Michèle Morançais

Long-term photoacclimation of Haslea ostrearia (Bacillariophyta): effect of irradiance on growth rates, pigment content and photosynthesis

Haslea ostrearia, the peculiar diatom that develops in oyster-ponds and synthesizes a water-soluble blue pigment (marennine), was grown in a semi-continuous mode over a wide range of irradiances (20–750 μmol m−2 s−1). Growth, photosynthesis and pigment content were determined for algae maintained in exponential growth phase by regular dilution with fresh medium. Increasing the growth irradiance resulted in a decrease in the chloroplast length, but had no clear influence on the size of the cellular compartments pigmented by marennine accumulation. Growth rates increased with irradiance from 20 to 100 μmol m−1 s−1 and were constant from 100 to 750 μmol m−2 s−1. Increasing growth irradiance caused a decrease in the cellular content of chlorophylls a and c and fucoxanthin, in contrast to diadinoxanthin. Algae acclimated to high irradiance had lower maximum photosynthetic rate (P n m) and maximum light utilization coefficient (α) when expressed on a per cell basis. On a chlorophyll a basis, the higher the grow...

Optimization of hydrolysis conditions of Palmaria palmata to enhance R-phycoerythrin extraction

In this study, response surface methodology was applied to optimize R-phycoerythrin extraction from the red seaweed Palmaria palmata, using enzymatic digestion. Several algal treatments prior to digestion were first investigated. The extraction yield and the purity index of R-phycoerythrin, and the recovery of proteins and reducing sugars in the water-soluble fraction were then studied in relation to the hydrolysis time, the temperature and the enzyme/seaweed ratio. Enzymatic digestion appears to be an effective treatment for R-phycoerythrin extraction. Moreover, using the seaweed roughly cut in its wet form gives the most interesting results in terms of extract quality and economic cost. The R-phycoerythrin extraction yield is 62 times greater than without enzyme treatment and 16 times greater than without optimization. Enzymatic optimization enhanced the purity index up to 16 times.

Physicochemical factors affecting the stability of two pigments: R-phycoerythrin of Grateloupia turuturu and B-phycoerythrin of Porphyridium cruentum.

Phycoerythrin is a major light-harvesting pigment of red algae, which could be used as a natural dye in foods. The stability of R-phycoerythrin of Grateloupia turuturu and B-phycoerythrin of Porphyridium cruentum in relation to different light exposure times, pHs, and temperatures was studied. Regarding the light exposure time, after 48h, the reduction in concentrations of B-phycoerythrin and R-phycoerythrin were 30±2.4% and 70±1%, respectively. Phycoerythrins presented good stability from pH 4 to 10. At pH 2, the reduction in concentration was 90±4% for B-phycoerythrin and 40±2.5% for R-phycoerythrin while, at pH 12, the phycoerythrins were degraded. Phycoerythrins showed good stability toward temperature, up to 40°C. At 60°C, the reduction in concentrations of B-phycoerythrin and R-phycoerythrin were 50±3.4% and 70±0.18%, respectively. Moreover, the best conditions of storage (-20°C) were determined.

Antioxidant and Free Radical Scavenging Properties of Marennine, a Blue-Green Polyphenolic Pigment from the Diatom Haslea ostrearia (Gaillon/Bory) Simonsen Responsible for the Natural Greening of Cultured Oysters

Among microalgae, the marine diatom Haslea ostrearia has the distinctive feature of synthesizing and releasing, into the surrounding environment, a blue-green polyphenolic pigment called marennine. The oyster-breeding industry commonly makes use of this natural phenomenon for the greening of oysters grown in the ponds of the French Atlantic coast. This article reports the in vitro antioxidant properties of pure marennine. Two kinds of evaluation systems were adopted to test the antioxidative activity of marennine: antioxidant capacity assays (beta-carotene and thymidine protection assays and iron reducing power assay) and free radical scavenging assays (DPPH*, O2*-, and HO*). In almost all cases, marennine exhibited significantly higher antioxidative and free radical scavenging activities than natural and synthetic antioxidants commonly used in food, as shown by comparing median effective concentration (EC 50) values, for each test independently. This medium molecular weight polyphenol (around 10 kDa) from microalgae is thus a potentially useful natural antioxidant. Because of its blue-coloring property and water solubility, it could also be used as a natural food-coloring additive.

Finding new diatoms for intensive rearing of the pacific oyster (Crassostrea gigas): energy budget as a selective tool

Abstract Intensive shellfish rearing in the polders of Bourgneuf Bay on the French Atlantic coast (46–47°N, 1–2°W) relies on the diatom Skeletonema costatum (Grev.) Cleve produced in nutrient-rich saline ground water. An episodic reduction in biomass production caused by an unidentified protozoon stressed the need to find new microalgal species as substitutes for S. costatum and diversify the microalgae used by the bivalve industry. Three species met the requirements for year-round availability in Bourgneuf Bay coastal waters and growth potential in saline ground water: Nitzschia acicularis (Kutzing), Nitzschia closterium (Ehrenberg) Wm Smith and Nitzschia gandersheimiensis Krasske= Nitzschia tubicola Grunow. These microalgae were tested as food sources for adult Pacific oysters ( Crassostrea gigas ) by comparing short-term bivalve physiological responses with those obtained with S. costatum as reference. Suspended particulate matter concentrations in experimental diets ranged from 9.3 to 18.6 mg l −1 and particulate organic matter concentrations from 3.3 to 5.7 mg l −1 . Significant differences were observed, with clearance rates ranging from 4.0 l h −1 g −1 for N. acicularis to 7.3 l h −1 g −1 for N. gandersheimiensis . The filtration rate for organic matter was significantly higher for N. gandersheimiensis than the other species, but this algae was also significantly more rejected in pseudofaeces. No differences were found among the four mean faeces production rates. Net energy balance differed significantly among the four microalgae tested, ranging from 282 J h −1 g −1 for S. costatum to 27 J h −1 g −1 for N. closterium . However, no differences were found between S. costatum , N. acicularis and N. gandersheimiensis . These results suggest that N. acicularis and N. gandersheimiensis could be tested on a larger scale involving the production of microalgae in 50-m 3 outdoor tanks in association with Pacific oyster growth experiments.

Preliminary characterisation of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen

Haslea ostrearia is a common marine tychopelagic diatom which has the particularity of synthesizing a blue-green hydrosoluble pigment called “marennine”. This pigment, when released into the external medium, is known to be responsible for the colour of oyster gills. Here we present results for main biophysical and biochemical characteristics of pure intra- and extracellular marennine. Tests for chemical determination show that the nature of the two forms of marennine cannot be distinguished and could be related to a polyphenolic compound. Nevertheless, based on spectral properties and the molecular weight, which is about 10751 ± 1 and 9893 ± 1 Da, for the intracellular and extracellular forms respectively, we assess that the pigment accumulated in the apex of the cell and the one released in the external medium have probably distinct molecular structures.

Biological activities of purified marennine, the blue pigment responsible for the greening of oysters

Marennine, the blue pigment produced by the diatom Haslea ostrearia , exists in two different forms, the intra- and extracellular forms. We investigated the antibacterial, antiviral, and antiproliferative properties of both of these forms. Both forms of marennine inhibited the development of marine bacteria, in particular the pathogenic organism Vibrio aesturianus , at concentrations as low as 1 μg/mL, but they did not display any effect on a wide range of pathogenic bacteria that are relevant for food safety. Both forms of the pigment produced by H. ostrearia also exhibited antiviral activity against the HSV1 herpes virus, with intra- and extracellular marennine having EC(50) values of 24.0 and 27.0 μg/mL, respectively. These values are 2 orders of magnitude higher than the value for the reference drug, Zovirax. Moreover, both forms of marennine were effective in slowing or inhibiting the proliferation of cancer cells. This study confirms the potential of marennine as a biologically active organic molecule, which could have a protective effect on bivalves, which filter seawater and fix the pigment on their gills. Moreover, marennine could be used in food engineering and chemistry as a natural blue pigment. However, despite that it is eaten and possibly assimilated by green oyster consumers, it also deserves in depth evaluation before being considered for use as a nutraceutical.

Effect of enzymatic digestion on thallus degradation and extraction of hydrosoluble compounds from Grateloupia turuturu

The extraction of metabolites of industrial interest from seaweed tissue is difficult due to the presence of anionic polysaccharides in the cell walls. We studied enzymatic digestion of Grateloupia turuturu (Rhodophyta) to facili- tate extraction of the desired metabolites from this sea- weed. Four polysaccharidases (Onozuka R-10 cellulase, agarase, k- and i-carrageenases) were used alone and in combination. We studied the efficiency of the enzy- matic process in degrading the seaweed thallus; we also investigated the effect of the enzymatic treatment on solubilization of R-phycoerythrin and reducing sugars. The best degradation conditions for Grateloupia thalli were obtained with cellulase alone (0.48 U ml -1 ), or with a combination of cellulase/k-carrageenase. However, only the combination cellulase (0.08 U ml -1 )/k-carrageen- ase (0.01 U ml-1), or the individual enzymes wagarase or i-carrageenase at low concentration (0.55 U ml -1 and 2.5=10-3 Um l-1)x improved solubilization of R-phycoery- thrin compared to the control conditions. The simulta- neous use of polysaccharidases enabled solubilization of a large quantity of reducing sugars (40 mg glu g -1 dw). Therefore, this condition appears suitable for the pro- duction of oligosaccharides. This result also confirms indirectly the presence of agar, cellulose, k- and i-car- rageenans in the cell wall of Grateloupia turuturu.

Purification of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen

The diatom Haslea ostrearia that lives in oyster ponds has the distinctive feature of synthesizing “marennine”, a blue-green pigment of which the chemical nature still remains unknown. This pigment is responsible for the greening of oyster gills. Here, we report a new method for extraction and purification of intracellular (accumulated in the apex of the cell) and extracellular (released into the external medium) forms of the pigment. Intracellular marennine is obtained by extraction from blue algal pellets with a carbonate buffer. The extract is then centrifuged and filtered. Extracellular marennine is obtained by clarification of blue-coloured culture medium. Both extracts are then purified by a semi-preparative process, using ultrafiltration through membranes and anion-exchange chromatography. This procedure allows us to produce native pigment displaying the degree of purity required to enter upon the molecular characterisation of marennine. By this process, about 35% of the initial amount of pigment can be recovered. If necessary, this method could be easily scaled up to a larger production system to accommodate potential industrial applications.

A (2)H solid-state NMR study of the effect of antimicrobial agents on intact Escherichia coli without mutating.

Solid-state nuclear magnetic resonance (NMR) is a useful tool to probe the organization and dynamics of phospholipids in bilayers. The interactions of molecules with membranes are usually studied with model systems; however, the complex composition of biological membranes motivates such investigations on intact cells. We have thus developed a protocol to deuterate membrane phospholipids in Escherichia coli without mutating to facilitate (2)H solid-state NMR studies on intact bacteria. By exploiting the natural lipid biosynthesis pathway and using perdeuterated palmitic acid, our results show that 76% deuteration of the phospholipid fatty acid chains was attained. To verify the responsiveness of these membrane-deuterated E. coli, the effect of known antimicrobial agents was studied. (2)H solid-state NMR spectra combined to spectral moment analysis support the insertion of the antibiotic polymyxin B lipid tail in the bacterial membrane. The use of membrane-deuterated bacteria was shown to be important in cases where antibiotic action of molecules relies on the interaction with lipopolysaccharides. This is the case of fullerenol nanoparticles which showed a different effect on intact cells when compared to dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol membranes. Our results also suggest that membrane rigidification could play a role in the biocide activity of the detergent cetyltrimethyammonium chloride. Finally, the deuterated E. coli were used to verify the potential antibacterial effect of a marennine-like pigment produced by marine microalgae. We were able to detect a different perturbation of the bacteria membranes by intra- and extracellular forms of the pigment, thus providing valuable information on their action mechanism and suggesting structural differences.