Marine Diatom Haslea Ostrearia Research Articles

Electrochromic Properties and Electrochemical Behavior of Marennine, a Bioactive Blue-Green Pigment Produced by the Marine Diatom Haslea ostrearia.

Marennine has long been known as the unique peculiar pigment responsible for the natural greening of oysters. It is specifically produced by the marine diatom Haslea ostrearia and it is a natural blue molecule indeed promising for food industry because of the rarity of such non-toxic, blue-colored pigments. In the search for its still not defined molecular structure, investigation of the color changes with the redox state has been carried out combining different approaches. Reducing and oxidizing chemicals have been added to purified marennine solutions and a stable blue-green color has been confirmed for the oxidized state, while a yellow color corresponded to the reduced unstable state. Raman spectroscopy has been used to monitor changes in the Raman spectra corresponding to the different colored states, and cyclic voltammetry has allowed the detection of a redox system in which protons and electrons are exchanged. These findings show that marennine is a suitable stable blue pigment for use in food applications and help in the elucidation of the chromophore structure.

Not only greening: The effects of marennine produced by Haslea ostrearia on physiological traits of three bivalve species

The marine diatom Haslea ostrearia is known for its ability to produce marennine, a blue-green water-soluble pigment, which is responsible for the oyster greening phenomenon in the French Atlantic Coast. Marennine can be considered a bioactive extracellular compound (BEC) as it exhibits activities against detrimental pathogens in bivalve aquaculture. However, marennine could also be toxic to animals. The aim of this experimental study was to assess the effect of marennine produced by H. ostrearia upon three commercially important bivalve species, the American oyster Crassostrea virginica, the blue mussel Mytilus edulis and the giant scallop Placopecten magellanicus. Effect of this BEC was also assessed on different ontogenic stages of scallops. The animals were exposed to 3 mg L−1 of marennine and physiological response associated with clearance rates (CR), oxygen consumption and condition index (CI) were measured after 16 h of exposure (day 0), and after one subsequent week of recovery (day 7). In the short term, CR and oxygen consumption on mussels and oysters were significantly altered by marennine but not for scallops. CR recovery was observed in oysters after seven days, but no depuration was observed in mussels. Nevertheless, oxygen consumption in both mussels and oysters recovered on day 7. CI in the marennine-exposed group for both adult oysters and scallops was not significantly different, yet CI of mussels was lowered by 18.2 % compared to control. Scallop adults seemed largely resistant to marennine, but juveniles were more susceptible with a reduced CR and oxygen consumption by 71.9 % and 24.5 %, respectively, and no depuration activity observed until day 7. Additionally, CI in marennine-exposed juvenile scallops was 27 % lower than that of control. This study demonstrates species- and age-specific effect of marennine on bivalves, thus highlighting the importance of further assessment on the utilization of this pigment as a natural product decreasing pathogenicity of bacteria in shellfish aquaculture systems.

Effect of the blue pigment produced by the tropical diatom Haslea nusantara on marine organisms from different trophic levels and its bioactivity

Marennine is the blue-green pigment produced by the temperate ubiquiste marine diatom Haslea ostrearia. This pigment is water-soluble and excreted in seawater during H. ostrearia growth and ageing. Previous studies demonstrated the adverse effects of H. ostrearia culture supernatant that contains marennine (Blue Water, BW) against marine organisms. In tropical waters in the Southern hemisphere, a new diatom species has recently been discovered, Haslea nusantara, which shares a peculiarity with H. ostrearia in producing a blue-green, marennine-like pigment. However, little is known about the characteristics of the marennine-like pigment from H. nusantara and its bioactivity. The present study aimed to determine spectral characteristics of BW from H. nusantara in comparison to BW containing marennine of H. ostrearia, using UV–vis spectrophotometry, and to investigate the effects of BWs from these two species on several marine organisms from different trophic levels including autotrophs (Isochrysis galbana) and heterotrophs (Artemia salina). Additionally, the antibacterial activities of these BWs were also evaluated on two bacterial pathogens that are relevant to aquaculture, Vibrio harveyi and Staphylococcus aureus. The results show that both BWs from H. ostrearia and H. nusantara at 0.5 μg mL−1 significantly reduce the growth of I. galbana. At similar range of concentration, toxicity tests reveal that the both BWs from blue Haslea species significantly increase mortality rate of A. salina to a different extent. Additionally, both BWs from H. nusantara and H. ostrearia can inhibit the growth of tested bacterial pathogens. This study provides new insight into the different toxicity in the bioactivity of pigments obtained from the two species of blue diatoms from the same genus. Our results also demonstrate the need to determine concentrations that optimize beneficial effects and critical concentrations not to be exceeded before considering the use of marennine as natural antibacterial agents in the field of aquaculture.

Complex Relationships between the Blue Pigment Marennine and Marine Bacteria of the Genus Vibrio.

Marennine, the water-soluble blue pigment produced by the marine diatom Haslea ostrearia, is known to display antibacterial activities. Previous studies have demonstrated a prophylactic effect of marennine on bivalve larvae challenged with a pathogenic Vibrio splendidus, suggesting that the blue Haslea is a good candidate for applications in aquaculture as a source of a natural antimicrobial agent. Indeed, the genus Vibrio is ubiquitous in aquaculture ecosystems, and regular events of pathogenic invasion cause some of the biggest losses worldwide. To better characterize the effects of marennine on Vibrios, a panel of 30 Vibrio strains belonging to 10 different species was tested, including bivalve pathogenic species (e.g., Vibrio crassostreae and Vibrio harveyi). Vibrio strains were first exposed to 10 and 25 µg mL−1 of Blue Water (BW), a concentrated culture supernatant of H. ostrearia containing marennine. This screening evidenced a great diversity in responses, from growth stimulation to a total inhibition, at both the interspecific or intraspecific level. In a second series of experiments, 10 Vibrio strains were exposed to BW at concentrations ranging from 5 to 80 µg mL−1. The highest concentrations of BW did not systematically result in the highest growth inhibition as hormetic responses—opposite effects regarding the concentration—were occasionally evidenced. The relationships between marennine and Vibrio strains appear more complex than expected and justify further study—in particular, on the mechanisms of action—before considering applications as a natural prophylactic or antibiotic agent in aquaculture.

Harmful or harmless: Biological effects of marennine on marine organisms

Marennine is a water-soluble blue-green pigment produced by the marine diatom Haslea ostrearia. The diatom and its pigment are well known from oyster farming areas as the source of the greening of oyster gills, a natural process increasing their market value in Western France. Blooms of blue Haslea are also present outside oyster ponds and hence marine organisms can be exposed, periodically and locally, to significant amounts of marennine in natural environments. Due to its demonstrated antibacterial activities against marine pathogenic bacteria (e.g. Vibrio) and possible prophylactic effects toward bivalve larvae, marennine is of special interest for the aquaculture industry, especially bivalve hatcheries. The present study aimed to provide new insights into the effects of marennine on a large spectrum of marine organisms belonging to different phyla, including species of aquaculture interest and organisms frequently employed in standardised ecotoxicological assays. Different active solutions containing marennine were tested: partially purified Extracellular Marennine (EMn), and concentrated solutions of marennine present in H. ostrearia culture supernatant; the Blue Water (BW) and a new process called Concentrated Supernatant (CS). Biological effects were meanwhile demonstrated in invertebrate species for the three marennine-based solutions at the highest concentrations tested (e.g., decrease of fertilization success, delay of embryonic developmental stages or larval mortality). Exposure to low concentrations did not impact larval survival or development and even tended to enhance larval physiological state. Furthermore, no effects of marennine were observed on the fish gill cell line tested. Marennine could be viewed as a Jekyll and Hyde molecule, which possibly affects the earliest stages of development of some organisms but with no direct impacts on adults. Our results emphasize the need to determine dosages that optimize beneficial effects and critical concentrations not to be exceeded before considering the use of marennine in bivalve or fish hatcheries.

First insight on interactions between bacteria and the marine diatom Haslea ostrearia: Algal growth and metabolomic fingerprinting

A first insight on the interactions between bacteria isolated from monospecific cultures of the marine diatom Haslea ostrearia and this microalgae is described in this work. From 28 bacterial isolates, the maximal growth rate and the maximal cell concentration were on average 58% and 27% higher (max. 67% for isolate B22). Isolates B5, B10 (both Saccharospirillum sp.) and B22 (Rhodobacter sp.) were the most efficient. The geographical origin of H. ostrearia from which bacteria were isolated had no impact on the algal growth performances, and a given bacterial isolate led to the same algal growth irrespective of the geographical origin of H. ostrearia. To explain the results obtained, a comprehensive integrative strategy was conducted that analyzed the variations in the thousands of metabolites present in our culture extracts. Beyond a holistic view in which all organic substances of natural origin derived from the metabolism of a living organism were analyzed, we focussed on metabolites specific to each partner of the bacteria-H. ostrearia co-cultures, and relating to a particular biological function. To this end, a high throughput analytical technique, mass spectrometry was used, along with uni- and multivariate association statistical methods. Metabolic fingerprinting (untargeted approach) enabled compounds specific to each association to be identified, especially those concerning the positive bacterial effect on the growth of H. ostrearia.

Bacterial community structure of the marine diatom Haslea ostrearia

Haslea ostrearia produces a water-soluble, blue-green pigment, called marennine, with proven economic benefits (as a bioactive compound used to green oysters, which improves their market value). Incomplete knowledge of the ecological features of this marine diatom complicates its cultivation. More specifically, the ecology of bacteria surrounding H. ostrearia in ponds is what remains unknown. The structure of this bacterial community was previously analyzed by means of PCR-TTGE before and after isolating H. ostrearia cells recovered from 4 localities in order to distinguish the relative parts of the biotope and biocenose and to describe the temporal dynamics of the bacterial community structure at two time scales (2weeks vs. 9months). The bacterial structure of the phycosphere differed strongly from that of bulk sediment. The level of similarity between bacteria recovered from the biofilm and suspended bacteria did not exceed 10%. On the other hand, similarities among the bacterial community structures in biofilms were above 90% regardless of the geographic origin of the algal isolates, while the percentages were lower for suspended bacteria. The differences in bacterial structures of two H. ostrearia isolates (HO-R and HO-BM) resulted in specific metabolomic profiles. The non-targeted metabolomic investigation revealed more distinct profiles in the case of this bacteria-alga association than for the H. ostrearia monoculture. At the culture cycle scale under laboratory conditions, the bacterial community depended on the growth stage. When H. ostrearia was subcultured for 9months, a shift in the bacterial structure was observed as of 3months, with the bacterial structure stabilizing afterwards (70%–86% similarities), in spite of the size reduction of the H. ostrearia frustule. Based on these results, an initial insight into the relationships between H. ostrearia and its surrounding bacteria could be drawn, leading to a better understanding of the ecological feature of this marine diatom.

Comparison of cryopreservation methods for the long term storage of the marine diatom Haslea ostrearia (simonsen)

Long term maintenance of microalgal strains by serial subculturing is often expensive and time-consuming. Alternative methods, such as cryopreservation, present several benefits and thus seem more relevant. Our study aimed at comparing two cryopreservation procedures applied to the marine diatom Haslea ostrearia (Simonsen): (1) a two-step freezing method in liquid media using 5%, 10% and 20% MeOH, Me2SO or Glycerol, and (2) an immobilization-dehydration method consisting in an algal cell entrapped in 0.7M sucrose dehydrated and air-flow desiccated calcium alginate beads before “direct” or “two-step” freezing. Our results showed that the cryopreservation of H. ostrearia was feasible. With the two-step freezing protocol only Me2SO maintained cell viability without contamination but the low percentage of viability (<10%) prevents its use. Conversely, the immobilization-dehydration methods tested in this study were effective. Average viability of 57% and 77% were obtained with the “direct” and the “two step” cooling assays respectively, ensuring preservation of the genetic traits of H. ostrearia.

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.

Method for the quantification of the blue-green pigment “marennine” synthesized by the marine diatom Haslea ostrearia (Gaillon/Bory) Simonsen using HPLC gel-filtration and photodiode-array detection

This paper describes a new approach for quantifying marennine, a blue-green pigment synthesized by the marine diatom Haslea ostrearia, which is known to be responsible for the greening of cultured oysters in French coastal areas. The method uses gel-filtration HPLC interfaced with a photodiode-array detector (PDA). Under the chromatographic conditions applied, the peak of marennine is identified on the dextran pattern at 674 nm by its elution time (ET = 21–22 min) and its UV-Visible absorption spectrum. After calibration with pure marennine sample as external standard, consisting in plotting peak area as a function of marennine concentration, one can back-calculate concentrations of unknown samples. Results for quantitative determination of marennine using this procedure are validated and discussed in relation to those obtained by the spectrophotometric methods, the most often employed until now.

Microalgal cell immobilization for the long-term storage of the marine diatom Haslea ostrearia

To preserve the characteristics of the marine diatom Haslea ostrearia during long term storage, particularly size and shape, the algal cells were immobilized in alginate beads and stored at 4 ∘C at reduced irradiance up to 4 months. Two clones of different size (Ho34, 63 μm and Ho40, 78 μm) were studied. With Ho34, a 10.4% decrease of the size was shown after 120 days, by using the conventional storage management, while it did not exceed 2.2% with immobilized cells. Consequently, H. ostrearia would have auxosporulated after 9 months compared to 52 months. At the same time, the rate of distortion (aberrant valve structure) free Ho34 cells reached 86% while no distorted immobilized cells were observed. Chorophyll content in cells showed that all the cells were alive up to 60 days and after this time cells immobilized in the core of the beads most probably suffered from the poor light diffusion. Culturability of the immobilized cells was tested immediately after their immobilization and after 60 and 120 days of storage. The delay (at least 5) before immobilized cells released from the beads decreased with the time of storage, because of the embrittlement of the beads during the storage. Once in fresh medium, the cells actively multiplied. We concluded that immobilization strongly slowed down the decrease in frustule size with time and allowed the storage of concentrated and calibrated inocula which could be inoculated directly in liquid culture medium without needing to dissolve the beads.

Enhancement of marennine production by blue light in the diatom Haslea ostrearia

The marine diatom Haslea ostrearia Simonsen produces a blue pigment, marennine, which is used for greening oysters. This microalga is cultured industrially indoors with artificial light. The influence of light quality on marennine production by cultures of H. ostrearia was investigated in the laboratory and at a semi-pilot scale (300 L tanks). In the first series of experiments in the laboratory, a clone of H. ostrearia was cultured under light of different colors (white, blue, green, yellow, and red) and at two irradiances (‘low’ and ‘high’, 20 and 100 μmol photons m−2 s−1, respectively). Compared to the white light controls, growth was increased in blue light at the ‘low’, but not at the ‘high’ irradiance, and marennine production at the end of the exponential phase was the highest in cells grown under blue light, regardless of the light quality or intensity during growth. Increased marennine production during growth was also observed, whichever color of light (blue or white) was used during the acclimation phase. In a second series of experiments, intraclonal differences were studied by comparing marennine production in seven clones differing with regard to their mean cell size. The total marennine expressed either per cell or per culture volume, was higher in blue light for all the clones. Complementary experiments carried out under semi-industrial conditions confirmed this effect of blue light, which could be relevant for the industrial, indoor production of marennine.

Acclimation of Haslea ostrearia to light of different spectral qualities – confirmation of `chromatic adaptation' in diatoms

The marine diatom Haslea ostrearia was cultured under light of different qualities, white (WL), blue (BL), green (GL), yellow (YL), red (RL), and far-red (FRL) and at two irradiance levels, low and high (20 and 100 μmol photons m −2 s −1, respectively). The effects of the different light regimes were studied on growth, pigment content, and photosynthesis, estimated by the modulated fluorescence of chlorophyll, as relative electron transport rate (rETR). For all the light qualities studied, growth rates were higher at high irradiance. Compared to the corresponding WL controls, growth was higher in BL and lower in YL at low irradiance, and lower in YL and GL at high irradiance. Except for YL, almost all the pigment contents of the cells were lower at high irradiance. At low irradiance, cell pigment contents (chlorophyll a and c, fucoxanthin) and pigment ratios (in function of chlorophyll a) were lower in YL, RL, and FRL. Whatever the irradiance level, the maximum PSII quantum efficiency ( F v/ F m) remained almost constant for WL, BL, and GL. Other fluorescence parameters (photochemical quenching, rETR max, and α, the maximum light utilization coefficient) were lower in GL, YL, RL, and FRL, at low irradiance. Although not statistically significant, BL caused an increase in these fluorescence parameters. These findings are interpreted as evidence that inverse chromatic acclimation occurs in diatoms.

A new photobioreactor for continuous marennin production with a marine diatom: influence of the light intensity and the immobilised-cell matrix (alginate beads or agar layer).

In oyster ponds, the marine diatom Haslea ostrearia synthesises and excretes a hydrosoluble pigment of commercial interest called marennin. During the benthic stage, when algal cells are naturally immobilised in their own polysaccharides, marennin production is higher. To optimise this production, axenic cultures of H. ostrearia were immobilised in a polysaccharidic matrix (alginate or agar) and introduced into a new photobioreactor device for continuous marennin production. Solute diffusion was improved using an alginate beads monolayer, leading to higher levels of cell growth (a 2-fold higher cell concentration) and marennin productivity (7.57-8.80 mg day(-1) l(-1)). An increase in the light intensity (from 3.0 to 8.5x10(16) quanta cm(-2) s(-1)) led to an earlier and 1.3-fold higher production of marennin. However, the higher light intensity led to a higher rate of cell death [0.29 instead of 0.40 ng chlorophyll a (10(6) cells)(-1)]. Due to the secondary nature of marennin metabolism, it would be necessary to alternate between culture conditions favouring cell growth (moderate light intensity and no limiting nitrate supply) and those promoting marennin production (high light intensity and limiting nitrate supply).

Continuous marennin production by agar-entrapped Haslea ostrearia using a tubular photobioreactor with internal illumination.

The marine diatom Haslea ostrearia was immobilized in a tubular agar gel layer introduced into a photobioreactor of original design with internal illumination for the continuous synthesis of marennin, a blue-green pigment of biotechnological interest. Marennin was produced for a long-term period (27-43 days) and the volumetric productivity was maximum (18.7 mg day(-1) l(-1) gel) at the highest dilution rate (0.25 day(-1)) and lowest agar layer thickness (3 mm). Heterogeneous cell distribution in the agar layer revealed diffusional limitation of light and nutrients. However, the 3 mm gel thickness led to a more homogeneous cell distribution during incubation and to an increase of the whole biomass in the agar gel layer.

Production of exocellular pigment by the marine diatom Haslea ostrearia Simonsen in a photobioreactor equipped with immersed ultrafiltration membranes

A new photobioreactor coupled with an ultrafiltration system (immersed membranes) was investigated for the continuous culture of the microalga Haslea ostrearia in order to improve pigment (marennine) production and recovery. The system presents a commercial interest, because energetic costs were minimized, and the cells were not submitted to any shear stress due to pumping or circulation. To obtain this, since the photobioreactor was of cylindrical type, a membrane module was placed at the bottom of the reactor and the hydrostatic pressure (the height of the water column) used as driving force both for the permeation and periodical backflushing steps. The production of biomass and marennine was stable for a three-week period, with marennine specific productivity ≈30–35 mg 10 −9 cell day −1, marennine concentration ≈3 times higher than in a conventional batch photobioreactor. The permeation flux obtained was acceptable (3–10 l h −1 m −2, 3 kPa, 15°C), but for such applications, this type of integrated process needs further improvements. Owing to its simple design, the concept “photobioreactor – ultrafiltration with immersed membranes” has good possibilities in biotechnology and aquaculture for continuous extraction of exocellular metabolites.

Antiviral and anticoagulant activities of a water-soluble fraction of the marine diatom Haslea ostrearia.

A water-soluble fraction from the marine diatom Haslea ostrearia was capable to inhibit the in vitro replication of HSV-1 in Vero cells with 50% inhibitory concentration (EC50) of 14 micrograms/ml at a multiplicity of infection of 0.01 ID50/cells. In addition, this fraction delayed the HIV-1-induced syncitia formation on MT4 cells. At concentrations up to 200 micrograms/ml, no cytotoxicity was observed for both the Vero and MT4 cells. The fraction only inhibited the blood coagulation process at concentrations considerably exceeding the EC50.

Concentration and desalting by membrane processes of a natural pigment produced by the marine diatom Haslea ostrearia Simonsen

The marine microalga Haslea ostrearia, also called ≪blue navicula⪢, presents the unique peculiar property among the diatoms, to produce at its extremities a blue hydrosoluble pigment called ≪marennine⪢. It is presented the concentration and the desalting of the exocellular pigment by membrane processes (ultrafiltration, nanofiltration, reverse osmosis). Nanofiltration is particularly developed given the potential of this type of application both for the concentration of molecules and for desalting. It is shown the effect of velocity and pressure on performances of nanofiltration membranes. Permeation flux superior to 100 l h −1 m −2 (at 14.10 5 Pa) are obtained with the Kiryat Weizmann membrane MP 20 (polyester coated with a polyacrylonitrile layer, cut-off 450 Da). For the desalting of the blue pigment solution, nanofiltration membranes present a few advantages: a low salt rejection (less than 10% at 14.10 5 Pa) and a high pigment rejection (the nanofiltration membrane MP 20 retains more than 95% of the pigment). This membrane used in diafiltration mode allows an acceptable speed of desalting (700 g of salt eliminated per hour and per m 2 at 25.10 5 Pa for a concentration of 18 g of salt per litre of solution).

Continuous high-pressure disruption of marine diatom Haslea ostrearia. Assessment by laser diffraction particle sizer

In order to recover the intracellular blue-green pigment, marennine, synthesized by the microalga Haslea ostrearia, a continuous flow high-pressure disrupter was evaluated in the range: 30–270 MPa. Cells were partly broken from 30 MPa, but a pressure of 100 MPa (1 cycle) was required to obtain optimal pigment release. The latter was directly linked to the physical cell breakage dependent upon the applied pressure and the number of disintegration cycles. Granulometric analysis by laser diffraction technology (0.04–2000 μm) revealed a size reduction of cell fragments when increasing these two operating parameters.

Artificial cell-immobilization: a model simulating immobilization in natural environments?

A study of the marine diatom Haslea ostrearia was performed in controlled culture conditions. Biofilm formation by cells grown in liquid medium was studied through macroscopic events and extracellular polysaccharides (EPS) production. Alcian blue staining and image analysis methods were used to quantify EPS production in regard to the usual pattern (lag, exponential and stationary phases) of microalgal growth. Simultaneously, an artificial immobilization of H. ostrearia cells was carried out using an agar layer. Samples from liquid medium and artificially immobilized cultures were studied in scanning electron microscopy. They revealed the structure of the entrapping material and progressive changes of the natural biofilm along culture ageing. The use of artificially immobilized cultures for solute diffusivity investigations is discussed.