Biomarkers & microbial response to changing salinity

Salinity-controlled transitions between a halotolerant and halophilic biota can be seen, indirectly in their organic residues and subsequent biomarkers. Biomarkers can be thought of as molecular or chemical fossils with a basic carbon skeleton derived from once-living organisms. Biomarkers are found in all modern sediments, as well as in petroleum and rock extracts and can provide information about species diversity, depositional environment, thermal maturity, migration pathways and hydrocarbon alteration (Table; Brocks and Summons, 2003; Peters et al., 2005; Warren 2016).

Commonly accepted environmental generalisations based on biomarker distributions include the notion that variation in the ratio between the isoprenoids pristane and phytane indicates oxidising versus reducing conditions (this assumes pristane and phytane are both breakdown products of chlorophyll). In an oxidising environment, the cleavage of the phytol side chain of chlorophyll is followed by decarboxylation to produce phytane (Figure). In a reducing environment, the sidechain cleavage of chlorophyll is followed by its reduction to ultimately produce pristane. Low pristane/phytane ratios are thought to indicate reducing conditions, while higher values (>1) indicate oxidising conditions (Table). 

Biomarker table (from Warren, 2016)

Philp and Lewis (1987) have shown that the chemistry of chlorophyll breakdown is much more complicated in many natural systems and that variations in the ratio may also indicate varying inputs from the archaea, which contain much higher levels of phytane chains than bacteria (Figure A). Values of pristane/phytane less than 0.5 may indicate hypersaline conditions where archaea dominated and 13C values can be elevated (Grice et al., 1998). Differences in the distribution of n-alkanes are also thought to indicate depositional differences in the organic contributors. Waxes in higher plants have significant concentrations of the long-chain C22-36 alkanes, with a pronounced odd/even distribution. Such organics tend to be relatively rare in evaporitic source rocks, unless surface waters were freshened by periodic runoff from the land. Much of this terrestrial organic material is oxidised and biodegraded before it makes it to the final site of deposition on the brine pool floor. In contrast, a high algal input is characterised by n-alkanes in the C16-18 region.

At its simplest, much of the utility of biomarkers in saline environments comes from salinity-related differences in contribution to organic matter of the general categories of primary producers (autotrophs), namely prokaryotes (cyanobacteria and bacteria) and eukaryotes (higher plants, algae) and archaea. Most triterpenes are associated with prokaryotic sources, whereas steranes tend to be produced by eukaryotes. Thus, the triterpene/sterane ratio can be a rough measure of the prokaryote/eukaryote contribution to the organic material.g

Various molecular structures. A) Chlorophyll a structure showing porphyrin ring and phytol tail. Alternate structures from bacteriochlorophyll a and chlorophyll b. B) Phosphatidylanol-amine forms a bi-layer lipid in bacterial cells. The acyl chain is usually, but not always straight. Some bacterial lipids have a methyl branch of a cyclohexyl group at the end of the acyl chain, other lipids have one or more unsaturated bonds and the connection of glycerol to the acyl chain is via an ester linkage. C) Lipids are monolayers in Archaea, and the phytanyl chain contains isoprenoid-like branches. An ether linkage connects the phytanyl chain. Archaeal membranes also contain bilayer-forming diether lipids.

As salinity increases, the less salinity-tolerant eukaryotic organisms (mostly green algae) give way to more halotolerant bacteria and cyanobacteria (tricyclic and hopane producers) with a corresponding increase in the triterpene/sterane ratio (e.g. Summons et al., 1999). Thus high alkalinity/salinity settings are characterised by tricyclics (C20-C24;m/z 191), β-carotene (C40H56 compound; m/z 125) and gammacerane (C30 triterpene; m/z 191), all with prokaryote sources. Hence, high levels of gammacerane and β-carotene in ancient organic signatures are typically associated with nonmarine highly saline environments (Peters and Moldowan, 1993; Peters et al., 2005).

Lipids, entrained as organic residues in modern evaporitic carbonates and gypsums from modern saline pans (mesohaline and lower penesaline waters), are mostly derived from heterotrophic bacteria acting on cyanobacteria and green algae (see what lives in saline waters). The resulting n-alkane distributions show a high predominance of n-docosane. In the modern evaporitic carbonate domain (mesohaline), the C20 highly-branched isoprenoid olephines, tetrahymanol (from bactiverous ciliates) and large amounts of phytol are precursors to most lipids found in buried evaporitic sediments.

In contrast, the main lipid contributors to organics preserved in modern halites and bittern beds are the extremely halophilic archaea and their organic signatures are enriched in the isoprenoids, especially phytane (Table; Barbé et al., 1990; Wang, 1998). Likewise, Waples et al. (1974) and ten Haven et al. (1986), noted that Tertiary sediments deposited in many saline evaporitic lagoons retain high concentrations of regular C25 isoprenoids. They related it to the activities of the photolithotrophic Chlorobiaceae sp., an anaerobic green sulphur bacteria, known to flourish at the halocline of modern saline lakes. It is thought to have flourished in similar stratified settings in ancient mesohaline to hypersaline seaways. Its biochemistry leads to the preservation of a series of 1-alkyl-2,3,6-trimethyl benzenes, thought to be derived from the breakdown of its aromatic carotenoids in sulphate- and sulphide-rich brines (Summons and Powell, 1987). Ten Haven et al. (1988) went on to propose that there are a number of biomarkers indicating likely hypersalinity at the time of deposition including; short sidechain steranes, 5α(H), 14β(H), 17β(H) pregnanes and homopregnanes, as well as high gammacerane indices (gammacerane/C30 hopane). Gammacerane is a pentacyclic C30 triterpene thought to be a diagenetic alteration product of tetrahymenol (Table). The principal source of tetrahymenol is bactiverous ciliates, such as protists like Tetrahymena. Thus, the presence of tetrahymenol is not restricted to any particular environment but the presence of tetrahymenol (and its daughter gammacerane) in large amounts suggests the presence of a stratified water column and possibly hypersalinity in the depositional setting (Table; Sinninghe Damste et al., 1995; Peters et al., 2005 p.576).

For Archaea, the cell membrane itself, due to its high thermal stability, is a good candidate for a biomarker. Archaeal cytoplasmic membranes do not contain the same lipids that prokaryotes and eukaryotes do. Instead, their membranes are formed from isoprene chains (ether lipids) made up from C5 isoprenoid units (as for the side chains of ubiquinone) rather than C2 units (ester lipids) in the normal fatty acids of the non-archaea (Figure B). Halophilic Archaea do not have fatty acids. Instead, the cell wall is constructed of lipids, mainly of glycerol, connected to phytanyl chains some twenty carbons in length by ether bonds to form phytanylic diether. Typically these are organised in bilayers that make up the cell wall. In the case of archaea living in extreme conditions, two glycerol molecules can be connected to a double chain of phytanol to create a tetraether structure of forty carbons (Figure C). Phytane-rich isoprene derivatives indicative of ancient archaea have been found in Mesozoic and Palaeozoic evaporitic sediments, and more generally in older Precambrian sediments (Hahn and Haug, 1986).

Interestingly, Archaeal chemical traces have even been tentatively identified in sediments from the Isua district of west Greenland, the oldest known sediments on Earth, some 3.8 billion years old. Biomarker evidence for green and purple sulphur bacteria is found in Palaeoproterozoic marine sediments from northern Australia in the form of a new carotenoid biomarker - okenane (Brocks et al., 2005). Bacterioruberin constitutes the purple patches in the archaeal cell wall (Warren, 2016; Chapter 9). It may well represent an excellent as yet unrecognised biomarker for haloarchaea in ancient evaporitic sediments that were precipitated in moderate to extreme salinities (Brocks and Summons, 2003). The likely fossil equivalent of bacterioruberin is perhydro-bacterioruberin, but it has yet to be discovered in geological samples. However, it is worth bearing in mind that some high-molecular-weight biomarkers may have escaped detection because they are challenging to analyse using conventional GC-MS methods.

Do biomarkers indicate hypersalinity?

Work in modern hypersaline systems clearly shows that archaea and bacteria can both flourish as heterotrophs in upper penesaline and supersaline waters at salinities higher than 200-250‰ and that cyanobacteria and some green algae can still survive and grow at these salinities (Caumette, 1993; Ollivier et al., 1994). Hence, respective biomarker signatures in ancient counterparts will not be mutually exclusive, and none of the biomarker indicators listed in the table above are unique indications of hypersalinity.

Also, some of the widely used biomarker-derived maturity indicators for low maturity oils, such as low levels of diasteranes, Ts/Tm ratios <1, somewhat low homohopane and sterane isomerization values, all historically derived from studies in ancient marine settings, are not as reliable when applied to relatively young saline-lacustrine oils, such as those sourced from hypersaline Oligocene laminites in the northern Qaidam Basin, China (Hanson et al., 2001). These sediments and their outcrop equivalents have vitrinite reflectance values in the range 0.68 - 1.0, all within the oil window.

The discrepancy between mature indications coming from vitrinite studies and the immature indications derived from various sterane isomerization ratios in these young hypersaline-sourced oils are perhaps explained by a lack of time for various sterane equilibria to develop. Some of these hypersaline oils come from hydrogen-rich type I lacustrine source sediments in the Qaidam Basin fill that may be as little as 3 million years old. But some other so-called maturity indicators, such as the low levels of diasterane probably are better tied to deposition of the source organics in noncarbonate shales accumulating in hypersaline waters (Philp et al., 1991).

The fluctuating salinities that control life in the depositional setting of all modern and ancient evaporites means that there is no simple single biomarker indicator for hypersalinity in any ancient sediment. For example, the 2-methyl hopanoids are though to indicate a cyanobacterial association (Table) and cyanobacteria are commonplace in mesohaline waters. But the presence of cyanobacteria and hence 2-methyl hopanoids are not limited to this setting. Likewise, the pentacyclic triterpenoid gammacerane is present in trace amounts in almost all bitumens and oils, but tend to be more abundant in sediments that were deposited under a stratified water column. This is a hydrological condition that is typical of, but not exclusive to, hypersaline lacustrine settings (Sinninghe Damste et al., 1995). A similar argument can be made for low pristane/phytane ratios that are used as indicators of both reducing conditions (assumes a chlorophyll precursor) and halophilia (assumes an archaeal precursor). 

All biomarker signatures are indirect and somewhat ambiguous indicators of hypersalinity via assumptions of the relative make-up the original primary producers. In addition, all traces of biomarkers from the primary producers preserved in ancient strata have been overprinted by the effects of syndepositional decomposers, as well as subsequent mixing from various sources during catagenesis. The overprinting effects of the decomposers in microbial laminites are clearly seen when molecular characteristics of modern hypersaline sediment from the Ejinur salt lake (northern China) are compared to Tertiary (Eocene) core samples from Qianjiang Formation (hypersaline lacustrine) of Jianghan Basin, central-eastern China (Figure A; Wang et al., 1998). N-alkanoic acids in saline sediments from both areas (Ejinur and Jianghan) show a pronounced even-over-odd predominance (EOP) and a bimodal distribution. In the lower molecular weight range, the C16 and C18 components are prominent, with the former dominant. For higher homologues (greater than or equal to C20), docosanoic (C22), and tetracosanoic (C24) acids dominate the n-alkanoic acid homologues in the Jianghan and Ejinur samples, respectively. Alkanoic acids with an isoprenoid skeleton are more abundant in Jianghan samples, including C20, C21, C24, C25 and C30 homologues, with a C25 component (3,7,11,15,19-pentamethyleicosanoic acid) most pronounced in the lower part of the Qianjiang Fm. The carbon skeletons of these isoprenoid acids in both units are attributed to archaeal decomposers.

Biomarkers. A) Cross plot of Pristane/nC17 versus Phytane/nC18 ratios for various saline/hypersaline settings in China (after Wang, 1998). B) Structure and properties of representative bacteriochlorophylls, maleimides and porphyrins from source rocks in the northern and southern Zechstein basins, offshore NW Europe (after Pancost et al., 2002). C) Relationship between pristane/phytane and the methyltrimethyltridecylchhroman (MTTC) ratio in Jurassic Malm carbonates of eastern Bavaria (after Schwark et al., 1998). The MTTC ratio is defined as 5,7,8-trimethylchroman/total MTTCs. D) Variations in pristane/phytane ratio and the gammacerane index for oils from lacustrine source rocks in Angola (Inset shows gammacerane structure). Independent biomarker evidence suggests a marine source for the point that lies off the shaded trend (after Peters et al., 2005).

Iso and anteiso branched carboxylic acids are prevalent in both the Ejinur samples and in the upper portion of the Qiangiang Formation. They derive from bacteria, probably sulphate-reducing bacteria, and their abundance clearly shows once again the importance of bacterial decomposers, along with haloarchaea, to the biochemical signature of organic matter in modern and ancient saline lake sediments. The presence of hopanoid acids and a 3-carboxy steroidal acid further attest to contributions from bacterial and eukaryotic sources, respectively. The occurrence of particular carboxylic acids in the Jianghan samples illustrate these compounds, indicators of halotolerant and halophilic decomposers, can survive as biomarkers in hypersaline source rocks.

Across a broader time and geographic scale, acyclic isoprenoid hydrocarbons are the dominant components in organic matter extracted from sedimentary cores and oils sampling various hypersaline settings in China; the results encompass the Tertiary Janghan salt lake basin, the Cretaceous Taian salt lake basin, and the Triassic, Permian and Cambrian Yangtze evaporitic marine platforms (Wang, 1998). Inland saline lake basins in China, such as Jianghan and Songpu, are characterised by a tremendous predominance of phytane (C20), which ranges up to 15% of the total extract. The evaporitic marine sediments are also unique in retaining a complete series of super-long-chain acyclic isoprenoids, up to C40 (Figure ). These isoprenoids in the marine evaporites possess head-to-head, tail-to-tail or regular linkages that indicate a significant contribution from various archaea to the biomarker signature, i.e. a mixed archaeal contribution from halophiles, methanogens and acidothermophiles.

Based on the split in distribution and composition of isoprenoids between the inland salt lakes and the evaporitic marine platform settings, Wang (1998) concluded that the archaeal biota in modern and Cenozoic inland salt lake sediments of China are likely dominated by halophilic archaea, while the halobiotal signatures of Mesozoic and Palaeozoic evaporitic marine sediments were predominantly those of methanogens and acidothermophiles. He also found that the concentration of the various chlorine salts in the sediments is more directly proportional to the abundance of phytane than to sulphate, once again indicating the dominance of halophilic archaea in waters at the halite precipitation stage. He also noted that reduced species of sulphur, sulphide and organic sulphur compounds in the anoxic brine played a crucial role in the preservation and formation of the abundant phytane in these inland salt lake basins.

A predominance of degraded green algal and cyanobacterial biomarkers in mesohaline settings can be recognised in biomarkers preserved in even older, but organically immature, evaporitic source rocks. The degree of methylation of 2-methyl-2-trimethyl-tridecylchromans (MTTCs) and the abundance of maleimides and bacteriochlorophylls implies a euhaline to mesohaline (≥30-40‰) marine-fed setting for the accumulation and preservation of organics during Kupferschiefer sedimentation in the Permian of NW Europe (Figure B; Bechtel and Puttmann, 1997; Pancost et al., 2002). These biomarkers are thought to be derived from green/purple sulphur bacteria (decomposers) within organic-rich laminites and suggest that the bottom waters were saturated with H2S at the time of deposition. Maximum water depths were probably less than 100 metres (it was a stratified mesohaline drawdown basin at the onset of Zechstein salinity; see Chapter 5 in Warren, 2016). Decomposers probably lived near the brine boundary between the photic zone and the anoxic (euxinic) bottom water at typical water depths of 10-30 metres below the water surface, as in modern marine-fed density-stratified systems, much like in Lake Mahoney today. Primary production in the upper water mesohaline column was dominated by photosynthetic cyanobacteria or green algae, while sulphate reduction in the bottom sediment was tied to the availability of abundant sulphate and organic detritus from the overlying water column.

Methanogenesis was also active during Kupferschiefer deposition. This is reflected in the light carbon isotopic composition of organic matter that had originated via recycling of CO2 generated by methane-oxidising bacteria in the water column (Bechtel and Puttmann, 1997). Saccate pollen is the only morphologically preserved body fossil in the organic matter within this laminated Kupferschiefer sediment, all other traces of cellular /microbial morphologies/fossils are gone. Euxinic conditions were confirmed by Pancost et al. (2002) over a much larger area of Kupferschiefer deposition than studied by Bechtel and Puttmann. They concluded that almost all of the Kupferschiefer seaway was subject to periods of photic zone euxinia and stratification during the early mesohaline history of the Zechstein Sea.

By themselves, high pristane/phytane ratios, high amounts of gammacerane, and MTTC ratios are not reliable indicators of hypersalinity. More reliable determinations can be made when two or more of these biomarkers are cross plotted, and the resulting output shows a consistent trend. Figure C plots pristane/phytane ratio against the MTTC ratio in the Jurassic Malm Zeta laminites of eastern Bavaria in SW Germany (Schwark et al., 1998). The output shows a covariant decrease in the two measures, lipids from phototrophic organisms and shows 13C values consistent with the heterotrophic metabolism, which is interpreted as indicating a trend from normal-marine to mesohaline deposition.

A similar covariant trend can be seen in saline lacustrine source rocks when the pristane/phytane ratio is cross plotted against the gammacerane index in Mesozoic source rocks from the offshore of west Africa (Peters et al., 2005). Figure D plots variations in pristane/phytane (a redox and archaeal indicator) and gammacerane index (an indicator of salinity stratification in saline waters) for oils from a set of lacustrine source rocks. Increasing salinity in the depositional setting explains the covariant trend in the plot, whereby higher salinity was accompanied by density stratification and associated reduced oxygen levels in bottom waters. Independent biomarker evidence suggests a marine source for the point that plots off the shaded salinity trend in this figure (after Peters et al., 2005). Although the levels of entrained organics are very low, similar covariant trends in pristane/phytane and gammacerane occur in C21-25 isoprenoids, along with relatively heavy δ13C signatures, in biomarkers extracted from the Miocene halites of the halokinetic Sedom Formation in Israel. They are enriched by 7‰ compared to lipids from phototrophic organisms and show 13C values consistent with the heterotrophic metabolism of Halobacteriales. It indicates a predominance of halophilic archaea, possibly consuming the remnants of Dunaliella-like bloom when the salt was deposited in a CO2-limited system (Grice et al., 1998; Schouten et al., 2001). 

Stratigraphy plotted against the archaeol/caldarchaeol * 100 (ACE index) also with calculated salinity outputs based on the modern calibration of the ACE for two detailed hypersaline Messinian sections in Italy. A) Serra Pirciata and B) Torrente Vaccarizzo (after Turich and Freeman, 2011).

A recent development in biological indicators during salinity progression comes from the work of Turich and Freeman, 2011). In a wide-ranging study of archaeal biomarkers, they found a distinct evolution in archaeol molecules related to increasing salinities in hypersaline waters. They found the relative amounts of acyclic diether and tetraether membrane lipids synthesized by Archaea correlate with salinity from 0-250 practical salinity units (psu) in modern settings (Figure). They then examined the preservation of the lipid biomarker-salinity relationship in ancient sedimentary organic matter mostly using samples from two sequences of marls and diatomites deposited just before the Messinian Salinity Crisis in the Mediterranean. The archaeol ratio-based salinity estimates were consistent with expected absolute salinity, as well as the amplitude of isotope variations leading up to the Messinian Salinity Crisis. Their lipid biomarker approach to salinity reconstruction complements existing paleosalinity proxies, as listed in the table above, because: (i) Archaea survive and thrive over a broad salinity range from 7 - 340‰, which is well beyond that of haptophyte algae and other plankton, which define much of the microfossil record in mesohaline settings, and (ii) it provides better salinity resolution for the wide range of salinities broadly defined as hypersaline using existing biomarkers. Using their proxy, there is the potential to provide novel insights into salinity variation within desiccating basins in climatically sensitive seas (e.g. Dead Sea, Permian Delaware Basin), the evolution of brines, the timing of onset of hypersaline conditions and evaporite deposition, as they have done for the Messinian (Figure).

In addition to the signature from their particulate organics, modern brines can also contain large amounts of dissolved organic matter (DOM), with levels that increase with increasing salinity (Hite and Anders, 1991). This liquified organic is often entrained within brine inclusions in halite and other salt crystals and is not picked up in standard TOC determinations. Levels of volatile fatty acid (VFA), especially acetic acid, are also high in these saline brines and possibly reflect a higher bacterial contribution (Jørgensen et al. 2008). The relative contribution of DOM to the source potential is poorly understood in subsurface evaporitic systems where crossflowing basinal brines are dissolving buried halite and may also have significance in the survival of Archaean “proto-life” (Pasteris et al., 2006).