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Occurrence of 3-hydroxyalkanoic acids in sediments from the Guaymas basin (Gulf of California) J. Guezennec a; *, F. Rocchiccioli b , B. Maccaron-Gomez c , N. Khelifa e , J. Dussauze d , A. Rimbault e a
e
IFREMER, Centre de Brest, Department DRV/VP/BMH, P.O. Box 70, F-29280 Plouzaneè, France b INSERM U 342, Hoêpital Saint-Vincent de Paul, F-75674 Paris Cedex 14, France c Institut de Biologia Fonamental, U.A.B. Ballatera, E-08193 Barcelona, Spain d Laboratoire Municipal de Brest, F-29280 Brest, France è Faculte des Sciences Pharmaceutiques et Biologiques, Laboratoire de Microbiologie/UMA, Universiteè Reneè Descartes, F-75270 Paris Cedex 06, France Received 10 April 1997; revised 12 February 1998 ; accepted 4 June 1998
Abstract The Guaymas basin (Gulf of California) provides a particularly interesting extreme environment. Temperatures ranged from 3³C to 11³C at the organic-rich sediment surface and increased with depth, ranging from 59.7³C to 150³C from 20 cm to 30 cm. After either acidic or alkaline hydrolysis of rock and sediment samples collected near active hydrothermal mounds, 3-hydroxyalkanoic acids have been detected by gas chromatography and gas chromatography-mass spectrometry with 3-hydroxyoctanoic and 3-hydroxydecanoic acids predominating. These acids appear to be of microbial origin, arising from the endogenous storage polymers poly(3-hydroxyalkanoates). With respect to the microbial community structure as previously determined from lipid biomarkers, these 3-hydroxyalkanoic acids seem to be associated with either anaerobic bacteria and/or type II methanotrophs. Conversely these polymers were not associated with Beggiota mats present on the surface of the sediments. The presence of 3-hydroxy-3-methylbutanoic acid in trace amounts as well as of 2-hydroxyoctanoic acid is reported. The occurrence of these hydroxyalkanoic acids raises interesting questions about their origin. z 1998 Published by Elsevier Science B.V. All rights reserved. Keywords : Poly(3-hydroxyalkanoate) ; 3-Hydroxyalkanoic acid; Sediment; Microbial community
1. Introduction Poly(3-hydroxyalkanoates) (PHAs) are endogenous storage polyesters produced by various microorganisms. Their accumulation can serve as a meas* Corresponding author. Tel.: +33 (2) 98 22 45 26; Fax: +33 (2) 98 22 45 47; E-mail:
[email protected]
ure of unbalanced growth conditions when a suitable carbon source is present but one or more essential nutrients are absent [1]. It recently became of industrial interest to evaluate PHA polyesters as natural, biodegradable and biocompatible plastics for a wide range of applications [2]. The presence of well-adapted microorganisms of biotechnological interest in terms of new metabolites,
0168-6496 / 98 / $19.00 ß 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 4 9 - X
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new thermostable enzymes, or polymers can be anticipated in extreme environments. Previous studies have already demonstrated the presence near deepsea hydrothermal vents of unusual exopolysaccharides associated with heterotrophic mesophilic aerobic bacteria [3,4]. Regarding their chemical and rheological properties, some of these polysaccharides can be expected to ¢nd applications in the food industry as gelling or thickening agents, in the pharmaceutical industry as therapeutic agents for the treatment of heavy metal poisoning or in wastewater treatments [5]. The Guaymas basin, located in the Gulf of California, provides a particularly interesting extreme environment. This tectonically active basin extends from the East Paci¢c Rise to the San Andreas fault and is unusual among hydrothermal sites in that the ocean £oor consists of a deep layer (ca. 400 m) of unconsolidated sediment through which sea water circulates [6,7]. The hydrothermal £uid is emitted from smokers or reaches the sea£oor by slow percolation through the sediments, causing temperature gradients from 3³C at the surface to 180³C at a sediment depth of 80 cm. Furthermore, this organic-rich sediment contains large amounts of petroleum-like hydrocarbons as a consequence of the pyrolysis of organic diatomaceous residues [8,9] which can provide suitable carbon sources for a great variety of microorganisms [10^12]. The aim of this work was to determine if 3-hydroxyalkanoic acids were present, after a suitable hydrolysis step, in the sediments recovered from this active deep-sea hydrothermal area and, if so, to characterize them. The presence of 3-hydroxyalkanoic acids was tentatively related to the previously characterized microbial community structure in this hydrocarbon-rich sediment.
lyophilized and kept at 320³C until further analysis. The sediment samples were predominantly black with a strong odor of petroleum or hydrogen sul¢de. 2.2. Guaymas basin sample identi¢cation For the identi¢cation of samples, the following nomenclature system was used. GY stands for Guaymas and is followed by two digits indicating the dive number. The following letter is related to the nature of the sample (T = sediment, R = rock) followed by a digit giving a more precise location. The last digit between parentheses corresponds to the sub-bottom depth of sample in the sediment (1: 0^5 cm; 2: 5^10 cm; 3: 10^15 cm; 4: 15^20 cm; 5: 20^25 cm and 6:
2. Materials and methods 2.1. Samples Sediment and rock core samples from di¡erent locations were collected during the oceanographic cruise `Guaynaut' performed by IFREMER (1992) with the research vessel `Nadir' and the submarine `Nautile' in the Guaymas basin (Fig. 1). They were kept at 380³C and upon arrival at the laboratory,
Fig. 1. Location of the Guaymas hydrothermal site and sampling areas in the Gulf of California.
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25^30 cm). Thus GY06T8(1) corresponds to a Guaymas basin sample of sediment collected during dive number 6 in site 8 from 0^5 cm including the surface sediment. 2.3. Lipid extraction Lyophilized sediment and rock samples were extracted using a modi¢ed Bligh-Dyer method [13,14]. The extracted lipids were fractionated into neutral lipids, glycolipids, and polar ether and ester lipids by silicic acid column chromatography using appropriate volumes of chloroform, acetone and methanol, respectively. Bacterial community structure was determined on the methanol fraction [15]. Since poly(3hydroxybutanoic acid) (PHB) is soluble in hot chloroform [16], PHAs can be found in both the chloroform and acetone fractions. These two fractions were pooled for PHA analysis. The methanol and the pooled chloroform and acetone fractions were each reduced to dryness under a stream of nitrogen and stored under nitrogen until further analysis. 2.4. PHA analysis PHAs were hydrolyzed into their 3-hydroxyalkanoate monomers according to either an acidic (P1) or an alkaline (P2) hydrolysis procedure. In procedure P1, the pooled chloroform and acetone fraction was hydrolyzed at 100³C for 4 h in the presence of absolute ethanol and 12 M hydrochloric acid [17]. The resulting 3-hydroxyalkanoate ethyl esters were puri¢ed by thin-layer chromatography; 3-hydroxyundecanoic acid was added as internal standard to the pooled fraction prior to hydrolysis. Ethyl 3-hydroxyalkanoates were converted to the TMS derivatives by silylating the hydroxyl group as described below. In procedure P2, the residue was hydrolyzed at 80³C for 30 min after addition of 1 ml of water: methanol (1:1, v/v) containing sodium hydroxide (150 g l31 ) [18] and two internal standards, 2-hydroxybenzoic acid (IS1, 4.8 nmol ml31 ) and 3-hydroxyundecanoic acid (Larodan, Malmoë, Sweden; IS2; 9.6 nmol ml31 ). After extraction with hexane, the residual acidi¢ed aqueous phase was extracted with ethyl acetate (2 ml, twice). Triethylamine (0.4
337
ml) was added to the organic phase which was dried over anhydrous sodium sulfate and evaporated under nitrogen. 2.5. Formation of derivatives 2.5.1. Penta£uorobenzyl ester derivatives Regarding the low biomass found in some samples [15], 3-hydroxyalkanoic acids were also analyzed as penta£uorobenzyl (PFB) ester derivatives [18] by gas chromatography (GC) equipped with an electron capture detector (ECD). Free organic acids obtained using the mild alkaline hydrolysis (P2) were dissolved in acetonitrile and derivatized with 100 Wl of PFB bromide (Sigma, 3.5% in acetonitrile) and 100 Wl of triethylamine [19,20]. After 15 min at room temperature, the PFB derivatives were extracted with isooctane and puri¢ed on a short disposable silicic acid column to remove excess by-products and contaminants from the derivatization procedure. 2.5.2. PFB-hepta£uorobutyryl (HFB) and trimethylsilyl (TMS) derivatives for GC-MS analysis Acylation of PFB derivatives was performed at room temperature with hepta£uorobutyric anhydride (HFBA) in hexane [21]. TMS derivatives were obtained by addition of 0.1 ml of N,O-bis-trimethylsilyltri£uoroacetamide with 1% trimethylchlorosilane: pyridine and heating at 60³C for 10 min. For controls, two empty glass tubes were processed with the addition of reagents as previously described with, for one of them, addition of an internal standard-free hydrolysis reagent. 2.6. Gas chromatographic (GC) analysis Analyses were performed on a Carlo Erba (Rodano, Italy) HRGC 5300 gas chromatograph equipped with a CP-Sil 5 CB column (60 mU0.2 mm i.d.; ¢lm thickness, 0.25 Wm; Chrompack France, Les Ulis, France) and either a £ame ionization (FID) for TMS ethyl ester derivatives or an ECD detector (63 Ni foil; 10 mCi) for PFB-HFB derivatives. The temperature was programmed as follows: 100³C (1 min), 20³C min31 to 140³C, then 2³C min31 to 300³C. Hydrogen was used as the carrier gas at a velocity of 33 cm s31 .
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2.7. GC-mass spectrometric (MS) analysis A Carlo-Erba model 4165 gas chromatograph coupled to a quadrupole Nermag (Delsi, Argenteuil, France) R10-10C mass spectrometer with an INCOS (Finnigan Mat, San Jose, CA) data system was used. The sample (2 Wl) was introduced into a Ross injector and separation was achieved on a DB-5 capillary column (30 mU0.32 mm i.d; ¢lm thickness 0.25 Wm; JpW, Folsom, CA) with helium as the carrier gas. The column was temperature programmed from 120³C to 280³C at a rate of 5³C min31 . Ionization was performed using chemical ionization (CI; 70 eV; emission current, 0.2 mA) in the positive and negative modes (PICI and NICI, respectively) with ammonia as reactant gas. In PICI, the ammonia pressure was ca. 0.13 kPa. In NICI, this pressure was adjusted to ca. 6.5 Pa when introduction of per£uorotributylamine for signal optimization (m/z 633) was stopped. Selected ions were monitored (SIM) at m/z [M+1] for TMS derivatives, m/z [M+181] for PFB-HFB derivatives in PICI, and m/z [M-18] for PFB-HFB derivatives in NICI. 2.8. Organic acid nomenclature A shorthand nomenclature is used which is in the form of numbers separated by a colon. The number before the colon indicates the carbon chain length and that after the colon indicates the number of double bonds; 2-OH or 3-OH indicates an hydroxyl (OH) group on the second or the third carbon atom from the carboxyl end, respectively. Thus 3-OHC4:0, 3-OHC5:0, 3-OHC8:0, and 3-OHC10:0 correspond to 3-hydroxybutanoic, 3-hydroxypentanoic (3-hydroxyvaleric), 3-hydroxyoctanoic and 3-hydroxydecanoic acids, sometimes called in other papers HB, HV, HO and HD, respectively. 2.9. Standards Since some of the 3-hydroxyalkanoic acids were not commercially available, a series of 3-hydroxyalkanoic acids were synthesized by the Reformatsky reaction applied to bromoacetic acid ethyl ester and ketones of various chain lengths [22]. The resulting 3-hydroxyalkanoic acids were esteri¢ed to give
the ethyl esters which were characterized by 13 C nuclear magnetic resonance (NMR) and Fouriertransform infrared spectroscopy (FTIR). 3-OH-3MeC4:0 was obtained from Interchim (Montluc°on, France).
3. Results 3.1. Physical and chemical sample parameters Sediments from this cruise were characterized by high levels of hydrocarbons (up to 12000 ppm) and hydrogen sul¢de (0^300 ppb). Taken together carbon and sulfur represented up to ca. 20% (w/w) in some samples [23]. Hydrogen sul¢de and sulfur dioxide were also present in the water column and primarily in the emitted £uids at concentrations ranging from 0 to 300 ppb. Temperatures ranged from 3³C to 11³C at the sediment surface and increased with depth, ranging from 59.7³C to 150³C from 20 cm to 30 cm (Table 1). 3.2. Procedure optimization For bacterial mats and surface sediments, PHA hydrolysis products were analyzed as TMS ethyl ester derivatives and identi¢ed on the basis of their retention times and mass spectra compared to authentic standards. Other derivatization procedures, including formation of PFB-HFB or PFB-TMS derivatives, were required for subsurface or deep sediments. Excess by-product formation as observed in preliminary experiments made necessary a puri¢cation step performed on a disposable short silicic acid column. In that case, the use 3-OHC4:0, 3-OHC5:0 and 3-OHC10:0 as standards indicated that the recovery was good (up to 90%). Signal intensity in PICI for the PFB-HFB derivative of 3-hydroxyoctanoic acid (50 nmol nl31 ) was about double the intensity in NICI. For its di-TMS and PFB-HFB derivatives, the intensities were quite similar in PICI (Table 2). It has been previously reported that the signal intensity is highly dependent on the mass spectrometer setting [18]. Consequently, TMS derivatives were used and analyzed with ammonia PICI for P2-hydrolyzed samples.
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Table 1 Description of samples collected from the Guaymas basin Sample
Sampling site
Temperature (³C)
Carbon (%)
Sulfur (%)
Hydrocarbons (Wg g31 dry weight)
GY06T6 (1)
Bacterial mat 0^30 cm Boundary Bacterial mat 0^5 cm 5^10 cm 10^15 cm 15^20 cm 20^25 cm Bacterial mat
Surface : 3^11³C nd Surface : 3^10³C as GY06T7 nd nd nd nd nd Surface : 6.7³C 30 cm: 59.7³C nd nd Surface : 3.5³C 20 cm: 107³C as GY12T2 10 cm: 139³C 30 cm: 150³C nd nd
0.49 nd nd
2.3 nd nd
nd nd nd
3.61 2.09 5.92 2.29 1.66
1.4 2.0 2.1 2.3 2.7
2 100 3 600 12 700 2 700 1 600
1.96 1.39
10.5 4.4
3 200 2 200
1.32
4.3
2 850
2.64 3.93 nd
25.8 20.3 nd
5 070 6 600 nd
GY06T7 GY06T8 (1) (2) (3) (4) (5) GY12T1 (1) (2) GY12T2
Bacterial mat
GY12T3 GY14T1
Bacterial mat Deposits with emanating £uids
GY09R1 GY10R1 GY12R1 GY14R1 GY14R2
Black smoker Black smoker Black smoker (top) Smoker (black area) Smoker (yellow area)
86³C nd
nd: not determined.
3.3. 3-Hydroxyalkanoic acid pro¢les The 3-hydroxyalkanoic acid pro¢les for sediments recovered from the Guaymas basin are listed in Table 3. Most surface sediment samples (i.e., GY12T1(1), GY12T1(2), GY12T2(1), GY12T3(1), GY12T3(2), GY06T7(1), GY06T7(2), GY06T6(2), GY06T8(1) and GY06T8(2)) showed the highest concentrations of 3-hydroxyalkanoic acids, ranging from ca. 10 to 60 ng g31 dry weight sediment. In deeper sediments, lower concentrations occurred, ranging from 0 to 2 ng g31 dry weight sediment. With the exception of sample GY06T7(5) and, to a lesser extent sample GY06T8(5), no 3-hydroxyalkanoic acids were found in sediments as deep as 20 cm. No unsaturated hydroxyalkanoic acids were detected. Controls did not show any signi¢cant peaks with the exception of reagents, by-products and internal standards when present. Components 3-OHC4:0, 3-OHC5:0, 3-OHC8:0 and 3-OHC10:0 predominated. Traces of 3OHC6:0 and 3-OHC9:0 were also found in some
samples. However, in most samples, 3-OHC8:0 and 3-OHC10:0 were the major 3-hydroxyalkanoic acids, with abundances up to 68% of the total hydroxyalkanoic acids. It is also interesting to note the presence in low amounts of a branched-chain 3-hydroxyalkanoic acid, 3-OH-3-MeC4:0, which was unambiguously identi¢ed on the basis of its retention time and its mass spectrum compared to the authentic standard.
Table 2 Comparison of the signal intensities in ammonia-NICI and ammonia-PICI GC-MS for HFB-PFB and di-TMS derivatives of 3hydroxyoctanoic acid (initial aqueous solution : 50 nmol l31 ) HFB-PFB derivative ammonia-NICI/ammonia-PICI m/z
355/554 [M-181] /[M+18] area 907 400/1 685 520
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di-TMS derivative ammonia-NICI 305 [M+1] 1 506 752
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4. Discussion Hydroxyalkanoic acid-rich samples were detected as TMS ethyl ester derivatives by GC and GC-MS analysis. However, for some deeper sediments, in which the biomass was very low, £uorinated derivative formation combined with ECD-GC was required. Sensitivity in the detection was increased and it is a useful alternative to FID-GC for the detection of trace microbial constituents since sample contaminants and chemicals used during sample processing can be the source of important background signals. Use of PICI-MS for quanti¢cation of hydroxyalkanoic acid PFB derivatives allows a detection limit in the range of pg ml31 or less. Components 3-OHC12:0 to 3-OHC20:0 are classical constituents of bacterial lipid A [24]. 2-OHC12:0 and 2-OHC14:0 have also been reported in some bacterial lipopolysaccharides [25]. The fraction analyzed in this study results from lipid extraction using a modi¢ed Bligh-Dyer method and did not include the lipid A-containing residual fraction. The amounts of PHA products, expressed as the total of 3-hydroxyalkanoic acids released, ranged from 2 to 60 ng g31 dry weight sediment. These values are low compared to those observed in methane-enriched soil (20^180 nmol PHB g31 dry weight) [26] but are of the same order of magnitude as those found with thermophilic methane-producing digesters [27]. Hydrothermal sediments recovered from the petroleum-rich Guaymas basin are characterized by a bacterial biomass primarily located near the surface, with sulfur-oxidizing bacteria predominating. Along with these bacteria, methanotrophs and sulfate reducers are present [15,28]. With the exception of some samples and on the basis of ether lipid analysis, the biomass showed little indication of the presence of archaea in the surface sediments [15]. High concentrations of 3-hydroxyalkanoic acids were found in the surface sediments associated with a high bacterial biomass. Moreover, highest concentrations were observed in sediments enriched with type II methanotrophs, based on the presence of speci¢c lipid biomarkers [15]. In some deeper sediments, the absence of 3-hydroxyalkanoic acids may be related to the high temperatures (up to 100³C). Even though PHA biosynthesis by Beggiatoa spp.
[29] has been previously reported under laboratory conditions, it has never been observed in the natural habitat. The high density of these microorganisms in this particular environment indicates favorable growth conditions rather than unbalanced conditions and hence absence of PHAs in these sulfur-dependent bacteria. This is consistent with the low PHA amounts in sediments associated with a high density of sulfur-oxidizing bacteria (Table 3). The samples showing large PHA amounts, including 3OHC8:0 and 3OHC10:0, are characterized with a complex bacterial community structure where methanotrophs and anaerobic bacteria (presumably sulfate reducers) co-exist. Moreover, lipid biomarker studies showed the predominance of type II methanotrophs over type I [15]. It is known that methylotrophic bacteria can accumulate PHA [30^32]. Methanotrophs, primarily those of type II, can produce PHAs in a methane-rich sediment [26] and several type II methanotrophs can convert methane to PHB [33]. Anaerobic bacteria, primarily sulfate reducers, are also present and contribute signi¢cantly to the overall bacterial biomass. The presence of PHAs has been reported in various anaerobes, e.g., Clostridium botulinum [34], Syntrophomonas wolfei when grown in the presence of alkanes [35], as well as in some sulfate-reducing bacteria (N. Pfenning, quoted in [2] as personal communication). For this reason, the occurrence of signi¢cant proportions of 3-hydroxyalkanoic acids in samples associated with sulfate reducers needs more attention. Although a large number of bacteria have been shown to accumulate PHB and related PHAs, the most common polymer producers encountered in the literature are members of the Pseudomonas or Alcaligenes genera. The bacterial community of the Guaymas basin sediments is dominated by a large population of methanotrophs and sulfur-oxidizing bacteria. Whether the former are the only PHA producers in these sediments is questionable. Other alkane utilizers able to accumulate PHAs could be present in the sediments and not detected in the present study because of a lack of speci¢c biomarkers. However, the relationship between the presence of a signi¢cant methanotrophic population and the presence of 3-hydroxyalkanoic acids need further attention. With the exception of a few samples, no signi¢cant
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5 107 1 290 164 104 19
15 383 277
12 783
3 329
9 953 299 31
GY06T8(1)c GY06T8(2) GY06T8(3) GY06T8(4) GY06T8(5)
GY12T1(1)c GY12T1(2)
GY12T2(1)c
GY12T2(2)
GY12T3(1) GY12T3(2) GY12T3(3) ?
MT+SRB as GY12T3(1) ?
SRB
MT+SRB
MT+SRB MT+SRB
Beggiatoa-like Beggiatoa-like+SRB ? ? Archaea
SRB SRB SRB ? Archaea
Beggiatoa-like Beggiatoa-like ? ?
Microorganismsb
ND
5.6 4.2 1.7
0.8
8.5
8.2 7.1
1.1 2.1 trace ND 1.1
1.2 0.8 1.1 ND 2.4
1.1 1.8 ND ND
3-OHC4:0
ND
8.2 5.6 2.1
1.2
10.2
7.6 5.3
trace trace trace ND 0.8
3.4 1.1 0.5 ND 4.6
1.8 3.2 ND ND
3-OHC5:0
ND
14.8 12.7 4.2
1.5
17.1
1.7 6.2
4.2 2.8 ND ND ND
5.3 11.3 0.5 ND 2.8
trace 5.3 ND ND
3-OHC8:0
See Section 2. b On the basis of speci¢c chemical biomarkers [15]. c As determined after P1 acidic hydrolysis procedure. All others determined after P2 alkaline hydrolysis procedure. SRB: sulfate-reducing bacteria; MT: methanotrophs. ND : not detected.
a
48
14 519 5 121 475 186 176
GY06T7(1)c GY06T7(2)c GY06T7(3) GY06T7(4) GY06T7(5)
GY09R1
2 329 2 538 525 25
PLFA (ng g31 dry weight)
GY06T6(1)c GY06T6(2)c GY06T6(3) GY06T6(4)
Samplea
Table 3 PHA acid pro¢le in hydrothermal sediments
ND
23.6 21.3 2.8
1.8
25.1
3.8 10.7
5.5 4.7 ND ND ND
20.8 7.4 ND ND 1.7
ND 2.6 ND ND
3-OHC10 :0
ND
4.5 7.1 trace
ND
5.5
ND 2.3
ND ND ND ND ND
12.9 2.5 ND ND ND
ND 5.6 ND ND
2-OHC8 :0
ND
3-OH-3-MeC4 :0 3-OH-3-MeC4 :0
3-OHC6 :0(1.2), 3-OHC9 :0(1.7) 3-OHC6 :0 (traces)
3-OHC6 :0 (0.8), 3-OHC9 :0(0.7)
3-OH-3-MeC4 :0 ND ND
3-OH-3-MeC4 :0, 3-OHC6:0 3-OHC6 :0 ND ND 3-OHC6 :0
3-OHC6 :0, 3-OHC9:0 ND ND
Other PHA monomers
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amounts of 3-hydroxyalkanoic acids were detected in the deep sediments. However, the presence of 3-hydroxyalkanoic acids in some of them could be associated with an archaeal biomass, as determined by the presence of glycerol di- and tetraethers. In these sediments, the archaeal biomass was low [15], with an approximate density of 105 cells g31 dry weight (Guezennec, unpublished data). Even though some archaea were isolated from this speci¢c hydrothermal site, the archaeal biomass represents only a low input to the overall surface bacterial community. To date, only a few archaea have been shown to be able to accumulate PHAs. Several halophilic archaea, particularly Haloferax mediterranei, produce intracellular PHB and PHV [36]. However, the presence of halophilic archaea is not expected in this extreme environment, with a salinity similar to that of surface seawater. Insu¤cient data on the accumulation of PHAs in deep-sea archaea prevent us from establishing a clear relationship between the presence of PHAs and archaea. Components 3-OHC4:0 and 3-OHC5:0 are the most common monomers found in microbial PHAs. The copolymer (PHB-PHV) produced by Alcaligenes eutrophus is marketed as a biodegradable plastic and can be an attractive alternative to oilderived thermoplastics. For most samples, 3OHC8:0 and 3-OHC10:0 predominated, accounting for 30% of the total hydroxyalkanoic acids. 3-Hydroxyalkanoic acids with up to 11 carbon atoms in polymers extracted from marine sediments were reported for the ¢rst time by Findlay and White [17]. Pseudomonas oleovorans was demonstrated to be able to accumulate poly(3-hydroxyoctanoate) (PHO) and poly(3-hydroxydecanoate) (PHD) when grown on noctane and n-decane respectively [37]. The presence of n-alkanes ranging from C6 to C16 in the Guaymas basin sediments [23] could therefore support the accumulation of PHO and PHD by in situ microorganisms. In some samples, analysis showed the presence of trace amounts of an unusual branched-chain 3-hydroxyalkanoic acid, 3-OH-3-MeC4:0. Branchedchain 3-hydroxyalkanoic acids have been recently reported as constituents of PHAs in Pseudomonas oleovorans when grown on mixtures of octanoate and methyoctanoates [38]. However, to our knowledge, 3-OH-3-MeC4:0 has never been identi¢ed in bacte-
ria. The presence of this acid along with 2-OHC8:0 raises interesting questions about their origin.
5. Conclusions The hydrocarbon-rich sediments of the Guaymas basin exhibit the presence of 3-hydroxyalkanoic acids which we suggest to be derived from microbial PHAs. Taken together, the phospholipid ester-linked fatty acid and 3-hydroxyalkanoic acid pro¢les support the hypothesis that conditions of unbalanced growth exist within the sediment upper layers. Methanotrophs are known to accumulate intracellular storage material and it is interesting to observe that the presence of higher concentrations of 3-hydroxyalkanoic acids is associated with high concentrations of these microorganisms. To our knowledge, mesophilic or moderately thermophilic bacteria are considered to be the major PHA producers. Temperatures in the sediments vary from 3³C to 150³C and some 3-hydroxyalkanoic acids have been identi¢ed in the sediments associated with high temperature. Whether moderately thermophilic bacteria can accumulate PHAs or extreme conditions nearby the vents, including pressure, could favor PHA biosynthesis needs more attention. In extreme environments, microbial mats and habitats of archaea could o¡er new ¢elds for screening new PHA producers. Acknowledgments The authors would like to thank all the participants to the Guaynaut oceanographic cruise for providing samples. We are also grateful to the Brittany region (Programme BRITTA) for its ¢nancial support. Parts of this study were supported by IFREMER Grant 92-2-470333 DRO/EP to one of us (A.R.)
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