans stones and shales. The dolomitic facies typically contain of the ‘mid-Proterozoic’ interval ( 1800 to 800 Ma ago). In 0.2–2% organic carbon, and locally up to 7% (Powell et al., a seminal paper in 1998, Canfield suggested that global 1987). The organic matter most commonly occurs in discrete oceans did not become widely oxygenated with the disap- bedding-parallel stringers or in continuous, sub-millimeter pearance of banded iron formation 1800 Ma ago, but re- planar laminated layers that represent deep-benthic micro- mained largely anoxic and additionally became sulfidic. bial mats or the episodic flocculation of suspended organic Such an euxinic world ocean would have offered ideal grow- matter. The kerogen-rich dolostones recovered from GR drill ing conditions for green and purple sulfur bacteria wherever cores are essentially undeformed and unmetamorphosed, euxinic conditions rose into the photic zone of the water and the organic matter is marginally mature. The H/C ratio column. According to Canfield’s scenario, global euxinia of kerogen from GR10 reaches values >1.6, and RockEval may have prevailed well into the Neoproterozoic (1000– data for drill core GR7 include Tmax = 435–450 C, Produc- 542 Ma). In a recent study, we tested the assumption of a tion Index PI = 0.07–0.50 and Hydrogen Index HI = 77–732 mid-Proterozoic ‘Age of Phototrophic Sulfur Bacteria’ by (Powell et al., 1987). In contrast to the Glyde River region, studying the biomarkers of the thermally moderately ma- organic matter in the central and northern regions of the Bat- ture, 1640 Ma Barney Creek Formation (BCF) in the ten Trough is thermally usually less well preserved. McArthur Basin, northern Australia (Brocks et al., 2005). The rocks from the BCF yielded abundant aromatic carot- 2.2. Sample preparation and analysis enoid derivatives from green sulfur bacteria, but also new carotenoid hydrocarbons that were interpreted as biomark- Samples were processed in batches of 10, including one ers for purple sulfur bacteria. Here, we present a detailed kerogen-free sample (‘blank-rock’) and one procedural discussion of the distribution, chemical structure and eco- blank (Brocks et al., 2003). Rock samples were cleaned by logical significance of 22 derivatives of carotenoids found repeated ultrasonication in distilled water for 10 s. For in the BCF, including several new structures. a selected set of samples, surfaces were trimmed with a clean precision wafering saw (Buehler Isomet 1000; blade 2. GEOLOGY AND METHODS thickness 340 or 460 lm), and the combined surface mate- rial and the remaining rock core were separately processed 2.1. The Barney Creek Formation (BCF) and analyzed. The dried rock samples were ground to smal- ler than 200 mesh grain size in an alumina ring-mill. The The 1640 ± 3 Ma (Page and Sweet, 1998) Barney Creek mill was cleaned between samples by grinding annealed Formation of the McArthur Group, northern Australia, quartz sand two to three times for 60 s. Twenty to 25 g rock was deposited in a north-south-trending rift basin, the Bat- powder were extracted with dichloromethane (DCM):meth- ten Trough, west of the present Gulf of Carpentaria. The anol (9:1 v/v) with a Dionex Automated Solvent Extractor. intracratonic basin presently extends over a known area of The extracts were reduced to 100–500 lL under a stream of 25,000 km2, but deposition probably continued to the north purified nitrogen gas and separated into saturated, and east (Bull, 1998). The highly variable depositional thick- aromatic and polar fractions using column chromatogra- ness of the BCF, with a local maximum of 1000 m in the phy over 12 g annealed (450 C/24 h) and dry-packed Glyde River sub-basin in the south, suggests a complex silica gel (Silica Gel 60; 230–400 mesh; EM Science). bathymetry and variable water depths. The Barney Creek Saturated hydrocarbons were eluted with 1.5 dead volumes Formation shallows out towards the western and southern (DV) n-hexane, aromatic hydrocarbons with 2 DV margins of the basin and is bounded in the north by the ear- n-hexane: DCM (1:1 v/v) and polars with 2 DV lier Paleoproterozoic Tawallah anticline. Older interpreta- DCM:methanol (1:1 v/v). After fractionation, 50 ng D4 Author's personal copy
1398 J.J. Brocks, P. Schaeffer / Geochimica et Cosmochimica Acta 72 (2008) 1396–1414
(d4-C29-aaa-ethylcholestane; Chiron Laboratories AS) was 1987) and by GC coinjection experiments on a 60 m DB-5 added as internal standard to the saturated hydrocarbon column using synthetic C14 trimethylaryl isoprenoid iso- fraction, and 0.5 lgD14 (d14-para-terphenyl, 98 atom-% mers with 2,3,4-, 2,3,6-, 2,4,5-, 2,3,5- and 2,4,6-trimethyl deuterium; Aldrich Chem. Co.) to the aromatic hydrocar- substitution patterns. The GC oven was programmed at bon fraction. For selected samples, the aromatic fraction 60 C (2 min), heated to 200 Cat4 C/min, and finally was further separated into monoaromatics, diaromatics heated to 315 Cat10 C/min. Isorenieratane I, chlorobac- and higher aromatics by thin layer chromatography on an- tane II, okenane III, renieratane IV and renierapurpurane nealed (120 C, 12 h) and pre-extracted silica gel glass plates V (=perhydrorenierapurpurin) were identified by their mass (Silica Gel 60, F254; 0.25 mm film thickness; Merck). The spectra and by GC coinjection experiments on a DB-5 col- plates were developed twice using n-hexane as mobile phase. umn (60 m) using synthetic standards. The GC oven was programmed at 60 C (2 min), heated to 240 Cat10 C/ 2.2.1. Gas chromatography–mass spectroscopy (GC–MS) min, and finally heated to 315 Cat4 C/min. The identity GC–MS analyses were carried out on a Micromass of chlorobactane II and okenane III was re-confirmed by AutoSpec Ultima equipped with a HP6890 gas chromato- coelution experiments on a 60 m DB-1 column. Lycopane graph (Hewlett Packard) and a DB-1 or DB-5 capillary col- VI and b-carotane VII were identified by their mass spectra umn (60 m · 0.25 mm i.d., 0.25 lm film thickness) using He and by GC coinjection experiments with synthetic standard as carrier gas. The MS source was operated at 250 C in EI- on a 60 m DB-5 column (standards courtesy Geoscience mode at 70 eV ionization energy and with 8000 V accelera- Australia). The GC oven was programmed at 60 C tion voltage. Samples were injected in pulsed splitless mode (2 min), heated to 100 Cat8 C/min, and finally heated into a Gerstel PTV injector at a constant temperature of to 315 Cat4 C/min. c-carotane VIII was identified by 300 C. For full-scan and selected ion recording (SIR) its mass spectrum and by GC coinjection experiments on experiments, the GC oven was programmed at 60 C a 60 m DB-5 column using synthetic c-carotane VIII. The (2 min), heated to 315 Cat4 C/min, with a final hold time GC oven was programmed at 60 C (2 min), heated to of 35 min. The AutoSpec full-scan duration was 0.8 s plus 250 Cat10 C/min, and finally heated to 315 Cat4 C/ 0.2 s interscan delay over a mass range of 50–600 Da. Aro- min. The identity of c-carotane VIII was re-confirmed by matic hydrocarbons were analyzed by SIR under magnet coelution experiments on a 25 m HP Ultra 1 column. control with a total cycle time of 1.2 s per scan for 23 ions. GC–MS analyses in the chemical ionization (CI) mode 2.2.3. c-Carotane synthesis (Fig. 1) were performed on a Varian CP-3800 gas chromatograph Trans-1-Bromo-3,7-dimethyl-2,6-octadiene (geranylbro- coupled to a Varian 1200L mass spectrometer using isobu- mide, 5.279 g, 24.3 mmol) and triphenylphosphine tene as ionization gas (gas pressure: 2.5 Torr) at 70 eV. The (5.808 g, 22.1 mmol) were dissolved in toluene (50 mL) temperature of the source was set at 180 C and the scan- and maintained under reflux in a nitrogen atmosphere. ning mass range was between 100 and 700 Da. Chromato- After 5 min, a white precipitate formed, and the reaction graphic separations were performed on a HP-5MS was cooled down to room temperature and filtered under column (30 m · 0.32 mm, 0.25 lm film thickness) using he- vacuum. The white precipitate was rinsed several times lium (32 cm/s at 40 C) as carrier gas and a temperature using hexane, yielding quantitatively the desired phospho- program of 70–200 C (10 C/min), 200–300 C(4 C/ nium salt. The phosphonium salt (883 mg, 2.9 mmol) was min), followed by isothermal at 300 C. suspended in anhydrous diethylether (20 mL). After addi- tion of 1 N n-butyllithium in hexane (1.5 mL, 1.5 mmol), 2.2.2. Identification of aryl isoprenoids and carotenoid the solution turned red and was stirred for 5 min. b-Apoc- derivatives arotenal (438 mg, 1.05 mmol) dissolved in anhydrous DCM The 2,3,4- and 2,3,6-trimethylaryl isoprenoid series were was then added. The evolution of the reaction was followed identified by their mass spectra (Summons and Powell, by TLC and stopped after 1 h. The solvent was removed
Φ P 3, toluene +Φ - Br P 3Br
1) n-BuLi, ether
+Φ - P 3Br O 2) H β ( -apocarotenal) H2, PtO2 AcOEt, AcOH
γ-carotane VIII
Fig. 1. Total synthesis of c-carotane VIII. Author's personal copy
Okenane and other carotenoid derivatives 1399 under vacuum, and silica gel liquid chromatography using a 2.2.4. Pseudo-Kovats indices mixture of hexane/DCM (8:2, v/v) as eluent yielded 320 mg Retention indices were computed using third order (sat- (0.6 mmol) c-carotene (60%). An aliquot of c-carotene urated hydrocarbons) or fourth order (aromatics) polyno- (13 mg) was catalytically hydrogenated using PtO2 in ethyl- mial regression equations that were fit to the elution times acetate under reflux and vigorous stirring. After 5 h, the of C31 to C45 n-alkanes as the reference compounds. The reaction was monitored by GC, indicating that several GC column was a 60 m DB-1 (0.25 mm i.d., 0.25 lm film partly hydrogenated carotenoids were still present. The thickness). Helium was used as carrier gas at a constant hydrogenation reaction was then carried out under more pressure of 27 psi. The GC oven was programmed from drastic conditions using a mixture of acetic acid/ethyl ace- 40 to 200 Cat10 C/min and then to 320 Cat4 C/min. tate (1:3, v/v) as solvent. After refluxing for an additional period of 5 h, pure c-carotane VIII (GC and GC–MS deter- 3. RESULTS mination) was obtained. 1H NMR analysis of the c-carotane standard VIII was 3.1. General biomarker distribution performed on a Bruker DPX 400 (Advance Series) spec- trometer operating at an observation frequency of Fig. 2A shows the GC–MS total ion current of the sat- 400.1 MHz. The preparation of c-carotane by catalytic urated hydrocarbon fraction of a thermally exceptionally hydrogenation of c-carotene led to the formation of a high well preserved sediment from the upper section of the number of (stereo)isomers that were not separated by GC. BCF in the Glyde River region. Although signs of biodeg- The 1H NMR spectrum of the isomeric mixture shows radation are absent, the saturated hydrocarbon fraction unresolved, superimposed signals at 1.5–1.0 ppm ( 50 pro- consists to >90% of unresolved complex mixture (UCM). tons, methine and methylene) and 1.0–0.75 ppm ( 30 pro- n-Alkanes were detected up to C34. Regular acyclic isopre- tons, mainly methyl groups). The chemical shifts are noids with 15–20 carbon atoms are abundant, including reported in ppm relative to tetramethylsilane with the sol- farnesane (i15), pristane (pr) and phytane (ph), and are 1 vent used as internal standard (CDCl3: d H 7.27 ppm). probably predominantly derived from the isoprenoid
A B C31 m/z 191
C32 C 33 C 34 C35
C VIII ph i25 m/z 125 C21 VII
sq
i16 D pr TIC VI
TIC C28
i15
C13 VI VIII VII
20 30 40 50 60 70 80 min
Fig. 2. (A) GC–MS total ion current (TIC) of the saturated hydrocarbon fraction of B03162cS, a representative sample of the uppermost
Barney Creek Fm. in the Glyde-River sub-basin. (B) Mass chromatogram of m/z 191 showing S and R isomers of C31 to C35 ab-hopanes. (C) Mass chromatogram of m/z 125 indicating the presence of unidentified cyclic carotenoid derivatives eluting directly after b-carotane VII and c-carotane VIII. (D) Magnification of the TIC of the elution range of C40 carotenoid derivatives. ., n-alkanes (Cx) with carbon number x; d, acyclic isoprenoids (ix) with carbon number x; pr, pristane; ph, phytane; sq, squalane. The dotted line indicates the base line and highlights the overlying unresolved complex mixture. Author's personal copy
1400 J.J. Brocks, P. Schaeffer / Geochimica et Cosmochimica Acta 72 (2008) 1396–1414 side-chain of (bacterio)chlorophylls. In some samples, regu- ever, aromatization, cyclization and cleavage products re- lar acyclic isoprenoids with 21–25 carbon atoms (i21 to i25) lated to b-carotene VII that are abundant in other are abundant (Fig. 2A). formations (Koopmans et al., 1997), are conspicuously ab- The distribution of pentacyclic terpenoids and steroids sent in the BCF. in the BCF was described by Brocks et al. (2005). In brief, C27 to C35 hopanes (Fig. 2B) and C28 to C36 3b-methylho- 3.3. m/z 134 series of mono- and diaromatic carotenoid panes are abundant in thermally moderately mature sec- derivatives tions of the BCF. The 3b-methylhopane index (Brocks et al., 2003) for the C31 homologue (C31-3b-MHI) is very The two quantitatively most important compounds in high (average 5.7%, n = 9; drill core GR-7) and probably sample B03162 are represented by a double peak in the indicates significant activity of Type I methanotrophic bac- m/z 134 mass chromatogram of the aromatic fraction teria. In contrast to 3b-methylhopanes, the concentration (Fig. 3B). Both compounds are characterized by a molecu- of 2a-methylhopanes relative to desmethylhopanes is very lar mass of 554 Da (Fig. 5C), consistent with the sum for- low (C31-2a-MHI <1%). The low abundances of 2a-meth- mula C40H74 of reduced monoaromatic carotenoid ylhopanes in the dolomitic mudstones of the BCF are in derivatives. The first eluting compound was identified as strong contrast to other Precambrian carbonates where chlorobactane II, while the second belonged to a hydrocar- these biomarkers are almost always very abundant and bon biomarker unknown from other sedimentary sequences commonly ascribed to cyanobacteria (Summons et al., except as a H2/PtO2-hydrogenation product of partly re- 1999). Although the thermal history of the BCF was evi- duced carotenoids from recent sediments from lake Cadag- dently mild enough for extended hopanes to be preserved no, Switzerland (Schaeffer et al., 1997; Hebting et al., 2006). in unusually high concentrations, thermally equally stable By coelution experiments with an authentic standard (Scha- C26 to C29 steranes were below detection limits in all sam- effer et al., 1997), we identified this compound as a 2,3,4-tri- ples where rock surfaces had been removed. In Phanerozoic methyl substituted isomer of chlorobactane, okenane III,a environments, C26 to C29 steranes are regarded as diagnos- potential hydrocarbon derivative of the carotenoid okenone tic markers for eukaryotes, particularly algae. The apparent IX. The mass spectrum of synthetic okenane III is shown in absence or extremely low concentration of steranes in the Fig. 4B. The chromatograms of almost all samples that studied sections of the BCF suggests that eukaryotic organ- contained okenane III also showed three compounds I, isms may have played only an insignificant role in the off- IV and V with a molecular mass of 546 Da (Fig. 5C). Using shore environment of the Paleoproterozoic McArthur coelution experiments with authentic standards (Schaeffer Basin. In contrast to steranes, triaromatic steroids were de- et al., 1997), we identified the first eluting compound as iso- tected in relatively high concentrations (60–130 ppm). renieratane I, the second as renieratane IV and the third as However, the aromatic steroids entirely lack side-chain renierapurpurane V (Schaefle´ et al., 1977). The m/z 134 alkylation at C-24 and are >90% methylated in position 4 mass chromatogram in Fig. 5C also shows two chromato- of ring-A. This steroid alkylation pattern is extremely rare graphic peaks d1 and d2 with molecular masses of 552 Da, in eukaryotes but commonly observed in Type I methano- indicative of monoaromatic bicyclic carotenoids. The elu- trophic bacteria (Bird et al., 1971; Volkman, 2003), giving tion position and mass spectrum of d1 is consistent with further evidence for the existence of these organisms in b-isorenieratane X (compare Pseudo-Kovats indices in Ta- the BCF. ble 1 with Table 2 in Hartgers et al. (1993); note that the Like the saturated hydrocarbons (Fig. 2), the aromatics indices in the 1993 publication are consistently offset by in the BCF are strongly dominated by a (UCM) (Fig. 3C). 1% relative to the new values, presumably due to slight In the upper sections of the Glyde River cores, aromatic polarity differences in GC columns). By comparing the carotenoid derivatives are the most abundant biomarkers chromatographic behavior of d1 and d2 with Fig. 1b in Beh- (Fig. 3B). The sum of C14 to C37 2,3,6- and 2,3,4-trimethyl- rens et al. (2000), we suggest that d2 represents carotenoid aryl isoprenoids constitute 0.1% of the mass of the entire XI with a 2,3,4-trimethyl substitution pattern (‘b- aromatic fraction. In the thermally least altered bitumens, it renierapurpurane’). was possible to distinguish a wide range of intact mono- In a magnification of the m/z 134 mass chromatogram and diaromatic carotenoid derivatives with molecular (Fig. 5C), a cluster of several additional chromatographic masses from 544 to 554 Da. peaks becomes visible eluting just before chlorobactane II. The compounds have a molecular mass of 540 Da consis- 3.2. Carotanes: lycopane, c-carotane and b-carotane tent with the sum formula C39H72 of monoaromatic isopre- noids. The m/z 540 mass chromatogram in Fig. 5B reveals The BCF is currently the only known Precambrian se- that the cluster comprises at least six compounds c1 to c6. quence that contains aliphatic carotenoid derivatives The compounds may represent derivatives of okenane III (Brocks et al., 2005). Based on mass spectra and GC coelu- and chlorobactane II after loss of one of the seven methyl tion experiments with synthetic standards, we identified groups along the aliphatic side-chain, even though this does lycopane VI, b-carotane VII and c-carotane VIII (Fig. 2). not appear to be a likely diagenetic reaction. The m/z 540 The mass spectrum of synthetic c-carotane is shown in mass chromatogram (Fig. 5B) also reveals a second cluster Fig. 4A. The m/z 125 mass chromatogram of the saturated of at least five signals b1 to b5 with the base ion m/z 119. We fraction also contains several unidentified compounds that suggest that b1 to b5 have structures XII–XVI, representing may represent isomers of c- and b-carotane (Fig. 2C). How- the five substitution patterns that may form through loss of Author's personal copy
Okenane and other carotenoid derivatives 1401
Fig. 3. Mass chromatograms of the aromatic fraction of sample B03162eA (drill core GR7; 45.35 m). (A) Mass chromatogram of m/z 173 showing a novel series of aromatic isoprenoids. For pseudohomologues >C27, molecular ions were below detection limits and carbon numbers were estimated by comparison with the elution position of m/z 134 series compounds (diagonal dotted lines). (B) Mass chromatogram of m/z 134 series aryl isoprenoids and aromatic carotenoids. (C) TIC revealing a dominant unresolved complex mixture (UCM). The dotted line indicates the base line and highlights the overlying UCM. d, m/z 173 series isoprenoids; j, 2,3,6-trimethylaryl isoprenoids; m, 2,3,4- trimethylaryl isoprenoids. Author's personal copy
1402 J.J. Brocks, P. Schaeffer / Geochimica et Cosmochimica Acta 72 (2008) 1396–1414
57 A 100
γ-carotane VIII % 83 125
560 196 0 50 100 150 200 250 300 350 400 450 500 550 m/z
B 100 134
133
% okenane III
57 554 71 119 0 80 120 160 200 240 280 320 360 400 440 480 520 560 m/z
173 C 100 173
R + ? ?
% m/z 173 xx R = H
(133) 158 C18H28 157 (119) 143 159 244 (77) (91) (105) 229 0 50 70 90 110 130 150 170 190 210 230 250 m/z
Fig. 4. (A) Mass spectrum of synthetic c-carotane, (B) synthetic okenane, and (C) the C18 pseudohomologue of m/z 173 series aryl isoprenoids (sample B03162eA). Fragment ions m/z 173, 159, 158, 157, and 143 are common to all members of the pseudohomologues series. Fragment ions in parentheses, particularly 119 and 133 Da, cannot be interpreted due to high signal noise from coeluting compounds. one methyl group from the aromatic ring of okenane III tion positions of the pseudohomologues of the m/z 173 ser- and chlorobactane II. ies (Fig. 3A) are very similar to the m/z 134 Da series (Fig. 3B). Moreover, in both series, the concentration of 3.4. m/z 173 series of aromatic carotenoid derivatives C17,C23,C28 and C33 pseudohomologues is significantly lower than those with other carbon numbers, suggesting A series of aromatic isoprenoids that is, to our knowl- that the parent compounds are acyclic isoprenoids with a edge, new, has the base ion m/z 173 (Figs. 3A, 4C and central tail–tail link. The mass of 173 Da corresponds to 5A). It comprises pseudohomologues with 14–37 carbon aC13H17 end-group, consistent with a bicyclic monoaro- atoms (Fig. 3A), three compounds a1 to a3 having a molec- matic structure. The high intensity of the m/z 173 fragment +Å ular mass of 552 Da (C40H72), four compounds a4 to a7 relative to M in the mass spectrum shown in Fig. 4C sug- þÅ with unknown molecular masses (Fig. 5A), and a8 with a gests that the C13H17 fragment forms by cleavage of the very weak molecular ion at m/z 544. In contrast to the m/ isoprenoid side-chain b to an aromatic ring. z 134 mass chromatogram that shows two well separated Two structures that have an isoprenoid skeleton isomers for each pseudohomologue (Fig. 3B), each carbon theoretically yield a m/z 173 base ion by b-cleavage of the number in the m/z 173 series is only represented by a single side-chain, the b-ionene end-group XVII and the chromatographic peak (Fig. 3A). However, the relative elu- tetramethyl indan moiety XVIII. b-Ionene is a known Author's personal copy
Okenane and other carotenoid derivatives 1403
a4 A (XXI ?) m/z 173 M + = ?
M+ = 552 M+ =544
a1 a2a3 a8 (XXIII?)
III M+ =?
a7 II (XXII?)
a5 a6
b+ = 119/120 b+ = 134 B (XII - XVI ?) c c5 b2 b c 4 m/z 540 4 2 c c6 b3 1 c C39H72 b 3 b1 5
554 II III C 552 M+ = 540 d 1 d2 m/z 134 568 (X?) (XI?) M+ = 546 V 554 554 IV I
74 76 78 80 82 84 86 88 90 92 min
Fig. 5. Detail of the high molecular weight range of Fig. 2A and B. (A) Mass chromatogram of m/z 173 showing signals of potential parent carotenoids a1 to a8 of the m/z 173 aryl isoprenoid series. (B) Mass chromatogram of m/z 540 showing carotenoid derivatives with the sum formula C39H72. (C) Mass chromatogram of m/z 134 showing aryl isoprenoids and aromatic carotenoids with molecular masses of 540– 568 Da. The peaks of chlorobactane II and okenane III are truncated. degradation product of b-carotene (Day and Erdman, 1963; library with at least one methyl group at the aromatic ring Byers and Erdman, 1981). Yet, structure XVII is not consis- have a mass fragment that is 30 mass units below the base tent with the carbon number distribution of the m/z 173 Da ion, corresponding to the loss of two methyl groups. series. The formation of the b-ionene end-group from a par- Formation of the five-membered ring of the indanyl end- ent carotenoid requires loss of methyl group C-19 (number- group, either as a biological product or during diagenesis, ing referring to structure VII). Consequently, pseudohomo- probably requires an unsubstituted carbon atom at the logues with 16, 22, 27 and 32 carbon atoms should appear six-membered ring in the ortho position to the side-chain in very low relative concentrations. However, in the BCF, (Fig. 6A). Therefore, aromatic carotenoids with the 2,3,6- these pseudohomologues are abundant (Fig. 3A). trimethyl substitution pattern, such as chlorobactene and Carotenoids with the indan end-group XVIII have, to isorenieratene, are not likely sources for indanyl isopre- our knowledge, not been described. However, their struc- noids. This may explain why the m/z 173 Da isoprenoid ser- ture appears to be fully consistent with the carbon number ies have not been observed among the large variety of distribution in the BCF, and the mass spectrum of the C18 diagenetic products of isorenieratene that are frequently de- pseudohomologue (Fig. 4C) shows characteristics also ob- tected in Phanerozoic bitumens and oils (e.g. Grice et al., served in indans in the NIST 2002 mass spectral library. 1996; Koopmans et al., 1996a; Sinninghe Damste´ et al., For instance, all compounds from the m/z 173 series in 2001), and have not been reported as diagenetic products the BCF possess fragments with m/z 157, 158 and 159. of b-carotene (Koopmans et al., 1997). Suitable precursors, NIST library spectra of 1,1-dimethylated indans XIX with on the other hand, are aromatic carotenoids with a 2,3,4- no, one and two additional methyl groups at the aromatic trimethyl substitution pattern such as okenone IX.As ring show corresponding triplets at 15, 16 and 17 (and some okenane III is abundant in the BCF, we suggest structure cases 18) mass units below the base ion. All compounds in XX with a 1,4,5,6-tetramethyl substitution pattern for the the m/z 173 series and all 1,1-dimethylindans in the NIST indanyl isoprenoid series. Author's personal copy
1404 J.J. Brocks, P. Schaeffer / Geochimica et Cosmochimica Acta 72 (2008) 1396–1414
A R R diagenesis
?
B diagenesis 1 2
2,3,6-trimethyl-
C
2,3,4-trimethyl-
Fig. 6. (A) Hypothetical formation of m/z 173 aryl isoprenoids from 2,3,4-trimethylaryl isoprenoids. (B) Diagenetic formation of 2,3,6- trimethylaryl isoprenoids from b-carotene by shift of a single methyl-group and subsequent aromatization. (C) The diagenetic formation of 2,3,4-trimethylaryl isoprenoids from b-carotene would require a specific shift of two methyl groups and is, thus, unlikely.
Fig. 5A shows the elution range of the m/z 173 series position to the side-chain. During diagenesis, the free ortho having the highest molecular masses. The chromatographic positions should facilitate ring-closure to an indanyl carot- trace contains at least eight peaks a1 to a8 with the base ion enoid XXIV similar to the reaction shown in Fig. 6A. +Å m/z 173. a1 to a3 have a molecular ion M = 552, consis- tent with C40 carotenoid derivatives with one indanyl end- 3.5. m/z 235 and 237 series of aromatic carotenoid derivatives group. However, the high intensity of M+Å (>40% relative to the base ion) is atypical of the m/z 173 pseudohomo- Koopmans et al. (1996a) describe cyclization products logues series. In low molecular weight members of the m/z of isorenieratene that are characterized by a m/z 237 base 173 series, M+Å has a maximum intensity of 10%, declines ion and a biphenyl end-group (e.g. XXVI). The BCF con- with increasing molecular mass and is below detection lim- tains the same, or a similar, series of compounds. They also its for compounds with more than 27 carbon atoms. Thus, have the m/z 237 base ion and possess the sum formula a1 to a3 are not members of the regular series and their CnH2n 14 with n = 18–40 (Fig. 7B). The parent compound structure remains unknown. of the series has the sum formula C40H66 and thus, In contrast to a1 to a3, the molecular ions of a4 to a7 re- possesses a biphenyl moiety at one terminus and is mained obscure, even in chemical ionization (CI) experi- open-chained at the other. Carotenoids with two biphenyl ments. However, we can attain preliminary information end-groups, or one biphenyl and one phenyl end-group about the structures of some of these compounds by com- (Koopmans et al., 1996a) were not detected. In the series, paring relative elution times with those of the m/z 134 ser- pseudohomologues with 23, 28 and 33 carbon atoms are ies. In the gas chromatograms, members of the indanyl missing, consistent with a carotenoid branching pattern. isoprenoid series XX elute consistently earlier relative to The mass spectra are characterized by minor fragment ions aryl isoprenoids with the m/z 134 base ion (dotted diagonal at m/z 222 (8–20%), 209, 208, 207 and 192, closely resem- lines in Fig. 3). Thus, based on relative elution positions, we bling the published mass spectrum of the C19 pseudohomo- suggest that signal a4 is the indanyl equivalent XXI of oken- logue of XXVI (Fig. 10a in Koopmans et al., 1996). ane III, and a7 and a8 correspond to structures XXII and The m/z 237 series in the BCF could have formed by XXIII, the mono-indanyl equivalents of renieratane IV cyclization and aromatization of either chlorobactene or and renierapurpurane V, respectively. Structure XXIII is okenone IX, yielding biphenyl carotenoids XXVI or XXVII, also consistent with a weak molecular ion m/z 544 of a8 respectively. However, XXVII can be ruled out by the and a strong m/z 133 fragment (Table 1) indicative of the observation that the chromatographic signals of the m/z trimethyl aryl end-group. 237 series are broadened or split into atropisomer doublets Carotenoids with indanyl end-groups have not been ob- for C20 pseudohomologues and higher (inset Fig. 7B). As served in extant organism, and a diagenetic origin from described by Koopmans et al. (1996a), biphenyl isoprenoids cyclization of 2,3,4-trimethyl substituted aromatic carote- XXVI with two methyl groups in ortho position of the ter- noids seems likely. This hypothesis could be tested by minal phenyl group possess an axially chiral centre caused searching for 1,5,6,7-tetramethylated indanyl isoprenoids by restricted rotation around the phenyl–phenyl C–C bond XXIV in samples that contain diagenetic products of palae- (atropisomerism). For C20 homologues and higher, the renieratane XXV, such as Devonian oils from Belarussia presence of this axially chiral centre causes splitting of chro- (Clifford et al., 1998). Palaerenieratane XXV has a 3,4,5- matographic signals into symmetrical double peaks, or, trimethylaryl end-group and, therefore, possesses two where chromatographic separation is incomplete, broad- unsubstituted carbon atoms in the aromatic ring in ortho ened signals (Koopmans et al., 1996a). The biphenyl Author's personal copy
Okenane and other carotenoid derivatives 1405
Table 1 Mass spectral data and pseudo-Kovats indices (I0) for carotenoids of the Barney Creek Formation Peak Compound Structure Mass fragments m/za I0 Saturated carotanes Lycopane VI 3505 c-Carotane VIII 57(100%), 69(82), 83(42), 97(33), 111(38), 125(42), 3623 196(5), M+Å = 560(15) b-Carotane VII 3745 Aromatic carotenoids b C40, 237 series XXVII ? 207(5), 208(5), 209(10), 222(7), 237(100), 238(20), 3577 M+Å = 546(7) +Å a3 173 series — 173(100), 174(14), M = 552(43) 3657 a4 173 series XXI ? 157(1), 158(3), 159(3), 173(100), 174(15) 3714 +Å C40, 235 series XXVIII ? 220(9), 235(100), 236(18), M = 544(13) 3734 Chlorobactane II 119(9), 120(13), 133(56), 134(100), M+Å = 554(16) 3782 Okenane III 119(9), 120(9), 133(67), 134(100), M+Å = 554(16) 3797 b-Isorenieratane X? 133(56), 134(100), M+Å = 552(16) 3908 b-Renierapurpurane XI ? 133(66), 134(100), M+Å = 552(20) 3926 a7 173 series XXII ? 173(100), 174(18) 4005 a8 173 series XXIII ? 133(40), 157(2), 158(5), 159(3), 173(100), 174(14), 4019 M+Å = 544(3) Isorenieratane I 133(70), 134(100), M+Å = 546(13) 4071 Renieratane IV 133(100), 134(78), M+Å = 546(10) 4086 Renierapurpurane V 133(52), 134(100), M+Å = 546(16) 4100 a Intensities are given in % relative to the base ion. b ‘?’ indicates that the compounds were not identified using synthetic standards. isoprenoid XXVII only possess one methyl group in ortho than the m/z 237 series (Fig. 7) as the carbon bridge forces position of the terminal phenyl group, and atropisomers the two phenyl rings into a near-planar conformation, sig- that are stable at GC temperatures are not expected (see nificantly increasing the energy of interaction with the sta- Fig. 7 in Koopmans et al., 1997). Thus, XXVI is the most tionary phase of the GC column. likely structure for the parent compound of the m/z 237 ser- Following Clifford et al. (1998), we suggest that XXVIII, ies in the BCF, and chlorobactene the predominant precur- and not XXIX, is the correct generic structure for the m/z sor. This opens the question why okenone IX formed no 235 series in the BCF. Clifford et al. (1998) observed isopre- detectable diagenetic products with a biphenyl end-group. noids with a m/z 235 base ion in petroleum of the Devonian One possible explanation is that okenone IX underwent Duvernay Formation. The petroleum also contained 13C- similar diagenetic reactions but with products that form a enriched palaerenieratane XXV. Like precursors of oken- slightly different end-group. These products might be repre- ane III, precursors of palaerenieratane XXV possess a free sented by the m/z 235 series described below. Alternatively, ortho position, permitting the cyclization reaction either cyclization of aromatic carotenoids with a 2,3,4-trimethyl to a 9,9-dialkyl fluorene or 9,10-dihydrophenanthrene. substitution pattern may have predominantly resulted in However, the Duvernay Formation also contained a single 13 the formation of indan derivatives (see above) rather than C5-alkylated phenanthrene enriched in C. The isotopically biphenyl derivatives. enriched phenanthrene is almost certainly the degradation The distribution pattern of pseudohomologues in the m/z product of the Devonian m/z 235 series, indicating a 9,10- 235 series is similar to the m/z 237 series. It has the sum for- dihydrophenanthrene end-group. mula CnH2n 16 with n = 19–40 (Fig. 7A). Pseudohomo- logues with 23, 28 and 33 carbon atoms were not 3.6. Carbon isotopes detected, again consistent with a carotenoid branching pat- tern. Although the mass spectra had low signal to noise ra- The biological sources of aromatic carotenoid deriva- tios, it was possible to detect ions at m/z =M+Å, 236, 235, tives can often be distinguished by their diagnostic carbon 220 and 205 in all members of the series. The m/z 235 base isotopic compositions (e.g. Summons and Powell, 1987; ion represents an end-group with nine double-bond equiva- Schaeffer et al., 1997; Sinninghe Damste´ and Ko¨ster, lents, consistent with a biphenyl group bridged by one addi- 1998). For the BCF, carbon isotopic measurements on indi- tional ring. Two structures that may form by cyclization of vidual aromatic carotenoid derivatives have been unsuc- okenone IX (but not chlorobactene because of obstructing cessful due to partial coelution of chromatographic peaks methyl groups in ortho position; Clifford et al., 1998) are and a sloping baseline caused by the unusually large under- the 9,10-dihydrophenanthrene XXVIII and the 9,9-dialky- lying UCM (area above dotted line in Fig. 3C). Separation lated fluorene XXIX. The structures of both of these com- of compounds by a combination of column chromatogra- pounds are also consistent with the observation that the phy, thin layer chromatography and preparative GC did m/z 235 pseudohomologue series elutes significantly later not sufficiently reduce the UCM. Author's personal copy
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Fig. 7. Partial ion chromatograms of the aromatic fraction of sample B03162eA showing (A) m/z 235 series aryl isoprenoids, and (B) m/z 237 series aryl isoprenoids. The inset in (B) on the left shows the m/z 238 partial mass chromatogram with the C18 pseudohomologue of the 237 Da series (M+Å = 238). Percentages indicate signal heights relative to the m/z 134 trace in Fig. 3.
4. DISCUSSION 1988), and c-carotene and hydroxy c-carotene from Chlo- robiaceae (Vogl et al., 2006). As b-carotane VII only has 4.1. Origin of lycopane, c-carotane and b-carotane minor precursors in purple and green sulfur bacteria, the most likely source in the BCF are cyanobacterial carote- Biological precursors of VI, VII and VIII comprise noids such as b-carotene, echinenone, canthaxanthin and nearly the entire range of carotenoids found in nature and zeaxanthin. exist in numerous autotrophic and almost all phototrophic organisms in the three domains of life. As the abundance of 4.2. Origin of okenane steroids in the BCF is very low (except 4-methylated aro- matic steroids), the aliphatic carotenoids are probably lar- In principle, okenane III could be a diagenetic product gely derived from prokaryotes. Carotenoids with the of a non-aromatic aliphatic precursor. Koopmans et al. lycopane skeleton VI are particularly common in purple (1996b) observed that diagenetic aromatization of b-caro- sulfur bacteria, and it is likely that this group of organisms tene may yield 2,3,6-trimethyl substituted aryl isoprenoids. contributed significantly to lycopane VI in the BCF. Poten- This reaction involves shift of a geminal methyl group from tial sources of c-carotane VIII are carotenoids such as C-1 to C-2 of the forming aromatic ring under oxic condi- myxoxanthophyll found in cyanobacteria, c-carotene from tions (Fig. 6B). However, the selective generation of the green non-sulfur bacteria (Chloroflexi) (Palmisano et al., 2,3,4-trimethyl alkylation pattern in okenane III would Author's personal copy
Okenane and other carotenoid derivatives 1407 require isomer-specific rearrangement of two, not one, Table 3 summarizes the depth of the chemocline of 33 methyl groups (Fig. 6C), a reaction that almost certainly re- stratified water systems where okenone producing Chro- quires enzymatic activity and would not occur as an abio- matiaceae were observed. Blooms occurred at 1.5 to logical transformation. Therefore, it is likely that the 24 m depths, and 75% occurred at less than 12 m depth precursor of okenane III has a direct biological origin. (n = 33, average = 8.4 m) (Table 3). For comparison, green As carotenoids are intensely yellow to red colored pig- Chlorobiaceae with chlorobactene were observed at 2to ments, our knowledge about their distribution in living 16 m depth, with 75% of all occurrences at 6 m or less organisms is far better than for most other lipid classes. (n = 28, average = 5.7 m). Brown pigmented Chlorobiaceae However, despite the large number of known functionalized containing isorenieratene are adapted to conditions of low- carotenoids, the only monocyclic carotenoid with a 2,3,4- est irradiance and are observed between 2 and 80 m depth, trimethylaryl substitution pattern is okenone IX. Okenone with 75% of all observations at less than 17 m (n = 19, aver- is a purple-red pigment that is exclusively known from pur- age = 14.8 m; Van Gemerden and Mas, 1995). The abun- ple sulfur bacteria of the family Chromatiaceae (Imhoff, dance of okenane III and chlorobactane II suggests that 1995). Chromatiaceae are anoxygenic phototrophs with a the oxic/anoxic boundary in the McArthur Basin physiology similar to green sulfur bacteria. Blooms occur 1640 Ma ago was (temporarily) very shallow, probably less in a wide variety of anoxic environments in the presence than 12–25 m. of light and reduced sulfur compounds. Habitats include The wavelength distribution of light in the chemocline of stratified lakes and fjords, coastal lagoons, estuaries and modern stratified lakes has a strong influence on the relative coastal microbial mats. Okenone has been described from abundance of different groups of anoxygenic phototrophic 12 species of Chromatiaceae that belong to at least eight bacteria. If light is strongly filtered by dense populations genera. All other species within these genera, and all other of oxygenic phototrophs in the overlying water column, genera, contain acyclic carotenoids such as lycopene, rhod- then brown pigmented Chlorobiaceae have a competitive opin and spirilloxanthin. advantage over green species. Conversely, green pigmented The physiology of the 12 known okenone producing Chlorobiaceae become dominant when oxygenic photo- Chromatiaceae is summarized in Table 2. While Chlorobi- trophs are less abundant and the wavelength distribution aceae are strictly anaerobic and obligately phototrophic, is determined by overlying purple sulfur bacteria (Montesi- okenone producing Chromatiaceae are more versatile. With nos et al., 1983). During particularly dense blooms of pur- simple substrates, some species survive chemoorganotro- ple sulfur bacteria, brown pigmented Chlorobiaceae may be phically or chemolithoautotrophically under microoxic almost excluded from the ecosystem (Overmann et al., conditions in the dark (Table 2), and several species can 1991). These different ecological requirements of green even grow permanently under chemolithotrophic condi- and brown Chlorobiaceae can explain the relative concen- tions at oxygen concentrations of 11–67 lM(Overmann trations of aromatic carotenoids in the BCF. In representa- and Pfennig, 1992). However, as pigment production is re- tive sample B03162, the ratio of okenane III to pressed during prolonged chemotrophic growth, okenone chlorobactane II to isorenieratane I is 100:50:0.8 IX strictly indicates anoxic conditions in light. In addition (Fig. 3B). The high ratio of chlorobactane II to isoreniera- to sulfide and sulfur, most okenone producing species ac- tane I suggests that Chlorobiaceae were strongly dominated cept thiosulfate, sulfite, hydrogen and simple organic sub- by green species most of the time (and this is particularly strates as electron donors. However, although sulfide is true as the abundance of pigments in brown Chlorobiaceae not strictly required as electron donor, all species in Table is commonly much higher than in green Chlorobiaceae and 2 still rely on sulfide as a sulfur source as they are not capa- also Chromatiaceae). Thus, the low abundance of isoreni- ble of assimilative sulfate reduction. Therefore, despite the eratane I suggests an ecosystem where irradiance conditions metabolic versatility of Chromatiaceae, okenone IX proba- are strongly determined by dense blooms of Chromatia- bly always signifies sulfidic conditions. ceae. Dense blooms of oxygenic phototrophs, on the other Chromatiaceae can be found in benthic environments hand, probably did not occur during periods of stratifica- beneath the surface of photosynthetic microbial mats or tion, and this is also consistent with the absence of diagnos- in planktonic environments below the anoxic–oxic interface tic algal biomarkers. of stratified lacustrine to hypersaline waters. As light absorption by okenone reaches its maximum at the yel- 4.3. Origin of renieratane, renierapurpurane and low-green part of the spectrum around 520 nm, it appears b-renierapurpurane to be ideally suited to capture light under planktonic condi- tions, and it is the most common carotenoid of Chromati- Until recently, the only known carotenoid precursors of aceae in these ecosystems (63% of all cases; Van renieratane IV and renierapurpurane V were the diaromatic Gemerden and Mas, 1995). Conversely, all mat species pro- carotenoids renieratene and renierapurpurin isolated from duce carotenoids with a lycopane VI skeleton, and okenone marine sponges (Yamaguchi, 1960; Schaefle´ et al., 1977; Lia- has not been detected in microbial mats (Table 2). This aen-Jensen et al., 1982). Sponges, like all animals, are not observation is significant, as it opens the possibility to use capable of de novo biosynthesis of carotenoids. Therefore, okenone and its derivatives to distinguish between plank- renieratene and renierapurpurin in sponges are either derived tonic organic debris generated in an anoxic water column from unknown bacterial sponge symbionts or generated by and allochthonous organic matter derived from mat com- the sponge through modification of dietary carotenoids (Lia- munities that was transported to deeper sediments. aen-Jensen et al., 1982). However, renieratane IV and reni- Author's personal copy 48JJ rcs .Shee eciiae omciiaAt 2(08 1396–1414 (2008) 72 Acta Cosmochimica et Geochimica / Schaeffer P. Brocks, J.J. 1408
Table 2 Physiological and ecological data of okenone-producing Chromatiaceae
o p Species BChl Caroten- Color of Width * Flagel- Gas Chemo- Chemoorgano- Photoorgano- Photoautotrophic Max.H2S Strictly Opt. salinity Opt. N- Assimil. Benthic/ Habitat Ref. a k 2 oids cell length lated vacuoles lithotrophic trophic trophic e donors tolerated anaerobic range [%] pH fixation SO4 planktonic c e f suspension (lm) growth growth growth 2 0 2 [mM] red. S S S2O3 H2
Chromatium weissei a o — 3.5–4.0 * 7–9 y n n n n y y n n — y — — n n p f [1,2] Chromatium okenii — o — 4.5–6.0 * 8–15 y n n n n y y n n — y — — n n p f [1,3] ‘Kaiike strain’ a o Purplish-red 3–4 * 3–6 y n n n — y y — — — y 0.3–2 7.5–8.5 y — p b [4] Lamprocystis purpurea a o Purple-red Ovoid 1.9–4.5 n y y — — y y y n 7.5 n (4.5) 7.0–7.3 — n p f, s [5,6,7] (Amoebobacter purpureus) m Lamprobacter a o Purplish-pink 2–2.5 * 2.5–5 y y y — — y y y y — n 1–2(10) 7.4–7.6 y n b , p s, m [8] modestohalophilus g n Marichromatium purpuratum a o Purple-red 0.9–1.8 * 2.5–4 y n — n y y y y — — — 2–7 7.2–7.6 — — p b, m, s [3,9] (Chromatium purpuratum) ‘Thiocapsa purpurea’ — o — — — — — — — — — — — — — — — — — — b, m [10] b m Thiocapsa marina a o,(s, k) Purple-red Cocci 1.5 * 3.0 n n y y — y y y y — n 1–2(8) 7.5 y n b , p b, m [11,12] Thiocystis gelatinosa a o Purple-red Cocci 2–10 y n nd n—yyn–j — y 1–2 6.5–7.6 — n p f, b [13,14] (Thiothece gelatinosa) i j Thiocystis minor a o — 2–4 * 2.5–6 y n y — n y y y – <10 n — — — n p f [3] (Chromatium minus) Thiohalocapsa halophila a o, (s, k) Purple-red Cocci 1.5–2.5 n n y y — y y y y 6 n 3–7(<25) 6.0–8.0 — n bm h [15] (Thiocapsa halophila) Thiopedia rosea a o Purple-red Ovoid 1.0–2.5 n y n n yh y y n n 0.6 y — 7.3–7.5(9)l — n p f [16]
a o, carotenoids of the okenone series; s, spirilloxanthin series; k, R.g.-ketocarotenoids; minor carotenoids in parentheses. b 50–80% okenone, 10–15% Thiothece-OH-484 (=demethylated okenone), trace of anhydrorhodovibrin, 9–14% spirilloxanthin, 8% R.g.-ketocarotenoids. c Chemolithoautotrophic growth by oxidation of sulfide and thiosulfate under microoxic conditions in the dark. d Table 4 in Behrens et al. (2000) suggests T. gelatinosa can grow chemolithotrophically. e Chemoorganotrophic growth under microoxic conditions with simple substrates such as acetate and pyruvate. f ‘y’ indicates anaerobic phototrophic growth with simple organic substrates as sole electron donors (as opposed to mere photoassimilation of organic substrates; all Chromatiaceae can photoassimilate simple organic chemicals under anaerobic conditions. However, metabolically specialized species can only use acetate and pyruvate in the presence of carbon dioxide and sulfide. Other species such as T. marina are more versatile and may photoassimilate a wider variety of organic species including malate, lactate and fructose, but also only in the presence of sulfide as electron source and carbon dioxide as additional carbon source). g Organic substrates as photosynthetic electron donors: acetate, lactate, pyruvate, succinate, fumarate, malate, propionate, butyrate, valerate (measured with 1 mM cystein as sulfur source). h Organic substrates as photosynthetic electron donors: acetate, pyruvate, glucose, butyrate, valerate (measured with 70 lMH2S as sulfur source). i Thiocystis minor also growths photoautotrophically on sulfite. j Hydrogenase is present, but utilization of hydrogen as electron donor for phototrophic growth has not been demonstrated. k Values in parentheses represent the salinity tolerance maximum. l Value in parentheses represents the maximum tolerated pH. m As okenone was only detected in cultivated isolates, it remains unclear whether these species express okenone biosynthesis in benthic habitats. There are no publications that describe the identification of okenone directly in microbial mats. n M. purpuratum was also isolated from sponges and zooplankton inhabiting oxic marine environments. o f = freshwater, b = brackish, m = marine, s = saline lakes, h = hypersaline lakes. p Physiological and environmental data was compiled using the references listed below, and additional information was taken from the review Imhoff, 1995: [1] Tru¨per and Jannasch (1968), [2] Imhoff (1995), [3] Imhoff and Tru¨per (1980), [4] Michiro (2004), [5] Overmann et al. (1991), [6] Imhoff (2001), [7] Eichler and Pfennig (1988), [8] Gorlenko et al. (1979), [9] Casamayor et al. (1998), [10] Sueling, J., GenBank accession number AJ543327, [11] Caumette et al. (2004), [12] Caumette et al. (1985), [13] Lunina et al. (2005), [14] Pfennig et al. (1968), [15] Caumette et al. (1991), [16] Eichler and Pfennig (1991). Author's personal copy
Okenane and other carotenoid derivatives 1409
Table 3 XXX (18,180-v,v-carotenedioic acid), a newly discovered Depth distribution of okenone producing Chromatiaceae in carotenoid with the renierapurpurane skeleton V. However, modern environments a detailed examination of carotenoid degradation products Water system Deptha [m] Ref. in the BCF suggests that cyanobacterial synechoxanthin Arcas, Central Spain 8.5–9.3 [1,2] XXX is probably not the precursor of renierapurpurane in Banyoles III, Spain 14–18 [3–6] this particular formation. Synechoxanthin XXX is a dicar- Banyoles IV, Spain 11–13.5 [3–6] boxylic acid, and catagenesis should lead, at least partially, Banyoles V, Spain 13 [3–6] to decarboxylation. However, the expected decarboxylation Banyoles VI, Spain 12.5–14.5 [3–6] products, 2,3-dimethylaryl isoprenoids, were not detected in Belovod, Russia near Moscow 10–13 [7] m/z 119 selected ion chromatograms. The only demethyla- Cadagno, Switzerland 10–11.5 [8,9] tion products of aromatic carotenoids detected in the BCF Cassidy, N. America 6 [10] are derived from chlorobactane and okenane and reflect Ciso´, Spain 1.5–3 [3,5,11] random loss, not targeted loss, of one of three methyl groups Dagow, Germany 6.0 [12] (b to b , Fig. 5B). Moreover there is evidence from Lake Estanya, Spain 13 [3] 1 5 Fish, Wisconsin 10 [10] Cadagno, a meromictic lake that hosts okenone producing Hays Land and Cattle Company feedlot <2.5 [13] Chromatiaceae, that biomarkers with the b-renierapurpura- lagoon, Kansas ne skeleton XI can be derived from purple sulfur bacteria. Kaiike, Kamikoshiki Island 4–5 [14,15] Behrens et al. (2000) described b-renierapurpurane XI as a La Cruz, Spain 11–16 [5,16] major H2/PtO2-hydrogenation product of functionalized Little Mill, Great Lakes region 9 [10] lipids from surface sediments of the lake. Given its stable Mahoney, British Columbia 6.6 [17,18] carbon isotopic composition which was shown to be identi- Mittlerer Buchensee, Germany 8–9 [19] cal to that of H2/PtO2-hydrogenated okenone, it was sug- Mogil’noe, Kil’din Island, Barents Sea 8–10 [20] gested that b-renierapurpurane XI in Lake Cadagno Montcorte`s, Spain 16–24 [3,21] sediments originates from Chromatiaceae (Schaeffer et al., Monte Alegre, Brazil 4.0 [3,22] Nou, Spain 3.5–4 [22] 1997; Behrens et al., 2000). Most importantly, however, ren- Plußsee, Holstein, Germany 5 [22] ieratane, renierapurpurane and b-renierapurpurane have Puddledock Reservoir, NSW, Australia 4–6 [23] thus far only been detected in geological formations that Rotsee, Switzerland 6 [24] also contain evidence for green sulfur bacteria, the Devo- Schlachtensee, Germany 7–8 [25] nian Duvernay Formation of the Western Canada Basin Shira, Khakasia (south central Siberia) 13.5 [26] (Hartgers et al., 1993), Toarcian shales from the Paris Basin Silver I, Great Lakes region 12 [10] (Schaefle´ et al., 1977) and the Paleoproterozoic BCF Tarpon Blue Hole, Andros Island, 4.5–5.0 [27] (Brocks et al., 2005). In these cases, purple sulfur bacteria Bahamas appear to be the most likely source of 2,3,4-trimethylaryl Vechten, Netherlands 6 [28] substituted carotenoid derivatives. Therefore, although a Vilar, Northeastern Spain 3.8–5 [3,5,11,29] Wintergreen, Great Lakes region 4.25 [10] cyanobacterial origin of renierapurpurane, b-renierapurpu- Zaca, California 6 [30] rane and renieratane is possible, in the BCF, these biomark- ers are more likely derived from purple sulfur bacteria. a Lowest and highest level of the oxic/anoxic boundary when Although the discovery of synechoxanthin XXX in cyano- okenone producing Chromatiaceae were reported (not the depth range at which Chromatiaceae were observed). References: [1] bacteria requires a re-evaluation of 2,3,4-trimethylaryl carot- Camacho and Vicente (1998), [2] Camacho et al. (2000), [3] enoid derivatives in the geological record, it does not Montesinos et al. (1983), [4] Casamayor et al. (2001), [5] necessarily diminish the value of aromatic carotenoids as Casamayor et al. (2000), [6] Garcia-Gil and Abella (1992), [7] diagnostic biomarkers. It should become possible to distin- Sorokin (1970), [8] Schanz et al. (1998), [9] Tonolla et al. (1999), guish the diagenetic products of cyanobacteria and purple [10] Vila et al. (1998), [11] Guerrero et al. (1985), [12] Glaeser and sulfur bacteria through specific carbon isotopic compositions Overmann (2003), [13] Wenke and Vogt (1981), [14] Michiro (Schaeffer et al., 1997) and distinct diagenetic pathways. (2004), [15] Ohkouchi et al. (2005), [16] Romero et al. (2006), [17] Overmann et al. (1991), [18] Overmann et al. (1996), [19] Overmann 4.4. Origin of chlorobactane, isorenieratane and and Tilzer (1989), [20] Lunina et al. (2005), [21] Serra et al. (2003), b-isorenieratane [22] Van Gemerden and Mas (1995), [23] Banens (1990), [24] Kohler et al. (1984), [25] Gervais (1997), [26] Pimenov et al. (2003), [27] Marano Briggs (2000), [28] Steenbergen et al. (1989), [29] A review of biological sources of chlorobactane II, iso- Casamayor et al. (2002), [30] Folt et al. (1989). renieratane I, and b-isorenieratane X was given by Brocks and Summons (2004). One potential microbial source for erapurpurane V in the BCF are certainly not derived from isorenieratane I in the BCF are bacteria of the order Actin- sponges or sponge symbionts as these organisms must have omycetales. Actinomycetales used to be regarded as a micro- evolved much later in Earth history (Peterson and Butter- bial group that is restricted to terrestrial habitats. However, field, 2005). Therefore, renieratane IV and renierapurpurane modern microbiological techniques reveal that the group is V must have a free microbial source. also ubiquitous, and occasionally abundant, in marine envi- A potential microbial source was recently discovered by ronments (Ward and Bora, 2006). However, actinomycetes Graham and Bryant (2007). The authors observed that are only known to contain isorenieratene and its derivatives, many cyanobacteria can biosynthesize synechoxanthin but not precursors of chlorobactane II or any of the other Author's personal copy
1410 J.J. Brocks, P. Schaeffer / Geochimica et Cosmochimica Acta 72 (2008) 1396–1414 aromatic carotenoids detected in the BCF. As isorenieratane 1000 million years. They include several parent biomarkers I was one of the least abundant aromatic carotenoids in the and series that are, to the best of our knowledge, new BCF (Fig. 3B), actinomycetes evidently did not contribute (okenane III, b-renierapurpurane XI, and series with m/z significantly to the carotenoid pool. 173 and 235 base ions). The quantitatively most important biological source of (1) The BCF is currently the only known formation that 2,3,6-trimethyl substituted aromatic carotenoids in the contains okenane III. Based on the physiology of extant BCF are probably Chlorobiaceae. The major carotenoids species, we interpret okenane III as a biomarker for plank- in brown pigmented strains are isorenieratene and b-isoreni- tonic purple sulfur bacteria of the family Chromatiaceae. eratene, the potential diagenetic precursors of isorenieratane Although several species can grow permanently under I and b-isorenieratane X, respectively. Known biological pre- microoxic conditions in the dark, or use hydrogen or simple cursors of chlorobactane II are the pigments chlorobactene organic substrates as electron donors, the presence of oke- and hydroxychlorobactene from green pigmented Chlorobi- none IX (or okenane III) probably always indicates sulfidic aceae. Chlorobiaceae are strictly anaerobic, obligately conditions in the presence of light. The presence of okenane phototrophic organisms solely operating on photosystem I. III in the BCF suggests that the anoxic/oxic transition was Most Chlorobiaceae require hydrogen sulfide or other re- (temporarily) less than 12–25 m deep. Okenane III is the duced sulfur species as electron donor. Therefore, the pres- first unambiguous indicator for purple sulfur bacteria in ence of I, II and X confirms (intermittent) photic zone the geological record. euxinia in the McArthur Basin during deposition of the BCF. (2) The m/z 173 (XX) and 235 (XXVIII) series are likely diagenetic cyclization products of okenone IX and are also 4.5. Apparent absence of other diagenetic carotenoid products interpreted as biomarkers for Chromatiaceae. (3) Cyanobacteria are the only group of organisms Immature to moderately mature bitumens and oils from where biosynthesis of carotenoids with the renierapurpura- the Phanerozoic with high concentrations of carotenoids fre- ne skeleton V was directly observed. However, to date, ren- quently contain a large variety of carotenoid cyclization and ieratane, renierapurpurane and b-renierapurpurane have degradation products (Grice et al., 1996; Koopmans et al., only been detected in formations that also contain evidence 1996a, 1997; Clifford et al., 1998; Sinninghe Damste´ et al., for green sulfur bacteria. In these cases, including the BCF, 2001). These products include aryl isoprenoids with addi- purple sulfur bacteria appear to be the most likely source of tional mid-chain aromatic and aliphatic rings, biphenyl-, 2,3,4-trimethylaryl carotenoid derivatives. However, a naphtyl- and phenyl-naphtyl end-groups, and C32 and C33 cyanobacterial origin is possible, and the biosynthesis and diaryl isoprenoids with shortened isoprenoid chains. How- diagenetic fate of synechoxanthin XXX and renieratene re- ever, in the BCF, we were not able to identify any of these quires further scrutiny. common diagenetic products apart from the 237 Da series (4) Isorenieratane I and b-isorenieratane X indicate the discussed earlier, and two new series with base ions m/z presence of brown pigmented species of Chlorobiaceae in 235 and m/z 173. In particular, we failed to detect diagenetic the BCF, and chlorobactane II of green pigmented species products of aryl isoprenoids with major fragment ions at m/z of Chlorobiaceae. In modern environments, green pig- 183, 169, 258, 273, 287, 291, 313, 379, 325 and 351, or with mented species inhabit water depth of less than 16 m, giving molecular ions at m/z 420, 426, 434, 440 and 520 (see Appen- further evidence for particularly shallow stratification of the dices A and B in Koopmans et al. (1996a) for structures and McArthur Basin. In the BCF, the very high concentrations MS characterization). In principle, it is possible that the of okenane and chlorobactane relative to isorenieratane are above products were destroyed due to higher thermal matu- typical for an ecosystem where lighting conditions are dom- rity of the BCF bitumens. However, the thermal stability of inated by purple sulfur bacteria and where dense blooms of most of the above products is comparable to C40 carotenoids oxygenic phototrophs are uncommon. derivatives, and those are preserved in high concentrations (5) The BCF also contains c-carotane VIII, b-carotane in the BCF. Therefore, it is possible that the vast majority VII and lycopane VI. c-Carotane VIII was identified for of diagenetic alteration products of carotenoids found in the first time using an authentic standard. Potential precur- the Phanerozoic never formed in detectable concentrations sors of the three carotanes occur in numerous autotrophic under the diagenetic conditions that prevailed during depo- and almost all phototrophic organisms in the three domains sition of the BCF. The chemical and biological controls on of life. In the BCF, where eukaryotic organisms were generation and destruction of different diagenetic products apparently rare, c-carotane VIII is most likely derived from of carotenoids are as yet unresolved (Sinninghe Damste´ cyanobacteria, and possibly green non-sulfur bacteria et al., 2001). However, significant information about differ- (Chloroflexi) and Chlorobiaceae. Lycopane VI may be pre- ences in Paleoproterozoic and Phanerozoic water chemistry dominantly derived from purple sulfur bacteria, while cya- and ecology may reside in their solution. nobacteria are the most likely source of b-carotane VII. The discovery of intact carotenoid biomarkers of more 5. CONCLUSIONS than 1 billion years age opens the possibility of testing hypotheses concerning long term variations in the impor- Bitumens from the 1.64 Ga old Barney Creek Formation tance and distribution of different groups of phototrophic in the McArthur Basin of northern Australia contain more bacteria in the Precambrian. Although a difficult challenge than 22 different C40 carotenoid derivatives, extending the at the time of writing, it might also become possible to iso- geological record of this compound class by more than late individual carotenoids from the associated hydrocarbon Author's personal copy
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UCM and determine their carbon isotopic composition. Research Council Grant DP0557499. We thank Janet Hope for Lipids of green and purple sulfur bacteria and oxygenic technical support, and GA and the Northern Territory Geological phototrophs often carry distinctly different carbon isotopic Survey for samples. We thank Pierre Adam, Roger Summons and signatures. Thus, carbon isotopic measurements might help GA for generously contributing synthetic aromatic isoprenoids, to confirm the interpretation of the biomarkers in the BCF and B. Ludwig (Hoffmann La Roche, Switzerland) for the generous gift of b-apocarotenal. We also thank Emmanuelle Grosjean for and, in comparison with kerogen isotopic data, might per- reviewing an earlier version of this manuscript, and we are partic- mit an estimation of the contribution of different classes of ularly grateful to J.E. Graham, J.T. Lecomte, and D.A. Bryant for phototrophic bacteria to total primary productivity. sharing their information about aromatic carotenoids in cyanobac- teria. We thank Jaap S. Sinninghe Damste´ for editing the manu- ACKNOWLEDGMENTS script and for providing a detailed review and many valuable comments, and Lorenz Schwark, Kliti Grice, Brendan Keely and This work was supported by the William F. Milton Fund of an anonymous reviewer for constructive comments and Harvard University, Geoscience Australia (GA) and Australian suggestions.
APPENDIX Author's personal copy
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