Application of a Specific and Sensitive Radiometric Assay for Microbial Lipase Activities in Marine Water Samples from the Lagoon of Nouméa

[³H]triolein lipase assay.[9,10-³H(N)]oleic acid ([³H]oleic acid, 15 Ci · mmol⁻¹) and [9,10-³H(N)]triolein ([³H]triolein, 22 Ci · mmol⁻¹) were obtained from Perkin-Elmer—NEN. Water samples were distributed into 50-ml polypropylene centrifuge tubes, and [³H]triolein (<1 to 60 nM³H-esterified fatty acid [EFA]) was added as a solution in ethanol (final concentration, 0.005 to<0.2%). Immediately after vortex homogenization, series (triplicates) of 0.8-ml subsamples were distributed into 15-ml polypropylene centrifuge tubes. The first series was stopped without delay by the addition of 0.5 ml of a 1 M solution of Tris (adjusted to pH 11 using NaOH) and 4.8 ml of a methanol-chloroform-heptane (38.6:34:27.4) solvent mixture, followed by vigorous vortex agitation. The other series were placed in the dark, in an incubation bath (in situ temperature, 25 ± 1°C), until the end of their incubation period and then treated as described above. Stopped samples were centrifuged (for 3 min at 5,445 × g)to allow separation of phases. A portion (2 ml) of the upper-aqueous phase was transferred to polypropylene scintillation vials containing 10 ml of Hionic-Fluor liquid scintillation counting cocktail (Packard Bioscience) for radioactivity counting. At time zero, the distribution of fatty acids between the two phases was estimated with the same protocol, using [³H]oleic acid instead of [³H]triolein. No incubation was performed in this case. Finally, the expression of lipase activity was calculated from the radioactivity in the total volume of the aqueous phase, the proportion of released [³H]oleic acids contained in this phase, and the specific activity of the radiolabeled probe. It is noteworthy that the transformation products of the [³H]oleic acid (including ³H-fatty acids and ³H₂O) also end up in the aqueous phase. Autoclaved samples were used as controls for abiotic substrate degradation.

MUF-oleate lipase assay.Samples (50 ml) were incubated in the dark (25 ± 1°C) with MUF-oleate (Sigma) fluorogenic substrate (22). At selected incubation times, substrate hydrolysis was assayed on triplicate aliquots by using a TD-700 fluorometer (Turner Designs) that was equipped with an ammonium filter set (excitation, 365 nm; emission,> 410 nm) and calibrated with standard solutions of MUF (Sigma). Autoclaved samples were used as controls for abiotic substrate degradation.

Expression of substrate [³H]triolein and MUF-oleate concentrations and hydrolysis rates.One mole of [³H]triolein corresponds to 3 mol of ester bonds, which may be hydrolyzed by lipase enzymes, whereas 1 mol of MUF-oleate corresponds to 1 mol of ester bonds. We chose to express substrate concentrations as their EFA equivalence, and we described activity rates in terms of the release of free fatty acids (FFA) to facilitate comparison between the two techniques.

Test for reproducibility of the [³H]triolein lipase assay.Lipase activity was measured at two different stations (M05 and M41) in five subsamples by using the [³H]triolein lipase assay at a fixed substrate concentration. Substrate concentrations were 2.49 ± 0.25 (mean± standard deviation [SD]) nM ³H-EFA and 2.23 ± 0.12 nM ³H-EFA at stations M05 and M41, respectively.

Bacterial production.Bacterial production was determined from [methyl-³H]thymidine (45 Ci · mmol⁻¹; Amersham) incorporation (16). Duplicate 10-ml subsamples were incubated in the dark at in situ temperature (25± 1°C) for 60 min with 15 nM [³H]thymidine and were then preserved with buffered formaldehyde and unlabeled thymidine (final concentrations, 2% and 1 mM, respectively), filtered through 0.2-μm-pore-size Nuclepore membranes, extracted by ice-cold 5% trichloroacetic acid (TCA), and rinsed four times with 5 ml of 5% TCA. Labeled TCA precipitates were heated with 0.5 ml of 0.5 N HCl at 100°C for 30 min to minimize self-absorption of beta radiation before determination of radioactivity. Blanks prekilled with 2% formaldehyde were processed in parallel. The biomass production rates were computed by using the average of empirically determined conversion factors at D01, D08, and M05 (2.9 × 10¹⁸ cells · mol⁻¹ of thymidine [unpublished data]) and 12.4 fg of C · cell⁻¹ (17).

Station properties and sample collection.Samples were collected in subsurface waters (depth, 3 m) in and outside the lagoon of Nouméa, New Caledonia, onboard the R/V Coris. Locations of the stations are presented in Fig. 1. Stations along a nearshore-to-offshore transect (D01 to D33; depth, 6 to 23 m) exhibited an increasing gradient of salinity (34.98 to 35.10 g · liter⁻¹), which was associated with a decreasing gradient of chlorophyll a concentration (0.83μ g · liter⁻¹ to 0.42μ g · liter⁻¹). A middle-lagoon station (M05; depth, 32 m) and an outside-lagoon reference station (M41; depth, >200 m) were also investigated. Seawater samples were collected in a 5-liter Niskin sampling bottle and processed back in the laboratory within 2 h after sampling. Whenever possible, hydrological data were collected along the whole water column.

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FIG. 1.

Map of the lagoon offshore Nouméa city. +, locations of sampling stations.

Bacterial activities along a trophic gradient.Bacterial production and lipase activities evaluated by both [³H]triolein and MUF-oleate assays at similar substrate concentrations (12.5 nM EFA) were measured along the trophic gradient (D01 to D33). Additionally, multiconcentration kinetics experiments were conducted at D01 and D33 using MUF-oleate (4 to 75 nM EFA) and [³H]triolein (2.62 to 76.26 nM³H-EFA). Kinetic parameters (V_m and K_m) were determined from a Michaelis-Menten model by using the Marquardt Levenberg minimization in SB's Kinetics software package, version 3.0 (11).

[³H]triolein hydrolysis.The hydrolysis of the [³H]triolein as a function of time was reflected by the appearance of ionized free [³H]oleic acids in the alkaline aqueous phase of the extraction medium. Different assays on autoclaved seawater samples showed that neither contamination of the aqueous phase by the nonhydrolyzed radiolabeled substrate nor adsorption of the fatty acids on the inner wall of the tubes occurred during extraction. Tests performed at various incubation times (ranging from minutes to days) and various substrate concentrations led us to fix optimal conditions for the assay, i.e., with incubation times ranging from 1 h to several hours and substrate concentrations ranging from <1 to 25 to 50 nM ³H-EFA. The highest [³H]triolein substrate concentration that could be used significantly was approximately 60 nM ³H-EFA (i.e., 20 nM [³H]triolein). The use of higher substrate concentrations led to poor reproducibility in replicate samples and loss of linearity over time.

Reproducibility of the [³H]triolein lipase assay.For each station, turnover rates were calculated as the percentage per hour of ³H-FFA released from the ³H-EFA substrate pool. Under these conditions, turnover rates ranged from 5.10 to 6.77%³H-FFA · h⁻¹ at M05 and from 5.38 to 6.76%³H-FFA · h⁻¹ at M41. Variability of the [³H]triolein lipase assay at the indicated stations was 10.7 and 9.7%, respectively.

Multiconcentration kinetics.The multiconcentration kinetics profiles at the D01 and D33 stations, determined with both the MUF-oleate lipase and the [³H]triolein assays, are presented in Fig. 2.

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FIG. 2.

Multiconcentration kinetics. The graphs show lipase activities at stations D01 (▪) and D33 (•), which were determined by using the MUF-oleate lipase assay (a) and the [³H]triolein lipase assay (b).

In the MUF-oleate assay (Fig. 2a), the V_m of MUF-oleate hydrolysis was 24.5 ± 3.4 nmol · liter⁻¹ · h⁻¹ and the K_m was 54.1 ± 13.9 nmol · liter⁻¹ (mean ± SD) at D01, while at D33 the V_m was 20.3 ± 3.8 nmol · liter⁻¹ · h⁻¹ and the K_m was 334 ± 74 nmol · liter⁻¹.

In the [³H]triolein assay (Fig. 2b), the V_m of [³H]triolein hydrolysis was 4.09 ± 0.24 nmol · liter⁻¹ · h⁻¹ and the K_m was 59.1 ± 6.5 nmol · liter⁻¹ (mean ± SD) at D01. Under our experimental conditions, results at D33 did not fit a simple Michaelis-Menten model, and thus it was not possible to calculate significant kinetic parameters.

Bacterial activities along the trophic gradient.Lipase activities measured along the trophic gradient by using MUF-oleate (Fig. 3a) and [³H]triolein (Fig. 3b) substrates decreased from 5.83 to 0.88 nmol of FFA · liter⁻¹ · h⁻¹ and from 0.76 to 0.23 nmol of³H-FFA · liter⁻¹ · h⁻¹, respectively. They varied similarly, with activity values decreasing from D01 to D33 (except for D16). Bacterial production (Fig. 3c) also similarly decreased from D01 (0.59 μg of C · liter⁻¹ · h⁻¹) to D33 (0.26 μg of C · liter⁻¹ · h⁻¹).

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FIG. 3.

Lipase activities and bacterial production along a trophic gradient (D01 to D33). Lipase activities were measured using MUF-oleate (a) and [³H]triolein (b) as substrates (12.5 nM EFA substrate concentrations). Bacterial productions (c) are also presented.

Correlation of MUF-oleate and [³H]triolein hydrolysis with bacterial production.The ratio of MUF-oleate to [³H]triolein activities (Fig. 4a) was constant at the three offshore stations (D16, D22, and D33; 3.8± 0.1 [mean ± SD]) but gradually increased from 4.1 to 7.6 at nearshore stations D08, D05, and D01. As shown in Fig. 4b, the ratio of [³H]triolein activities to bacterial production varied little between D33 and D01 [0.99 ± 0.17 nmol of [³H]FFA · (μg of C)⁻¹], while the ratio of MUF-oleate activities to bacterial production increased considerably over the six stations [3.3 to 9.8; mean, 5.0 ± 2.6 nmol of FFA · (μg of C)⁻¹].

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FIG. 4.

Ratio of MUF-oleate activities to [³H]triolein activities (a), ratio of MUF-oleate (•) and [³H]triolein (▪) activities to bacterial production (b), and scatter plot of MUF-oleate (•) and [³H]triolein (▪) ectoenzymatic activities versus bacterial production (c). Solid lines represent exponential-regression fits.

Both enzymatic activities were closely related to bacterial production by an exponential relationship (Fig. 4c). However, [³H]triolein activities turned out to be better linked to bacterial production (n = 6; P= 0.0016) than MUF-oleate activities (n = 6; P = 0.012) were.