Artificial Seawater Medium Research Articles

An experimental approach to study the biosynthesis of brominated metabolites by the red algal genus Laurencia

The production of labeled brominated metabolites with radioactive 82Br in Laurencia species was investigated as part of a study of the biosynthesis of halogenated metabolites from species belonging to the red algal genus Laurencia (Rhodomelaceae, Ceramiales). Radiobromide [ 82Br], thin-layer chromatography (TLC), and TLC–autoradioluminography (ARLG) were used. When cultured in artificial seawater medium (ASP 12NTA including Na 82Br) under 16:8 h light:dark (LD) illumination cycles for 24 h, each of the strains of Laurencia, Laurencia japonensis Abe et Masuda, Laurencia nipponica Yamada (laurencin-producing race and laureatin-producing race), and Laurencia okamurae Yamada, produced species- (or race-) specific 82Br-containing metabolites. In the case of the laurencin-producing race of L. nipponica, laurencin and deacetyllaurencin were found to be produced in approximately 1:1 ratio, though laurencin is the major metabolite in the wild sample. Furthermore, when cultured in the dark, the production rates of brominated metabolites in Laurencia spp. were found to be diminished. The present study strongly indicates that the use of radiobromine [ 82Br] in combination with the TLC–ARLG method is an effective approach for investigating the biosynthesis of brominated metabolites in Laurencia.

Influence of turbulent flow on the corrosion of Al‐Zn‐Mg galvanic anode in artificial seawater media

AbstractThe effect of hydrodynamics on the corrosion of Al(14 wt%)‐Zn(8 wt%)‐Mg alloy in artificial seawater media at room temperature was studied in a rotating cylinder electrode (RCE) system under turbulent flow conditions. Five different rotation rates were studied: 100, 1000, 3000, 5000, and 7000 rpm. The corrosion rates were measured by electrochemical impedance spectroscopy (EIS). For the system studied, the steady‐state corrosion potential increased with increase in rotation rate. The effect of increasing the rotation rate is to increase the availability of oxygen at the surface, which in turn will polarize the corrosion reaction in the more noble direction. The corrosion rate also increases with increase in RCE rotation rate. This reflects the fact that the rate of corrosion is controlled, at least in part, by the rate of mass transfer. In this case, the effect of increase in the rotation rate on the corrosion rate is to increase the interfacial concentration of the reactant (oxygen).

Salinisphaera hydrothermalis sp. nov., a mesophilic, halotolerant, facultatively autotrophic, thiosulfate-oxidizing gammaproteobacterium from deep-sea hydrothermal vents, and emended description of the genus Salinisphaera

A mesophilic, aerobic, facultatively chemolithoautotrophic bacterium, designated strain EPR70(T), was isolated from hydrothermal fluids from diffuse-flow vents on the East Pacific Rise at degrees 50' N 10 degrees 17' W. Cells were Gram-negative rods, approximately 0.8-1.0 microm long and 0.3-0.5 microm wide. Strain EPR70(T) grew at 20-40 degrees C (optimum 30-35 degrees C), 1-25 % NaCl (optimum 2.5 %) and pH 5.0-7.5 (optimum pH 5.5). The shortest generation time observed for strain EPR70(T) was 42 min. Growth occurred under aerobic chemolithoautotrophic conditions in the presence of thiosulfate and CO(2). Strain EPR70(T) grew heterotrophically with acetate or n-alkanes as sole carbon and energy sources, and in complex artificial seawater medium. Nitrate was not used as an electron acceptor. The G+C content of the genomic DNA was 64 mol%. Phylogenetic analysis of the 16S rRNA gene indicated that this organism is a member of the class Gammaproteobacteria, with Salinisphaera shabanensis E1L3A(T) as its closest relative (94 % sequence similarity). On the basis of phylogenetic analyses based on 16S rRNA, rbcL and alkB genes and physiological analysis, it is proposed that the organism represents a novel species within the genus Salinisphaera, for which the name Salinisphaera hydrothermalis sp. nov. is proposed. The type strain is EPR70(T) (=DSM 21483(T) =JCM 15514(T)).

Statistical Analysis of Microarray Data with Replicated Spots: A Case Study withSynechococcusWH8102

Until recently microarray experiments often involved relatively few arrays with only a single representation of each gene on each array. A complete genome microarray with multiple spots per gene (spread out spatially across the array) was developed in order to compare the gene expression of a marine cyanobacterium and a knockout mutant strain in a defined artificial seawater medium. Statistical methods were developed for analysis in the special situation of this case study where there is gene replication within an array and where relatively few arrays are used, which can be the case with current array technology. Due in part to the replication within an array, it was possible to detect very small changes in the levels of expression between the wild type and mutant strains. One interesting biological outcome of this experiment is the indication of the extent to which the phosphorus regulatory system of this cyanobacterium affects the expression of multiple genes beyond those strictly involved in phosphorus acquisition.

Effect of Flow on the Electrochemical Behavior of Zn Galvanic Anodes in Artificial Seawater Media

The effect of hydrodynamics on the corrosion of a commercial Zn galvanic anode in artificial seawater media at room temperature was studied in a RCE system under turbulent flow conditions. Five different rotation rates were studied: 0, 1000, 3000, 5000 and 7000 rpm. The corrosion rates were measured by Linear Polarization Resistance Technique. For the system studied, the steady-state corrosion potential increased with increasing RCE rotation rate. The effect of increasing the rotation rate is to increase the availability of oxygen at the surface, which in turn will polarize the corrosion reaction in the more noble direction. The corrosion rate increases with increasing velocity over the range of rotational rates studied. This reflects the fact that the rate of corrosion is controlled, at least in part, by the rate of mass transfer. In this case, the effect of increasing RCE rotation rate on the corrosion rate is to increase the interfacial concentration of the reactant (oxygen).

Aerobic biodegradation of polyethylene glycols of different molecular weights in wastewater and seawater

In order to distinguish between aerobic biodegradation of synthetic polymers in fresh and seawater, polyethylene glycols (PEGs) were systematically and comparatively investigated in inocula from municipal wastewater and seawater aquarium filters for the first time. The molecular weight (MW) of the PEGs, (HO(CH 2CH 2O) n H, n = 3–1350) as representatives of water-soluble polymers, ranged from 250 to 57,800 Da. The biodegradation was observed by removal of dissolved organic carbon and carbon dioxide production by applying standardized ISO and OECD test methods. Specific analyses using liquid chromatography mass spectrometry (LC–MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were performed. All PEGs selected were completely biodegradable in freshwater media within 65 d. PEGs with an MW up to 14,600 Da have a similar degradation pathway which is characterized by gradual splitting of C 2-units off the chain resulting in formation of short-chain PEGs. In artificial seawater media, full biodegradation of PEGs up to 7400 Da required more time than in freshwater. PEGs with MW 10,300 and 14,600 Da were only partially degraded whereas PEGs with MW 26,600 and 57,800 Da were not degraded for a period of 135 d. The biodegradation pathway of PEG 250 and PEG 970 in seawater is similar to that for freshwater. For PEGs having an MW from 2000 to 10,300 Da, the degradation pathway in seawater differs from the pathway of the shorter PEGs.

Lipid body formation by Thraustochytrium aureum (ATCC 34304) in response to cell age

The heterotrophic marine protist, Thraustochytrium aureum produces substantial amounts of polyunsaturated fatty acids (PUFAs). In the present investigation, changes in the lipid and fatty acid profiles of T. aureum were studied according to the culture age. T. aureum was grown in artificial sea water medium for 10 days at 25 °C in shake culture condition. One to 10 day old cell samples were analyzed for cell biomass production, total lipid content, fatty acid profile and lipid body formation. In all the samples tested, total lipid production was found to be directly proportional to the dry cell weight of T. aureum. In the early phase of cell growth, cell biomass production, lipid content and glucose consumption were found to be higher. Thin layer chromatographic analysis (TLC) of lipids showed the presence of triacylglycerol (TAG; 169 mg/g, 90%), phospholipids (PL; 83 mg/g, 66%) and sterol (ST; 6 mg/g, 5%), which were recorded at maximum levels in the early growth phase of the cells. The composition of PUFAs and saturated fatty acids (SFAs) of the cell biomass and lipid class components (TAG and PL) was identified by gas chromatographic analysis (GC). In the early phase of cell growth, production of PUFAs in the total fatty acids was found to have attained maximum levels (61.3%) in which docosahexaenoic acid alone showed higher content of occurrence (99.0 mg/g in total lipid; 65.2 mg/g in TAG and 41.0 mg/g in PL). In the middle phase of cell growth, palmitic acid production was found to be higher (36.7 mg/g in total lipid; 31.3 mg/g in TAG and 12.6 mg/g in PL). Transmission electron microscopic studies of the cells showed the presence of a membrane around the lipid bodies in the early phase of cell growth. TAG and PL were actively involved in the formation of lipid bodies in the cells of T. aureum. Large-sized lipid bodies accumulated in 3 day old cells which were then fragmented into smaller bodies in the late growth phase.

Rotating Cylinder Electrode Study on the Influence of Turbulent Flow and Temperature on the Corrosion of an Al-Zn-Mg alloy in Artificial Seawater Media

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Photolithotrophic cultivation of Laminaria japonica gametophyte cells in a silicone tubular membrane-aerated photobioreactor

Gametophyte cells of brown algae Laminaria japonica were employed both in a modified silicone tubular membrane-aerated photobioreactor (bubble-less cultivation mode) and a bubble-column photobioreactor (bubbling cultivation mode), to study different gas–liquid mixing modes on cell growth rate and cell physiological status. With an inoculum density of 50 mg DCW l−1, in modified artificial Pacific seawater (APSW) medium at 13°C, light intensity of 60 μE m−2 s−1, light cycle of 16/8 h L/D, and aeration rate of 60 ml min−1, the specific growth rates were 0.082 d−1 for bubble-less mode and 0.070 d−1 for bubbling mode with biomass, in the form of dry cell density, increasing 10.9 and 6.8 times, respectively, during the 36 days’ photolithotrophic cultivation. The specific oxygen evolution rate under bubble-less mode was 39.6% higher than under bubbling mode on the 18th day. The gametophyte cells grew in cell aggregates with clump sizes, at day 36, of 1.5 mm and 0.5 mm diameter under bubble-less and bubbling mode respectively and cell injury percentages of 5.1% and 21.1%, respectively. The silicone tubular membrane-aerated photobioreactor was better suited for the cultivation of fragile macroalgal gametophyte cells due to the absence of hydrodynamic shear stress caused by fluid turbulence and the presence of a bubble-less gas supply.

Culturing the marine cyanobacterium Prochlorococcus

Prochlorococcus is the numerically dominant primary producer in open ocean ecosystems. Analysis of Prochlorococcus genome sequences from cultured isolates and ocean samples has broadened interest in studying this tiny cell, and efforts are underway to develop it into a model system for studying marine microbial ecology. A critical component of these efforts has been the development of systematic culturing methods that will facilitate the distribution of Prochlorococcus to diverse labs that may be interested in studying it. This paper provides detailed methods for maintaining cultures of Prochlorococcus, including a comparison of growth rates of cells on two artificial seawater media and on a standard medium that uses a natural seawater base. Procedures for agar plating, cryopreservation, obtaining new isolates, and issues associated with culture volume and carbon limitation also are described.

Influence of microstructure on the corrosion resistance of the duplex stainless steel UNS S31803

Duplex stainless steels (DSS) are high strength corrosion resistant materials used in the chemical and petrochemical industries. When compared to conventional austenitic grades (AISI 304L, 316L, 317L), DSS are more expensive, but present higher strength levels and are more resistant to Cl − environments. In the present work it was investigated how microstructural variations can affect the corrosion resistance of a DSS UNS S31803 in artificial sea water media. Pitting corrosion resistance in 3.5%NaCl solution was evaluated by cyclic polarization tests at room temperature and 60 °C. The double loop electrochemical potentiodynamic reactivation test (DL-EPR) was also used to evaluate the degree of sensitization due to tertiary phases, such as σ and Cr 2N. Structures containing sigma phase were found to be the most susceptible to pitting and localized corrosion. Cr 2N, present in the as-melted sample, decreases the pitting potential but does not cause sensitization.

Calcium oxalate crystals in the marine red alga Spyridia filamentosa (Ceramiales; Rhodophyta)

C.M. Pueschel and J.A. West. 2007. Calcium oxalate crystals in the marine red alga Spyridia filamentosa (Ceramiales; Rhodophyta). Phycologia 46: 565–571. DOI: 10.2216/06-101.1Birefringent polyhedral crystals up to 8 µm on a side were found both within cells and on the thallus surfaces of several genetically distinct isolates of the red alga Spyridia filamentosa (Wulfen) Harvey. Diagnostic chemical solubility tests and silver nitrate–dithiooxamide staining identified the crystals as calcium oxalate. Calcium oxalate crystals were observed in all cell types of the vegetative erect axes: axial cells of indeterminate filaments, corticating cells of the main axes, and axial and corticating cells of the determinate lateral filaments (brachyblasts). Thalli cultured in natural seawater medium had few crystals close to the growing apices, whereas older portions of the thalli developed a surface coating of fine crystals and scattered larger extracellular and intracellular crystals. Thalli cultured in natural seawater medium whose calcium concentration was supplemented with 20 mM calcium chloride had an abundance of intracellular and extracellular calcium oxalate crystals. Thalli grown in artificial seawater medium having low calcium concentrations (1.0 and 2.5 mM calcium chloride) deposited few calcium oxalate crystals, but when these thalli were transferred to calcium-rich medium and stressed, crystals formed rapidly. The nonconstitutive nature of calcium oxalate deposition in Spyridia may explain conflicting previous accounts regarding presence of calcium oxalate crystals in this genus.

The Influence of the Marine Aerobic Pseudomonas Strain on the Corrosion of 70/30 Cu-Ni Alloy

Corrosion of 70/30 Cu-Ni alloy by marine aerobic bacteria Pseudomonas sp. NCIMB 2021 in an artificial seawater medium was investigated by electrochemical and microscopic methods. Polarization curve measurements demonstrated that the corrosion rate of the alloy increased notably in the presence of the Pseudomonas sp. by the acceleration of both the anodic and cathodic reactions. The impedance spectra and the corresponding equivalent circuits confirmed that the alloy surface in the sterile medium comprised of an outer organic compound conditioning layer and an inner compact and protective oxide film layer; while in the Pseudomonas inoculated medium, a duplex layer of an outer porous, heterogeneous and non-protective biofilm layer and an inner porous oxide film layer was present. Both the charge transfer resistance, Rct, and the resistance of the porous oxide film, Rpo, decreased with exposure time, indicating an acceleration of corrosion due to the marine bacteria. Bacteria attached to the alloy surface formed blotchy biofilms, as observed by fluorescent microscopy. The dynamic development of the biofilm and surface roughness were monitored by atomic force microscopy. Scanning electron microscopy images showed micro- pitting corrosion on the alloy surface after the biofilm remova

A highly specific PCR assay for detecting the fish ectoparasite Amyloodinium ocellatum

Amyloodiniosis, caused by the dinoflagellate ectoparasite Amyloodinium ocellatum, is one of the most serious diseases affecting marine fish in warm and temperate waters. Current diagnostic methods rely entirely on the microscopic identification of parasites on the skin or gills of infested fish. However, subclinical infestations usually go undetected, while no method of detecting the free-swimming, infective (dinospore) stage has been devised. Targeting the parasite's ribosomal DNA region, we have developed a sensitive and specific PCR assay that can detect as little as a single cell from any of the 3 stages of the parasite's life cycle (trophont, tomont, dinospore). This assay performs equally well in a simple artificial seawater medium and in natural seawater containing a plankton community assemblage. The assay is also not inhibited by gill tissue. Sequence analysis of the internal transcribed spacer region of 5 A. ocellatum isolates, obtained from fish in the Red Sea (Israel), eastern Mediterranean Sea (Israel), Adriatic Sea (Italy), Gulf of Mexico (Florida), and from an unknown origin, revealed insignificant variation, indicating that all isolates were the same species. However, 3 of these isolates propagated in cell culture varied in behavior and morphology, and these differences were consistent during at least 2 yr in culture. Thus, our findings do not eliminate the possibility that different strains are in fact 'subspecies' or lower taxa, which may also differ in pathogenic and immunogenic characteristics, environmental tolerance, and other features.

An in situ assay with the microalga Phaeodactylum tricornutum for sediment‐overlying water toxicity evaluations in estuaries

Given the urgent need for ecologically relevant toxicity tools specific to estuarine systems, the main goal of the present study was to develop and evaluate the potential of an in situ algal assay for estuarine sediment-overlying water toxicity evaluations using the microalga Phaeodactylum tricornutum immobilized in alginate beads. With its simple and cost-effective design, the assay apparatus, coupled with the use of microalgae immobilized in alginate beads, allowed rapid and easy deployment as well as retrieval of assay chambers. The effectiveness of the proposed in situ assay was investigated by deploying it at two reference and six contaminated Portuguese estuaries. By deploying open chambers both with and without an artificial nutrient source and closed chambers with an artificial seawater medium, it was possible to discriminate toxic effects from those caused by differences in temperature, light, and nutrient conditions across sites. A reduction in algal growth was found at contaminated sites relative to reference sites. The strontium alginate beads used for microalgae immobilization were found to be appropriate to perform in situ assays, showing no signs of disruption or dissolution after up to 8 d of exposure in estuarine sediment-overlying waters. The proposed in situ assay was shown to be a useful tool for estuarine sediment-overlying water toxicity testing to provide relevant data concerning primary production to estuarine ecological risk assessment studies.

Marine sponge Craniella austrialiensis-associated bacterial diversity revelation based on 16S rDNA library and biologically active Actinomycetes screening, phylogenetic analysis

The aim of this study was to investigate the bacterial diversity associated with the sponge Craniella australiensis using a molecular strategy and isolating Actinomycetes with antimicrobial potentials. The bacterial diversity associated with South China Sea sponge C. austrialiensis was assessed using a 16S rDNA clone library alongside restriction fragment length polymorphism and phylogenetic analysis. It was found that the C. austrialiensis-associated bacterial community consisted of alpha, beta and gamma-Proteobacteria, Firmicutes, Bacteroidetes as well as Actinobacterium. Actinomycetes were isolated successfully using seawater medium with sponge extracts. According to the BLAST and phylogenetic analysis based on about 600-bp 16S rDNA sequences, 11 of the representative 23 isolates closely matched the Streptomyces sp. while the remaining 12 matched the Actinomycetales. Twenty Actinomycetes have antimicrobial potentials, of which 15 are found to possess broad-spectrum antimicrobial potentials. The sponge C. austrialiensis-associated bacterial community is very abundant including Proteobacteria, Firmicutes, Bacteroidetes and Actinobacterium while Actinomycetes is not predominant. Artificial seawater medium with sponge extracts is suitable for Actinomycetes isolation. Most of the isolated C. austrialiensis-associated Actinomycetes have a broad spectrum of antimicrobial activity. This study revealed the diversity of the bacterial community and the isolated Actinomycetes with antimicrobial potentials associated with sponge C. australiensis.

Chemical defence against bacteria in the red alga Asparagopsis armata: linking structure with function

Although numerous algal products have antimicrobial activity, limited knowledge of metabolite localisation and presentation in algae has meant that ecological roles of algal natural products are not well understood. In this study, extracts of Asparagopsis armata had antibacterial activity against marine (Vibrio spp.) and biomedical (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus spp.) strains. The major natural products in both life-history stages of A. armata (as determined by gas chromatography-mass spectrometry analysis (GC-MS)) were bromoform (0.58 to 4.3% of dry weight (DW)) and dibromoacetic acid (DBA) (0.02 to 2.6% DW), and each compound was active against these same bacteria. To resolve whether this antibiotic activity was ecologically rele- vant, we examined the localisation of metabolites in the specialised cells of A. armata and observed a delivery mechanism for the release of metabolites to the surface. Bromoform and DBA were sub- sequently quantified in the surrounding medium of laboratory cultures, establishing their release from the alga. In a novel ecological test of algal natural products, halogenated metabolites in A. armata were manipulated by omitting bromine from an artificial seawater medium. Significantly higher densities of epiphytic bacteria occurred on algae that no longer produced halogenated metabolites. Both bromoform and DBA were more active against bacteria isolated from algae lacking brominated metabolites than algae producing normal amounts of these compounds. Taken together, these results indicate that halogenated metabolites of A. armata may be important in reducing epiphytic bacterial densities.

Copper requirements for iron acquisition and growth of coastal and oceanic diatoms

Centric diatoms isolated from open ocean environments require higher concentrations of Cu for growth than their coastal counterparts. In artificial seawater medium containing <1 nmol L™1 Cu, three coastal species maintained near maximum rates of growth, but the oceanic clones were unable to survive. Copper limitation was more severe in the diatoms grown in low- than in high-Fe seawater, suggesting that Cu and Fe were interacting essential resources. The interactive effect was in part the result of a Cu requirement for Fe transport. Thalassiosira weissflogii and Thalassiosira oceanica had lower Fe quotas and slower rates of Fe uptake when [Cu] was reduced in the medium. Brief exposure of Cu-limited cells to 10 nmol L−1 Cu increased the instantaneous Fe uptake rate by 1.5 times in T. oceanica. Steady-state uptake rates of both species at high, growth-saturating concentrations of Fe were also Cu dependent. Oceanic species appeared to have an additional Cu requirement that was independent of Fe acquisition and likely responsible for their higher requirements compared to coastal species. Evidence for the importance of Cu in natural communities of phytoplankton was obtained from an incubation experiment performed in the Fe-limited basin of the Bering Sea. Addition of 2 nmol L−1 Cu doubled the phytoplankton net growth rate compared to the untreated controls and, in the presence of extra Fe, increased the growth rate compared to the samples amended with Fe alone. The results suggest that Cu may be an important micronutrient for phytoplankton growth in low-Fe regions of the sea because of its role in Fe acquisition. Paradoxically, oceanic diatoms may be more susceptible to the effects of low Cu concentrations than coastal species.

Laboratory culture studies of Trichodesmium isolated from the Great Barrier Reef Lagoon, Australia

Cultures of Trichodesmium from the Northern and Southern Great Barrier Reef Lagoon (GBRL) have been established in enriched seawater and artificial seawater media. Some cultures have been maintained with active growth for over 6 years. Actively growing cultures in an artificial seawater medium containing organic phosphorus (glycerophosphate) as the principal source of phosphorus have also been established. Key factors that contributed to the successful establishment of cultures were firstly, the seed samples were collected from depth, secondly, samples were thoroughly washed and thirdly, incubations were conducted under relatively low light intensities (PAR similar to 40-50 mumol quanta m(-2) s(-1)). N-2 fixation rates of the cultured Trichodesmium were found to be similar to those measured in the GBRL. Specific growth rates of the cultures during the exponential growth phase in all enriched media were in the range 0.2-0.3 day(-1) and growth during this phase was characterised by individual trichomes (filaments) or small aggregations of two to three trichomes. Characteristic bundle formation tended to occur following the exponential growth phase, which suggests that the bundle formation was induced by a lack of a necessary nutrient e.g. Fe. Results from some exploratory studies showed that filament-dominated cultures of Trichodesmium grew over a range of relatively low irradiances (PAR similar to 5-120 mumol quanta m(-2) s(-1)) with the maximum growth occurring at - 40-50 mumol quanta m(-2) s(-1). These results suggest that filaments of the tested strain are well adapted for growth at depth in marine waters. Other studies showed that growth yields were dependent on salinity, with maximum growth occurring between 30 and 37 psu. Also the cell yields decreased by an order of magnitude with the reduction of Fe additions from 450 to 45 nM. No active growth was observed with the 4.5 nM Fe addition.

Biosynthesis of di-(2-ethylhexyl) phthalate (DEHP) and di- n-butyl phthalate (DBP) from red alga— Bangia atropurpurea

The contents of di-(2-ethylhexyl) phthalate (DEHP) and di- n-butyl phthalate (DBP) of red alga, Bangia atropurpurea, filaments cultured in artificial sea water medium were similar to those cultured in natural sea water medium. In the culture experiment, B. atropurpurea filaments were found to synthesize de novo phthalate esters. Additionally, DEHP and DBP contents in different species of algae grown in the same environment were different significantly, suggesting that it was due to the intrinsic nature of algae.