Jean-François Rontani

Phytol degradation products as biogeochemical tracers in aquatic environments

Phytol (the ester-linked side-chain of chlorophyll-a) is perhaps the most studied biomarker of those found in modern aquatic environments. This paper reviews recent studies of phytol degradation and provides an update on more classical studies. After a discussion of the biosynthesis and isotopic fractionation of phytol, we examine the different biotic and abiotic processes which may play a significant role during the diagenesis of this widely distributed isoprenoid alcohol: (i) photodegradation of the chlorophyll phytyl chain in senescent phytoplanktonic cells, (ii) hydroperoxide-induced oxidation of the chlorophyll phytyl chain in senescent phytoplanktonic cells, (iii) degradation of the chlorophyll phytyl chain during marine invertebrate feeding, (iv) aerobic and anaerobic biodegradation of phytol, (v) clay-catalysed degradative processes, and (vi) sulfur incorporation. Emphasis is given to the mechanisms of these processes and to the structures of the products formed. We conclude with some remarks on the potential and constraints of the main phytol degradation products as biogeochemical tracers and highlight some areas requiring further work.

Characterisation of photo-oxidation and autoxidation products of phytoplanktonic monounsaturated fatty acids in marine particulate matter and recent sediments

Oxidation products of phytoplanktonic monounsaturated fatty acids have been detected in particulate matter and recent sediment samples collected at the SOFI station (Gulf of Fos, Mediterranean Sea). The fluxes of these compounds are not negligible and correspond to 0.14–0.03% of the total organic carbon flux in the zone investigated. Oxidation of monounsaturated fatty acids results mainly in the production of isomeric allylic hydroperoxyacids, which are relatively stable in the water column. Saturated hydroperoxyacids, epoxyacids and dihydroxyacids, deriving probably from the addition of hydroperoxides to the double bond of monounsaturated acids, have been detected also. During early diagenesis, hydroperoxides undergo heterolytic cleavage to aldehydes and ω-oxocarboxylic or ω-hydroxycarboxylic acids, or homolytic cleavage and subsequent transformation to the corresponding alcohols or ketones. Due to the good preservation of their distribution pattern in Recent sediments, isomeric allylic hydroxyacids thus formed will constitute more useful and specific tracers of oxidative processes than ω-oxocarboxylic or ω-dicarboxylic acids (with C9 as the predominant species). These compounds will allow easy differentiation between autoxidative and photooxidative processes and could thus provide a useful indication of the residence time of particulate organic matter in the water column, the physiological state of phytoplanktonic communities and current environmental problems related to ozone depletion.

Photochemical oxidation and autoxidation of chlorophyll phytyl side chain in senescent phytoplanktonic cells: Potential sources of several acyclic isoprenoid compounds in the marine environment

Visible light-induced degradation of the chlorophyll phytyl side chain was studied in senescent cells of two phytoplanktonic strains (Skeletonema costatum and Thalassiosira weissflogii). Particular attention was paid to the induction of autoxidation processes on the phytyl chain and its photoproducts by photochemically produced hydroperoxides. The combination of photochemical oxidation and autoxidation reactions resulted in the production of several acyclic isoprenoid compounds that have been unambiguously identified by comparison of their retention times and mass spectra with those of appropriate standards. Various mechanisms are proposed to explain the formation of these oxidation products. These processes appear to be potential sources of numerous oxidized acyclic isoprenoids that previously have been detected in lacustrine and marine environments. Some oxidation products newly described or whose presence in natural samples was never reported in the literature were then sought in particulate matter, sediment, and microbial mat samples. The results obtained supported the significance of photochemical oxidation and autoxidation of phytoplanktonic chlorophyll phytyl side chain in the marine environment.

Stability of long-chain alkenones in senescing cells of Emiliania huxleyi: effect of photochemical and aerobic microbial degradation on the alkenone unsaturation ratio (Uk′37)

Abstract Alkenones were photooxidized in pyridine solution of haematoporphyrin and in killed cells of Emiliania huxleyi . These ketones seem to be protected from photochemical damage in phytodetritus; this photochemical recalcitrance has been attributed to their chemical structure and localization in the cells. These results indicate that U k′ 37 ratios are not likely to be strongly photochemically modified during senescence of Emiliania huxleyi (increase limited to 0.04 U k′ 37 unit). In contrast, the major sterol of this algae (24α-methylcholesta-5,22 E -dien-3β-ol) appeared to be very sensitive to photochemical processes. Non-sterile incubations of broken cells of Emiliania huxleyi showed a strong biodegradation of alkenones, indicating that aerobic microbial processes must play an important role during senescence of this species. However, this degradation takes place without dramatic variations in U k′ 37 values (decrease limited to 0.05 U ′ 37 unit). Several mechanisms are proposed in order to explain this strong and weakly selective biodegradation.

Visible light-induced oxidation of lipid components of purple sulfur bacteria: a significant process in microbial mats

Visible light-induced degradation processes were studied in vitro in senescent cells of two purple sulfur bacteria (Thiohalocapsa halophila and Halochromatium salexigens) isolated from microbial mats from Camargue (France). These reactions act on the phytyl side chain of bacteriochlorophyll-a and on palmitoleic and cis-vaccenic acids. The experiments also confirmed some previous results, i.e. the regiospecific enzymatic oxygenation of the allylic carbon 10 of cis-vaccenic acid in senescent cells of T. halophila. The subsequent detection of substantial amounts of photoproducts from the phytyl chain and mono-unsaturated fatty acids in the lipid extracts of the microbial mats from which these two strains were isolated clearly established the importance of photodegradation processes in this particular ecosystem. The presence of photoproducts of cis-vaccenic acid showed that under natural conditions photochemical reactions act extensively on senescent phototrophic bacteria. Significant amounts of 10-oxooctadecanoic and 10-hydroxy-11-oxooctadecanoic acids were also detected in the lipid extracts. The detection of these compounds, which result from the enzymatic oxidation of carbon 10 of cis-vaccenic acid, supports a strong T. halophila contribution to the microbial mats.

NaBH4 reduction of alkenones to the corresponding alkenols : a useful tool for their characterisation in natural samples

Abstract During a search for phytoplanktonic lipid photoproducts in sediment extracts that had been reduced with NaBH 4 , we observed that the alkenones present had been reduced quantitatively to the corresponding unsaturated alcohols (alkenols). The silylated alkenols display better chromatographic characteristics than the corresponding alkenones and very useful EI mass spectra, which show strong fragment ions at m/z 117 or m/z 131 due to cleavage α to the functional group, allowing methyl and ethyl alkenols (and hence the parent alkenones) to be readily differentiated. Calculations of U K′ 37 values using the alkenols formed by reduction agreed within experimental error (±0.01 units) with values calculated from the original alkenones, suggesting that this might be a useful method for samples containing low contents of alkenones or where co-elution is a problem. Although small amounts of alkenols have been found in some sediments their presence does not appear to affect U K′ 37 calculations. We have found large amounts of alkenols (up to 20% of the amount of the corresponding alkenone) in extracts of some sediments from Camargue (France), which we suggest are probably formed by a non-selective bacterial reduction of alkenones under anoxic conditions. The possibility of a natural contribution of alkenols from haptophytes was tested and we were able for the first time to detect traces (less than 1% of the amount of the corresponding alkenone) of alkenols in non-reduced extracts of Gephyrocapsa oceanica and Isochrysis galbana. We presume that their biosynthesis is closely related to that of the alkenones. Studies of other haptophytes (particularly those from benthic or coastal environments) are needed to ascertain whether other species might contain higher contents of alkenols. The reduction-silylation technique has also proven to be very useful for the characterisation of previously unreported alkenones in microalgae, sediments and seawater. A re-examination of extracts of several haptophytes after this treatment allowed us to detect small amounts of a 35:1 methyl alkenone and 2 isomers of a 35:2 alkenone in G. oceanica , and monounsaturated C 35 –C 38 alkenones in Emiliania huxleyi .

Re-examination of the double bond positions in alkenones and derivatives : Biosynthetic implications

By derivatizing with OsO4, we determined the double bond positions of all the diunsaturated alkenones present in different haptophytes and demonstrated that the positions in C37–C40 homologs occur at a fixed carbon number from the carbonyl group, a finding contrary to earlier speculation. These data have allowed us to recognize three “families” of alkenones, for which we propose different biosynthetic pathways. The proposed biosynthesis of classical C37–C40 alkenones starts with acetyl‐ or propionyl‐SCoA and involves classical chain elongation steps with malonyl‐ and methylmalonyl‐SCoA affording alkanoyl‐ACP intermediates, which undergo subsequent Δ14, 21‐desaturation and decarboxylation reactions. Unusual diunsaturated shorter‐chain (C35 and C36) ketones now being produced for yet unknown physiological reasons by our specimen of Emiliania huxleyi (Lohm). Hay et Mohler strain CCMP1742. These compounds exhibit ω15,22 and ω16,23 double bonds, which are at a shorter distance (two carbon atoms) from the carbonyl group than the higher homologs, implying that their biosynthesis must involve an additional chain‐shortening step after Δ14, 21‐desaturation of alkanoyl‐ACP. Alkenones exhibiting a very unusual double bond spacing (three methylene groups instead of five) were detected in Holocene Black Sea sediments, in particulate matter collected in the Ligurian Sea and in Gephyrocapsa oceanica strain JB‐02. The formation of these compounds seems to be linked to the biosynthesis of monounsaturated alkenones previously detected in several haptophytes. Our work demonstrates the value of determining the double bond locations of alkenones whenever possible.

“Bound” 3-methylidene-7,11,15-trimethylhexadecan-1,2-diol: a new isoprenoid marker for the photodegradation of chlorophyll-a in seawater

3-Methylidene-7,11,15-trimethylhexadecan-1,2-diol was detected after saponification of chlorophyll-ain vitro photoproducts. It is postulated that this isoprenoid compound, which probably results from an attack of singlet oxygen on the olefinic carbon 2 of the chlorophyll-a phytyl chain, could be a marker for the photodegradation of chlorophyll-a in the marine environment. This theory is supported by the detection of high quantities (0.1–2 μg g−1 dry wt) of “bound” 3-methylidene-7,11,15-trimethylhexadecan-1,2-diol in recent sediments from the Gulf of Fos sur mer (Mediterranean Sea).

Early diagenesis of the intact and photooxidized chlorophyll phytyl chain in a recent temperate sediment

Abstract Comparison of the stability of intact and photooxidized chlorophyll phytyl chain with depth in a core of recent sediment (where high bacterial activity was observed) allowed us to select potential markers for monitoring past photodegradation of chlorophyll. “Esterified + bound” 3-methylidene-7,11,15-trimethylhexadecan-1,2-diol appeared to be particularly suitable for such an application, but we excluded “esterified + bound” 6,10,14-trimethyl-pentadecan-2-one which is not sufficiently stable nor is it exclusively produced by photooxidation. For example, it can be produced during alkaline hydrolysis of tocopherols. Bacterial processes appeared to be particularly active on “free” isoprenoid compounds.

Comparison of the photodegradation rates of chlorophyll chlorin ring and phytol side chain in phytodetritus: is the phytyldiol versus phytol ratio (CPPI) a new biogeochemical index?

Abstract Rates of photodegradation of chlorophylls in their entirety and of chlorophyll phytyl chains, and time evolution of the Chlorophyll Phytyl side-Chain Photodegradation Index (CPPI) under visible light exposure were determined and compared in laboratory experiments. Killed phytoplanktonic cells of two widespread Bacillariophyceae (Thalassiosira rotula Meun. and Skeletonema costatum (Grev.) Cleve), one widespread Prymnesiophyceae (Emiliania huxleyi (Lohm.) May and Mohler) and one Chlorophyceae (Dunaliella tertiolecta Butch) were used as source of pigments (chlorophyll a, chlorophyll b and phaeophytin a). Chlorophyll and chlorophyll phytyl chain photodegradation and CPPI evolution showed apparent first-order kinetics with respect to light exposure. The in vitro determined degradation rates were approximately 3.5 times higher for the chlorophyll tetrapyrrolic structure than for the phytyl chain. Few variations of the relative photodegradation constant rates were observed between strains: less than 34% for chlorophyll a and 25% for chlorophyll phytyl chain at 20°C. Nonetheless, the photo-oxidation process of the phytyl chain (Q10=2.0) seems more affected by temperature than does chlorophyll a (Q10=1.4). Phytol loss in the dark controls have been attributed to bacterial degradation, and, in the case of S. costatum, chlorophyllase dephytylation seems to have enhanced the potential for bacterial biodegradation. From these results, it clearly appears that in small detrital particles not only chlorophyll a, chlorophyll b and phaeophytin but also their phytyl chains may be rapidly photodegraded in the upper portion of the euphotic zone. The use of the CPPI for monitoring the chlorophyll photo-oxidation process in the aquatic environment is discussed.