Aerobic bacterial metabolism of phytol in seawater:
eect of particle association on an abiotic intermediate
step and its biogeochemical consequences
Jean-FrancËois Rontani *, Patricia C. Bonin
Laboratoire d'OceÂanographie et de BiogeÂochimie (LOB), Centre d'OceÂanologie de Marseille (OSU), Campus de Luminy, Case 901, 13288-Marseille, France
Received 11 November 1999; accepted 9 March 2000 (returned to author for revision 24 January 2000)
Abstract
The biodegradation of phytol by a bacterial community isolated from the oxic zone of a Recent temperate marine sediment was studied in the presence, and in the absence, of a solid support. Analyses of isoprenoid wax esters pro-duced by condensation of bacterial metabolites with themselves, or with phytol, showed that bacterial metabolism of phytol was strongly modi®ed in the presence of the solid support. Aerobic metabolism of phytol involves the transient production of (E)-phytenal, which in turn can be quickly abiotically converted to 6,10,14-trimethylpentadecan-2-one or (E)-phytenic acid. Sorption on mineral particles appears to hinder the addition of water to the activated double bond of (E)-phytenal, which constitutes the ®rst step of its transformation to the C18isoprenoid ketone. Consequently, it is
concluded that in surface temperate sediments phytol must be metabolised mainly via (E)-phytenic acid and that results obtained from in vitro experiments with free cell cultures may not be comparable to processes occuring in marine sedimentary environments.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Phytol; Bacterial community; Aerobic metabolism; Abiotic intermediate step; Isoprenoid wax esters
1. Introduction
Degradation of chlorophyll-a is thought to be the major source of phytol in the marine environment (Volkman and Maxwell, 1986), since chlorophyll-a is the most abundant photosynthetic pigment in almost all species of phytoplankton (Sun et al., 1993). Although the ester bond between phytol and the tetrapyrrolic macrocycle of chlorophyll can resist hydrolysis, as shown by the isolation of intact phytyl esters from sediments several million years old (Baker and Smith, 1974), sig-ni®cant amounts of free phytol are present in Recent sediments (Rontani et al., 1996; Grossi et al., 1998). Free phytol is generated from chlorophyll by hydrolysis
during digestive processes of copepods (Prahl et al., 1984; Grossi et al., 1996), senescence of diatoms (Jerey and Hallegrae, 1987) and early diagenesis in sediments (Johns et al., 1980).
Bacterial degradation of phytol is often considered to be an important source of acyclic isoprenoid acids in marine sediments (Volkman and Maxwell, 1986). Although some early studies provided information on the biodegradation of phytol (Brooks and Maxwell, 1974; Brooks et al., 1978; Gillan et al., 1983), it is gen-erally agreed that there are still many aspects of these processes which remain to be elucidated. Consequently, over the last few years we have studied the biodegradation of phytol by mixed sedimentary bacterial communities under aerobic (Rontani et al., 1999a,b) and anaerobic (Grossi et al., 1998; Rontani et al., 1999b) conditions.
In sediment slurries under sulfate-reducing condi-tions, phytol was quickly biodegraded to phytenes via phytadiene intermediates (Grossi et al., 1998). More
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* Corresponding author. Tel.: 4-9182-9623; fax: +33-4-9182-6548.
recently, we could show that two bacterial communities, isolated from marine sediments under aerobic and denitrifying conditions metabolised eciently phytol via 6,10,14-trimethylpentadecan-2-one and (E)-phytenic acid (Rontani et al., 1999b). In this case, the ®rst step in both aerobic and anaerobic bacterial degradation of phytol involves the transient production of (E )-phyte-nal, which in turn can be biotically and abiotically con-verted in seawater to (E)-phytenic acid and abiotically to 6,10,14-trimethylpentadecan-2-one (Rontani and Acquaviva, 1993). The production of this ketone involves addition of water to the activated double bond of (E)-phytenal and a subsequent retro-aldol reaction. Most of the isoprenoid metabolites identi®ed in vitro could be detected in a fresh sediment core collected at the same site as the sediments used for the incubations under aerobic and denitrifying conditions, and the major part of phytol appeared to be biodegraded in situ via (E)-phytenic acid (Rontani et al., 1999b). This result was attributed to the fact than (E)-phytenal is less sen-sitive to abiotic degradation at the temperature of the sediment (15C), cf. the in vitro experiments (20C)
(Rontani and Acquaviva, 1993).
Since in sediments, most benthic bacteria are not sus-pended in interstitial water but are attached to sediment particles (Epstein and Rossel, 1995) and the major part of phytol is associated with particulate marine detritus (phytodetritus and faecal pellets), particle association could also have an eect upon the abiotic stability of (E)-phytenal. Consequently, in the present work we studied the eect of particle association upon aerobic phytol metabolism by a marine bacterial community.
2. Experimental
2.1. Bacterial isolation
The aerobic bacterial community was obtained from surface sediments of Carteau Bay (Gulf of Fos sur mer, Mediterranean Sea) by methods described previously (Rontani et al., 1999b).
2.2. Growth conditions
The basic growth medium consisted of autoclaved arti®cial seawater (Baumann and Baumann, 1981) (ASW) supplemented with iron sulfate (0.1 mM), potassium phosphate (0.33 mM), ammonium chloride (0.1 mM) and phytol (3 mM) as the source of carbon and energy. Cultures were maintained in 250 ml Erlen-meyer ¯asks containing 50 ml of medium and agitated on a reciprocal shaker at 96 rpm. For the immobilised cell cultures, 7.5 g of mineral support [sand facies (Mearl), 90% of grains higher than 1 mm] composed by skeleton fragments of calcareous algae (Lithothamnium)
was added to the medium before sterilisation. For each experiment, two ¯asks were inoculated: the ®rst for estimation of substrate degradation and identi®cation of the metabolites and the second for monitoring bacterial growth. Experiments carried out in the presence of mercuric chloride (10 mg lÿ1) served as abiotic controls
(autoclaved sterilisation was avoided since phytol can be easily dehydrated during such a treatment).
2.3. Adhesion kinetics of bacteria
Several immobilised cell systems (without phytol sub-strate) were inoculated and shaken for dierent times. The separation of solid and liquid phases of each system was obtained by ®ltration on Whatman qualitative ®l-ters. The mineral particles were then washed three times with a known volume of ®ltered/sterilised ASW and the washings were pooled with the aqueous phase. Free cells were counted by epi¯uorescence in the presence of ¯uorochrome (40,6-diamidine-20-phenylindole
dihydro-chloride; DAPI) as previously described (Rontani et al., 1999a).
2.4. Treatment of bacterial cultures
At the end of bacterial growth aqueous and solid phases were separated by decantation. The aqueous phase was continuously extracted with chloroform overnight, while the wet solid phase was extracted ultrasonically with isopropanol/hexane (4:1, v/v) (De Leeuw et al., 1977a). The chloroform and hexane extracts were combined, dried over anhydrous Na2SO4,
®ltered and concentrated by rotary evaporation to yield the residual substrate and the neutral metabolites. To recover acidic metabolites, the isopropanol/water phase was ®ltered, concentrated under vacuum, acidi®ed with HCl (pH 1) and then extracted with chloroform (three times). After evaporation of solvents, the dierent extracts were taken up in 400ml of a mixture of pyridine and BSTFA (3:1, v/v) and allowed to silylate at 50C for
1 h. After evaporation to dryness, the residue was taken up in ethyl acetate and analyzed by gas chromato-graphy/mass spectrometry (GC/MS).
Bacterial metabolites were identi®ed by comparison of their retention times and electron impact mass spec-tra with those of synthesised reference compounds and then quanti®ed by calibration with external standards. GC/MS analyses were carried out with an HP 5890 ser-ies II plus gas chromatograph connected to an HP 5972 mass spectrometer (Hewlett-Packard). The following operating conditions were employed: 30 m0.25 mm (i.d.) capillary column coated with HP 5 (Hewlett Pack-ard); oven temperature programmed from 60 to 130C at
30C minÿ1and then from 130 to 300C at 4C minÿ1;
from 1.05 to 1.5 bar at 0.04 bar minÿ1; injector (on
col-umn) temperature, 50C; electron energy, 70 eV; (Electron
impact mode); source temperature, 170C; cycle time, 1.5 s.
2.5. Abiotic degradation of (E)- phytenal
These experiments were carried out in sterile 250 ml Erlenmeyer ¯asks containing 50 ml of synthetic sea-water and supplemented or not with 7.5 g of mineral support. After addition of synthetic (E)-phytenal (50ml) and agitation in the dark at controlled temperatures, the contents of the ¯asks were extracted as described above. The dierent extracts were dried over Na2SO4, ®ltered,
concentrated and silylated before GC/MS.
2.6. Chemicals
(E)-Phytol was puri®ed from a commercially available mixture of isomers (Acros) by column chromatography [25 cm1 cm (i.d.)] on silica gel (Kieselgel 60+0.5% H2O) with n-hexane±ethyl acetate (9:1, v/v) as the
eluant. The syntheses of 6,10,14-trimethylpentadecan-2-one, (E)-phytenal, 4,8,12-trimethyltridecan-1-ol, phytenic, phytanic, 4,8,12-trimethyltridecanoic and 5,9,13-tri-methyltetradecanoic acids have been described pre-viously (Cason and Graham, 1965; Rontani and Acquaviva, 1993; Rontani et al., 1997). 4,8,12-tri-methyltridecyl-4,8,12-trimethyltridecanoate, phytylphytan-ate, phytylphytenphytylphytan-ate, phytyl-4,8,12-trimethyltridecanoate and phytyl-5,9,13-trimethyltetradecanoate were prepared from their parent alcohols and acids by the procedure described by Gellerman et al. (1975).
3. Results and discussion
The solid support model used in the present work appeared to be well suited to the study of the eects of particle association on phytol metabolism by marine bacteria, since it allowed rapid sorption of the major part of the substrate and immobilisation of a good pro-portion of the bacteria. The percentage of phytol sorbed to the mineral particles as a function of time was mea-sured in the sterile systems. After 24 h of shaking at 20C, it appears that 96% of the substrate was sorbed
by the particles (Fig. 1). The distribution of phytol between the water and the particles obtained with our experimental conditions must allow a good simulation of the sedimentary environment, where the major part of phytol is associated with marine detritus (phytode-tritus and faecal pellets). Free cells of immobilised cell systems (without phytol substrate) were counted at dif-ferent incubation times with the epi¯uorescence techni-que. After 30 min in the presence of the solid support, we were able to determine that about 65% of the bac-terial community was irreversibly adhered (Fig. 2).
As described previously (Rontani et al., 1999b), the aerobic bacterial community isolated from the sediments of Carteau Bay degrades phytol very eciently. Compared to sterile controls, we obtained 97% degradation after 10 days of incubation at 20C in the presence or in the
absence of mineral support (Table 1). The major part of the degraded substrate presumably gone o to respira-tion and microbial biomass. A number of metabolites, not formed in sterile controls, were identi®ed (Table 1). The concentrations of these compounds in free and immobilised cultures were similar, except for (E )-phyte-nal, which was present in higher quantities in the immobilised culture. This result suggests that this labile intermediate is perhaps more stable abiotically after sorption to mineral particles. However, further sterile incubations of (E)-phytenal in the presence and in the absence of mineral particles did not support this hypoth-esis. In fact, the abiotic half-life period of this aldehyde was not signi®cantly modi®ed in the presence of a solid support [T1/2(15C)=2.1 days in the absence of particles
and 1.9 days in the presence of particles, n=4 and
r2=0.984 and 0.995, respectively].
Fig. 1. Sorption of phytol from arti®cial seawater to the mineral particles used (20C). (error bar2standard
devia-tion, IC=95%,n=3).
Since bacterial metabolites are often fugitive species, it is sometimes dicult to determine the importance of one biodegradation pathway relative to another, simply on the basis of the analysis of the intermediates formed. To dierentiate the anaerobic metabolism of n-alkanes
by sulfate-reducing bacteria from that of the corre-spondingn-alkenes, Aeckersberg et al. (1998) have used the proportion of the linear cellular fatty acids with C-odd or C-even carbon chains formed during growth. These compounds have been also used with success to
Table 1
Metabolites detected during growth of the aerobic bacterial community on phytol at 20C for 10 days
Sterile control with support
Culture without support
Culture with support
Residual substrate (mg)a 43.0 1.4 1.3
Metabolites Amount (%)a,b Amount (%)a,b Amount (%)a,b
6,10,14-Trimethylpentadecan-2-one ± 0.03 0.01
4,8,12-Trimethyltridecan-1-ol ± 0.03 <0.01
4,8,12-Trimethyltridecanoic acid ± 0.01 0.01
(E)-3,7,11,15-Tetramethylhexadec-2-enal ± 0.06 0.17
(Z+E)-3,7,11,15-Tetramethylhexadec-2-enoic acids ± <0.01 0.01
3,7,11,15-Tetramethylhexadecanoic acid ± <0.01 <0.01
5,9,13-Trimethyltetradecanoic acid ± <0.01 <0.01
Total isoprenoid wax esters ± 0.27 0.26
a Average of duplicates.
b Based on the amount of degraded substrate (accuracy 0.01%).
compare the importance of the `C18 ketone' and `(E
)-phytenic acid' pathways during cultures of Acineto-bacter sp. PHY9 on phytol at dierent temperatures (Rontani and Acquaviva, 1993). During the aerobic metabolism of phytol by this strain, the `C18-ketone'
pathway leads in fact to the production of acetyl and propionyl-CoA (acetyl- and propionyl coenzyme A thioesters) and thus results in the biosynthesis of a higher proportion of odd straight-chain fatty acids than the `(E)-phytenic acid' pathway which produces only acetyl-CoA. Unfortunately, owing to the greater diversity of the pathways involved during the metabolism of phytol by the aerobic bacterial community employed in the present study (cf. Rontani et al., 1999b) (Fig. 3), such a dierentiation is not possible in the present case. Since the present bacterial community produces rela-tively high amounts of isoprenoid wax esters during growth on phytol (Rontani et al., 1999b) (Table 1), we intended to use these compounds to determine the eects of particle association upon phytol metabolism. These compounds are formed in fact by condensation of phytol metabolites with themselves or with phytol (Fig. 4) (Rontani et al., 1999a) and constitute energy storage components of bacteria (Fixter et al., 1986; Alvarez et al., 1997). We observed strong dierences between the pro®les of isoprenoid wax esters produced by this aerobic bacterial community in free (Fig. 5a) and immobilised (Fig. 5b)
cultures. In the case of immobilised cultures, we note in particular a strong decrease of the 4,8,12-trimethyl-tridecyl-4,8,12-trimethyltridecanoate (1) concentration and the concomitant production of phytylphytanate (7) and phytylphytenate (8).
4,8,12-trimethyltridecan-1-ol, which constitutes the alcohol moiety of ester 1, can only be formed from 6,10,14-trimethylpentadecan-2-one by a Baeyer±Villiger sequence (Rontani et al., 1997) (Fig. 4). Consequently, the decrease of this ester observed in the case of immo-bilised cultures suggests that the `C18 ketone' pathway
(pathway I in Fig. 4) must be inhibited in the presence of mineral particles in favour of the `(E)-phytenic acid' pathway (pathway II in Fig. 4), which results in the production of the esters 7 and 8. This result can be attributed to a modi®cation of the abiotic reactivity of the labile metabolic intermediate (E)-phytenal when this is sorbed to mineral particles. In fact, the sorption may hinder the addition of water upon the activated double bond of (E)-phytenal, which constitutes the ®rst step of its transformation to 6,10,14-trimethylpentadecan-2-one (Rontani and Acquaviva, 1993). In order to test this hypothesis, we carried out some sterile incubations of (E)-phytenal in the presence and in the absence of mineral particles. The results show that (E)-phytenal is mainly abiotically degraded through (E)-phytenic acid, when it is sorbed to mineral particles (Fig. 6). If, in
Fig. 5. Mass fragmentograms showing the distribution of 4,8,12-trimethyltridecan-1-yl (m/z196) and phytyl (m/z278) wax esters: (A) in free cell culture at 20C, (B) in immobilised cell culture at 20C and (C) in immobilised cell culture at 15C. The numbers refer to
contrast to the temperature, the sorption to mineral particles does not sensibly modify the abiotic degrada-tion rate of (E)-phytenal, it strongly favours its abiotic degradation through the `(E)-phytenic acid' pathway at the expense of the `C18ketone' pathway.
The pro®le of isoprenoid wax esters obtained after a manipulation carried out at 15C in the presence of
solid support (Fig. 5c) shows that in these conditions, which are closed to these observed in surface sediments of Carteau Bay, only a small part of phytol is metabolised via the 6,10,14-trimethylpentadecan-2-one. Conse-quently, we may conclude that in aerobic temperate marine Recent sediments phytol is probably metabo-lised mainly via (E)-phytenic acid. These conclusions are in good agreement with the previous detection of high amounts of (ZandE)-phytenic acids in the top layer of Recent sediments of Carteau Bay (Rontani et al., 1999b). These interesting results can be extended to the anaerobic degradation of phytol by denitri®ers since under denitrifying conditions the ®rst step of the anae-robic degradation of phytol also involves the production of (E)-phytenal (Rontani et al., 1999b).
There are numerous reports of free 6,10,14-tri-methylpentadecan-2-one in sediments (Ikan et al., 1973; Simoneit and Burlingame, 1974; Grimalt et al., 1991) and water column particulate matter (Volkman et al., 1983) samples. Biodegradation of phytol (Brooks and Maxwell, 1974; Brooks et al., 1978) and hydrolysis of photochemical products of the phytyl chain of chlor-ophyll (Rontani and Grossi, 1995; Rontani et al., 1996) have been proposed as the main potential sources of this ketone in the marine environment. The data obtained in the present work with sorbed phytol do not support the former hypothesis and suggest that most of this ketone detected in temperate sediments probably results from the hydrolysis of chlorophyll photoproducts.
These results demonstrate the key role played by the labile intermediate (E)-phytenal during the bacterial metabolism of phytol in seawater. Depending upon temperature (Rontani and Acquaviva, 1993) and sorp-tion to mineral particles (this work) dierent routes can be selected in the bacterial metabolism of this isoprenoid alcohol. Consequently, results obtained in vitro at rela-tively high temperatures and with free cell cultures may not be comparable to the marine environment.
Wax esters appeared to be particularly well suited to the study of the bacterial metabolism of isoprenoid compounds. It would be interesting to search for some of these esters, which may constitute useful markers of bacterial activity, in particulate matter and sediment samples. There are relatively few reports of isoprenoid wax esters in sediments (Boon and De Leeuw, 1979; Cranwell, 1986). This can be attributed to rapid hydro-lysis of these compounds in the sediments (De Leeuw et al., 1977b) or to an insucient fractionation of sedi-mentary organic extracts resulting in very complex chromatographic traces.
Acknowledgements
This work was supported by grants from the Centre National de la Recherche Scienti®que and the Elf Aquitaine Society (Research Groupment HYCAR 1123). Thanks are due to Mr. M. Paul for his careful reading of the English.
Associate EditorÐG.A. Wolfe
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