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

Marine microbiologists commonly assay lipase activities by using a synthetic fluorescent analog, 4-methylumbelliferyl (MUF)-oleate. The technique is convenient, but it is considered to be unspecific because of the structure of this analog. This study reports the design of a new specific and sensitive lipase assay based on the use of a radiolabeled triglyceride, [³H]triolein. Free fatty acids (FFA) resulting from its hydrolysis are isolated as a function of time in a one-step liquid-liquid extraction and then radioassayed. MUF-oleate and [³H]triolein techniques were compared by measuring lipase activities at similar substrate concentrations along a trophic gradient in the Southwest Lagoon of New Caledonia, near Nouméa. Hydrolysis rates decreased from the nearshore station to the offshore station and showed similar trends regardless of the technique used. Rates decreased from 5.83 to 0.88 nmol of FFA·liter⁻¹·h⁻¹ and from 0.76 to 0.23 nmol of ³H-FFA·liter⁻¹·h⁻¹, respectively. These results appeared to be consistent with bacterial production results, which also decreased similarly (from 0.59 to 0.26 μg of C·liter⁻¹·h⁻¹). However, the ratio of MUF-oleate activities to [³H]triolein activities, which was constant at the offshore stations (3.8 ± 0.1), gradually increased at the nearshore stations (from 4.1 to 7.6). This result shows that the two assays respond in different ways to changes in environmental conditions and validates the need to set up more specific enzymatic assays.     

The effect of temperature on eicosane substrate uptake modes by a marine bacterium Pseudomonas nautica strain 617: relationship with the biochemical content of cells and supernatants

Three hydrocarbon uptake modes (adherence, emulsification and solubilization) were identified and quantified in cells and supernatants of a mesophilic marine bacterium Pseudomonas nautica strain 617 grown on eicosane. The adherence capacity was related to the enrichment of cells with wax esters and glycolipids. The emulsifying activity was related to the presence of extracellular biosurfactants composed of proteins, carbohydrates and lipids (35:63:2). The intensity of substrate uptake modes was sensitive to temperatures currently found in the original environment of P. nautica (16°C, 20°C and 32°C). When temperature decreased, a significant increase in adherence and emulsifying activity was observed in relation to biochemical changes, whereas solubilizing activity decreased. The marine bacterium was able to degrade 53–59% eicosane at the end of exponential growth after 13, 5 and 3 days incubation at 16°C, 20°C and 32°C respectively.     

Effects of CO2 and nitrogen supply on the biochemical composition of Ulva rigida with especial emphasis on lipid class analysis

Lipid class composition was analysed in the green macroalga Ulva rigida grown under normal (350 ppm) and high (10,000 ppm) CO₂ levels, and in nitrate saturated and nitrogen limited conditions. A new protocol for the extraction of lipids has been defined. Culture conditions altered the fate of assimilated carbon, and significant changes were observed in protein and total lipid content in particular. A CO₂-enriched atmosphere conditioned the effects of nitrogen limitation on lipid class composition, revealing deep qualitative changes in carbon metabolism. Triglycerides accumulated at high CO₂ and under nitrogen limitation, while chloroplast-related lipids showed an inverse response. Changes in phospholipids could be related to carbon availability as they did not respond to nitrogen limitation. The ratio sterols/acetone-mobile polar lipids followed a negative linear relation with the optimum quantum yield for photosynthetic electron transport (F_v/F_m), and was considered as an index of the «light status» of the cell. The specificity of the response of lipid classes to growth conditions in U. rigida emphasizes the potential role of lipid class analyses as a diagnostic tool for environmental stress.     

Effects of a plume front on the distribution of inorganic and organic matter off the Rhône River

Hydrological and chemical structures off the Rhône River estuary resulting from the introduction of the river flow into the Mediterranean Sea are described. The effect of the fresh-water/sea-water interface on the distribution of inorganic and organic matter off the Rhône river is investigated. Strong vertical gradients of inorganic and dissolved organic matter such as lipids characterized the first few meters in this area (from 83.7 to 0.6 N-NO₃ µgat l^–1, from 6.39 to 0.92 N-NH₄ µgat l^–1 and from 299 to 73 µg l^–1 of total dissolved lipids). At the interface, substantial increases of particulate organic (PON: from 45 µg l^–1 at surface to 118 µg l^–1 at the interface, POC: from 462 to 876 µg l^–1, total particulate lipids: from 33 to 648 µg l^–1) and suspended matter in general (from 18 to 22.2 mg l^–1) were observed. High phytoplanktonic production may account for some of this enrichment, although passive accumulation might also be involved.     

Fast Growth May Impair Regeneration Capacity in the Branching Coral Acropora muricata

Regeneration of artificially induced lesions was monitored in nubbins of the branching coral Acropora muricata at two reef-flat sites representing contrasting environments at Réunion Island (21°07′S, 55°32′E). Growth of these injured nubbins was examined in parallel, and compared to controls. Biochemical compositions of the holobiont and the zooxanthellae density were determined at the onset of the experiment, and the photosynthetic efficiency (F_v/F_m) of zooxanthellae was monitored during the experiment. Acropora muricata rapidly regenerated small lesions, but regeneration rates significantly differed between sites. At the sheltered site characterized by high temperatures, temperature variations, and irradiance levels, regeneration took 192 days on average. At the exposed site, characterized by steadier temperatures and lower irradiation, nubbins demonstrated fast lesion repair (81 days), slower growth, lower zooxanthellae density, chlorophyll a concentration and lipid content than at the former site. A trade-off between growth and regeneration rates was evident here. High growth rates seem to impair regeneration capacity. We show that environmental conditions conducive to high zooxanthellae densities in corals are related to fast skeletal growth but also to reduced lesion regeneration rates. We hypothesize that a lowered regenerative capacity may be related to limited availability of energetic and cellular resources, consequences of coral holobionts operating at high levels of photosynthesis and associated growth.     

Hydrostatic pressure affects membrane and storage lipid compositions of the piezotolerant hydrocarbon‐degrading Marinobacter hydrocarbonoclasticus strain #5

A new piezotolerant alkane‐degrading bacterium (Marinobacter hydrocarbonoclasticus strain #5) was isolated from deep (3475 m) Mediterranean seawater and grown at atmospheric pressure (0.1 MPa) and at 35 MPa with hexadecane as sole source of carbon and energy. Modification of the hydrostatic pressure influenced neither the growth rate nor the amount of degraded hexadecane (≈ 90%) during 13 days of incubation. However, the lipid composition of the cells sharply differed under both pressure conditions. At 0.1 MPa, M. hydrocarbonoclasticus #5 biosynthesized large amounts (≈ 62% of the total cellular lipids) of hexadecane‐derived wax esters (WEs), which accumulated in the cells under the form of individual lipid bodies. Intracellular WEs were also synthesized at 35 MPa, but their proportion was half that at 0.1 MPa. This lower WE content at high pressure was balanced by an increase in the total cellular phospholipid content. The chemical composition of WEs formed under both pressure conditions also strongly differed. Saturated WEs were preferentially formed at 0.1 MPa whereas diunsaturated WEs dominated at 35 MPa. This increase of the unsaturation ratio of WEs resembled the one classically observed for bacterial membrane lipid homeostasis. Remarkably, the unsaturation ratio of membrane fatty acids of M. hydrocarbonoclasticus grown at 35 MPa was only slightly higher than at 0.1 MPa. Overall, the results suggest that intracellular WEs and phospholipids play complementary roles in the physiological adaptation of strain #5 to different hydrostatic pressures.     

Effects of light intensity, CO2 and nitrogen supply on lipid class composition of Dunaliella viridis

Lipid class composition of Dunaliella viridis Teodoresco was analysed using thin layer chromatography coupled with flame ionisation detection (TLC/FID technique). D. viridis was cultured under four different photon fluence rates and in darkness, and under two different conditions of CO₂ supply (atmospheric and 1%) with and without nitrogen sufficiency. Nine lipid classes were identified and quantified. Total lipids per cell and acetone-mobile polar lipids decreased with light, while the percentage of sterols and triglycerides increased with increasing irradiance. Total phospholipids increase was related with growth rate while hydrocarbons, wax esters and sterol esters accumulated in darkness. There were almost no changes in total lipids per cell because of nitrogen limitation; however, nitrogen limitation led to higher changes in lipid class composition under 1% CO₂ than under atmospheric CO₂ levels. The main reserve lipid, triglycerides, accumulated in high amounts under 1% CO₂ and nitrogen limitation, increasing from 1% to 22% of total lipids. The ratio sterols/acetone-mobile polar lipids could be an index of the 'light status' independently of nitrogen limitation, while the ratio triglycerides/total phospholipids could indicate any physiological stress uncoupling C and N metabolism and affecting the growth rate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morphological adaptation of Pseudomonas nautica strain 617 to growth on eicosane and modes of eicosane uptake

The transfer of Pseudomonas nautica 617 from a hydrophilic growth substrate (acetate) to a solid hydrocarbon substrate (eicosane) induced major morphological changes. The cell surface was covered by extracellular vesicles, and filamentous structures were observed. Adherence and emulsification were the two main modes of transfer of hydrocarbon to the cell surface. Growth on eicosane was associated with biosurfactant production.     

Diel and Seasonal Variations in Abundance,Activity, and Community Structure of Particle-Attached and Free-Living Bacteria in NW Mediterranean Sea

Diel and seasonal variations in abundance, activity, and structure of particle-attached vs free-living bacterial communities were investigated in offshore NW Mediterranean Sea (0–1000 m). Attached bacteria were always less abundant and less diverse but generally more active than free-living bacteria. The most important finding of this study was that the activity of attached bacteria showed pronounced diel variations in the upper mixed water column with higher activities at night. Under mesotrophic conditions, the contribution of attached bacteria to total bacterial activity increased from less than 10% at day time to 83% at night time. At high chlorophyll a concentration, the highest cell-specific activities and contribution to total bacterial activity were due to free-living bacteria at day and to attached bacteria at night. Under summer oligotrophic conditions, free-living bacteria dominated and contributed to the most important part of the bacterial activity at both day and night, whereas attached bacteria were much less abundant but presented the highest cell-specific activities. These diel and seasonal variations in activities were concomitant to changes in bacterial community structure, mainly in the upper layer. The number of attached ribotypes was fairly constant suggesting that particles are colonized by a relatively limited number of ubiquitous ribotypes. Most of these ribotypes were also free-living ribotypes suggesting that attached bacteria probably originate from colonization of newly formed particles by free-living bacteria in the upper layer. These results reinforce the biogeochemical role of attached bacteria in the cycling of particulate organic carbon in the NW Mediterranean Sea and the importance of diel variability in these processes.     

Application of a specific and sensitive radiometric assay for microbial lipase activities in marine water samples from the lagoon of nouméa

Marine microbiologists commonly assay lipase activities by using a synthetic fluorescent analog, 4-methylumbelliferyl (MUF)-oleate. The technique is convenient, but it is considered to be unspecific because of the structure of this analog. This study reports the design of a new specific and sensitive lipase assay based on the use of a radiolabeled triglyceride, [3H]triolein. Free fatty acids (FFA) resulting from its hydrolysis are isolated as a function of time in a one-step liquid-liquid extraction and then radioassayed. MUF-oleate and [3H]triolein techniques were compared by measuring lipase activities at similar substrate concentrations along a trophic gradient in the Southwest Lagoon of New Caledonia, near Nouméa. Hydrolysis rates decreased from the nearshore station to the offshore station and showed similar trends regardless of the technique used. Rates decreased from 5.83 to 0.88 nmol of FFA. liter-1. h-1 and from 0.76 to 0.23 nmol of 3H-FFA. liter-1. h-1, respectively. These results appeared to be consistent with bacterial production results, which also decreased similarly (from 0.59 to 0.26 micrograms of C. liter-1. h-1). However, the ratio of MUF-oleate activities to [3H]triolein activities, which was constant at the offshore stations (3.8 +/- 0.1), gradually increased at the nearshore stations (from 4.1 to 7.6). This result shows that the two assays respond in different ways to changes in environmental conditions and validates the need to set up more specific enzymatic assays.