Note
Occurrence of fatty acids linked to non-phospholipid
compounds in the polar fraction of a marine sedimentary
extract from Carteau cove, France
E. Aries
a,*, P. Doumenq
a, J. Artaud
a, J. Molinet
a, J.C. Bertrand
baLaboratoire de Chimie Analytique de l'Environnement ESA CNRS 6171, Faculte des Sciences de Saint-JeÂroÃme, Avenue Escadrille,
Normandie Niemen, case 312, 13397 Marseille Cedex 20, France
bLaboratoire d'OceÂanographie et de BiogeÂochimie UMR CNRS 6535, Centre d'OceÂanologie de Marseille, 13288 Marseille, France
Received 12 September 2000; accepted 18 October 2000 (returned to author for revision 5 October 2000)
Abstract
A surface sediment from Carteau cove, located at the western part of the Gulf of Fos (France), was collected and analysed to determine the lipid composition of its most polar fraction. Analyses by high performance liquid chroma-tography (HPLC) and thin-layer chromachroma-tography (TLC) of the polar fraction of the sediment extract revealed the presence of phospholipids (PL), with mainly phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and lyso-phosphatidylethanolamine (LPE), but also the presence of non-phospholipid polar compounds (NPC). Phospholipids and non-phospholipid polar compounds (NPC) were isolated by preparative thin-layer chromatography and trans-methylated. Phospholipid ester-linked fatty acids ranged from 12- to 20-carbon atoms and accounted for 34.8mg gÿ1of dry sediment whereas fatty acids derived from non-phospholipid polar compounds ranged from 12- to 28-carbon atoms and accounted for 39.1 mg gÿ1 of dry sediment. The occurrence of signi®cant amounts of fatty acids derived
from non-phospholipid polar compounds of a sediment polar fraction illustrates the importance of the preliminary analysis and puri®cation of phospholipids to avoid an overestimation of the microbial biomass when determined with a current phospholipid ester-linked fatty acids (PLFA) analysis.#2001 Elsevier Science Ltd. All rights reserved.
Keywords:Sediments; Phospholipids; Biomass; Microbial community; Phospholipid ester-linked fatty acids
1. Introduction
Analyses of the phospholipid ester-linked fatty acids (PLFA) is an established method for estimating micro-bial biomass and community structure in marine sedi-ments (White et al., 1988). The major strength of the method is that fatty acids in intact phospholipids are mainly derived from living biomass since the polar
group of phospholipids in dead organic matter is rapidly hydrolysed (White et al., 1979). A current PLFA analy-sis requires hydrolyanaly-sis of phospholipids recovered by preparative liquid chromatography in the most polar fraction of the sediment extract, without any pre-liminary analysis of intact phospholipids. A recent report highlights the fact that the analysis of the head groups of phospholipids provides more information for bacterial characterisation than that provided by a sim-ple PLFA analysis (Fang et al., 2000). From this point of view, the present investigation focuses on the analysis of the most polar fraction of a marine sediment col-lected in Carteau cove (Gulf of Fos, France) to deter-mine its phospholipid and PLFA compositions.
0146-6380/01/$ - see front matter#2001 Elsevier Science Ltd. All rights reserved. P I I : S 0 1 4 6 - 6 3 8 0 ( 0 0 ) 0 0 1 5 3 - 4
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* Corresponding author.
2. Experimental
2.1. Sampling
Sediment sampling, at a mean depth of 3 m, was car-ried out in February 2000 at Carteau cove, a semi-closed cove located at the western part of the Gulf of Fos. Sediments were collected by inserting a 10 cm diameter core into the sediment. The cores were extruded, the top 2 cm was sampled and stored at ÿ35C for analysis.
Triplicate sediment cores were collected and worked up separately as replicate samples.
2.2. Lipid extraction and fractionation
Freeze-dried sediments (ca. 10 g) were extracted by the modi®ed Bligh and Dyer method (White et al., 1979). Extracts were fractionated on glass columns (1.5 cm i.d.) packed with 6 g of 70±230 mesh silica gel deac-tivated with 5% of distilled water. Neutral lipids, med-ium polar and polar fractions were obtained by eluting with 100 ml of chloroform, 100 ml of acetone and 300 ml of methanol, respectively.
2.3. Polar fraction analyses
Polar fraction separation was accomplished by HPLC on a 2504.6 mm i.d. column packed with 5mm YMC PVA-Sil (Hichrom, Reading, UK). A Hitachi L-6200 A pump was used with a DDL 21 light scattering detector (Cunow, Cergy, France). The elution scheme consisted of a ternary gradient as previously described by Christie and Urwin (1995). Polar fractions were also analysed by TLC on precoated silica gel F plates (20 cm20 cm0.2 mm, Merck, Darmstadt, Germany). Plates were developed with CHCl3/MeOH/glacial acetic acid (65/25/8, v/v/v). Lipids were visualized using the Vaskovsky's reagent, spe-ci®c for phospholipids (Vaskovsky and Kotetsky, 1968).
2.4. Puri®cation of phospholipids and of non-phospholipid polar compounds
Preparative TLC was carried out for the one step puri®cation of phospholipids and non-phospholipids by using the same conditions described above. The silica gel band corresponding to phospholipids (0<Rf<0.85) and that corresponding to non-phospholipids (0.85<Rf<1.0) were scraped into a Te¯on-lined screw cap culture tube. Phospholipids and non-phospholipids were desorbed from the silica gel with 10 ml of methanol and analysed by HPLC using similar conditions to those described above.
2.5. Preparation of derivatives
Phospholipids and non-phospholipids were transes-teri®ed using BF3/MeOH as previously described by
Morrison and Smith (1964). Before transesteri®cation, the henicosanoic acid was added as an internal stan-dard. To con®rm the position of double bonds for monounsaturated fatty acids, dimethyldisul®de (DMDS) adducts of fatty acid methyl esters (FAME) were prepared using the method previously described by Nichols et al. (1988).
2.6. GC and GC/MS analysis
Fatty acid methyl esters were quanti®ed by GC with a CP 9000 Chrompack chromatograph, equipped with an on-column injector, using a DB-5MS column, (60 m0.25 mm i.d.; 0.25 mm) (J&W Scienti®c, Folsom, CA). Helium was used as carrier gas. The oven program was 60C for 2 min, then 20C/min up to 160C, 2C/
min up to 290C and held there for 20 min. FAME
samples and DMDS adducts were identi®ed on a Hew-lett-Packard 5890 GC equipped with the same column as used for GC analysis coupled to a Hewlett-Packard 5898A MS Engine mass spectrometer. The transfer line was held at 295C and the source at 240C. Samples
were injected using a splitless injector. Helium was used as carrier gas. For FAME and DMDS analyses, the oven program was 30C for 1 min, then 50C/min up to
70C, 10C/min up to 120C, 2C/min up to 290C, and
held for 10 min.
3. Results and discussion
with the results obtained by TLC, the two peaks, eluting between PG and LPE, were attributed to the unknown phospholipid previously designated as PLX. The pur-i®ed NPC fraction from the polar fraction revealed a very complex distribution of polar compounds. The chromatographic conditions employed for this analysis led to an unresolved complex mixture: a clean separa-tion of all compounds was not achieved Fig. 1(c). We believe this work provides the ®rst detailed analysis of a sedimentary polar fraction. Our results demonstrate the occurrence of both phospholipids and non-phospholipid polar compounds in a polar fraction extracted from a marine sediment. These latter compounds prevented us from identifying phospholipids unambiguously. Since the analysis of intact phospholipids could be a potential tool used to assess a microbial community structure within marine sediments (Fang et al., 2000), this work suggests the need of a preliminary puri®cation step of phospholipids in the sedimentary polar fraction to avoid interferences due to the occurrence of non-phospholipid polar compounds.
Phospholipids and non-phospholipids, isolated by preparative TLC, were also subjected to methanolysis to analyse and quantify their respective fatty acids con-tents. Quanti®cation of the fatty acids derived from phospholipids led a total PLFA content of 34.8 mg gÿ1
of dry sediment (Table 2). As shown in Table 2, PLFA ranged from 12 to 20 carbon atoms long and are char-acterised by large proportions of saturated fatty acids (62.5%) with mainly 16:0,i15:0,a15:0 and 18:0, by low level of monounsaturated fatty acids (30.8%) with 18:1!7c predominating and by polyunsaturated fatty
acids (6.7%). Fatty acids linked to non-phospholipids
(NPFA) accounted for 39.1mg gÿ1of dry sediment. In
the range C12±C20, the non-phospholipids exhibited a fatty acid pro®le nearly identical to the PLFA pro®le (Table 2), apart from a higher percentage of 14:0 (11.2%) and a lower percentage of 18:1!7c(4.7%). As
Table 1
Analysis of authentic phospholipid standards and of the polar fraction from Carteau cove by TLC using the solvent system CHCl3/MeOH/glacial acetic acid (65/25/8, v/v/v)
Polar lipid designation Polar
Lysophosphatidylcholine (LPC) ND 3 Blue Phosphatidylcholine (PC) ND 9 Blue Lysophosphatidylethanolamine (LPE) + 13 Blue Phosphatidylinositol (PI) ND 22 Blue
PLXc + 30 Blue
Phosphatidylethanolamine (PE) + 42 Blue Phosphatidylglycerol (PG) + 62 Blue Phosphatidic acid (PA) ND 76 Blue Diphosphatidylglycerol (DPG) ND 79 Blue
NPCd + 90 Green
aND, not detected; +,detected.
bA blue stain indicates a positive reaction to the reagent of Vaskovsky. cPhospholipid unidenti®ed.
dNon-phospholipid polar compounds.
Table 2
Fatty acid compositions, expressed as percentages of total fatty acids, of phospholipids and non-phospholipids isolated from the polar fraction of Carteau cove
Percentage composition
Total saturated fatty acidsc 62.5 79.3 Total monounsaturated fatty acidsc 30.8 16.9 Total polyunsaturated fatty acidsc 6.7 3.8 Total fatty acid concentrationd 34.8 39.1
a Tentative peak identi®cation.
b Determination of the methyl branching position and of the double bond location was not achieved.
c Expressed as % total fatty acids. d Expressed as
opposed to phospholipids in the range C20±C28, non-phospholipids exhibited a clear predominance of even-numbered straight chain fatty acids, mainly 20:0, 22:0, 24:0, 26:0 and 28:0, over the corresponding odd-num-bered straight chain fatty acids, 21:0, 23:0, 25:0, 27:0 (Table 2). This study also appears to be the ®rst to report the occurrence of signi®cant amounts of non-phospholipid fatty acids in the polar fraction of a sedi-ment extract. This ®nding, also observed on sedisedi-ments from various locations (E. Aries, unpublished data), asks few questions relative to the current PLFA method which takes into account the fatty acid content of the whole polar fraction to estimate the viable microbial biomass (White et al., 1979). Indeed, our results evi-dence the occurrence of two distinct pool of fatty acids in a sediment's polar fraction. With regard to the phos-pholipid pool, high levels of branched fatty acids, and a distribution in the range C12±C20, indicates a bacterial origin (Le Chevalier, 1977). The occurrence in the non-phospholipid pool of long chain fatty acids, with an even predominance, indicates an allochthonous terrige-nous contribution of polar lipids derived from higher plants (Shaw and Johns, 1986). As a result of these observations, we conclude that a preliminary puri®ca-tion of sedimentary phospholipids from the polar frac-tion should be systematically done before the analysis of their associated fatty acids to avoid an overestimation of the viable microbial biomass.
Associate EditorÐA.G. Douglas
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