Seeding nets with neutral spores of the red alga Porphyra umbilicalis (L.) Kutzing for use in integrated multi-trophic aquaculture (IMTA)

Nets in traditional Porphyra mariculture are seeded with conchospores derived from the conchocelis phase, and spend a nursery period in culture tanks or calm coastal waters until they reach several centimeters in length. Some species of Porphyra can regenerate the foliose phase directly through asexual reproduction, which suggests that the time, infrastructure, and costs associated with conchocelis culture might be avoided by seeding nets with asexual spores. Here, we present work from a short-term mariculture study using nets seeded with asexual spores (neutral spores) of a native Maine species of Porphyra. Porphyra umbilicalis (L.) Kutzing was selected for this proof of concept research because of its reproductive biology, abundance across seasons in Maine, and evidence of its promise as a mariculture crop. We studied the maturation, release, and germination of the neutral spores to develop an appropriate seeding protocol for nets, followed by development of a nursery raceway to provide an easily manipulated environment for the seeded nets. Neutral spores were produced throughout the year on the central Maine coast,however, there was a temporal variability in the number and survival of released neutral spores, depending upon thallus position in the intertidal zone. Small thalli were strictly vegetative, but most thalli reproduced by neutral spores- sexual reproduction was absent. Neutral spores germinated quickly at 10 and 15 'C, but germination was delayed at 5 degrees C. Unlike some algal zygotes and spores, neutral spores of R umbilicalis required light to germinate; however, irradiances of 25 and 100 mu mol photons M-2 S-1 were equally sufficient for germination. Rafts of seeded nets were deployed in Cobscook Bay, Maine, at two distances from salmon aquaculture pens and at a control site on a nearby, fallow aquaculture site (no salmon). There was no difference in nitrogen content of harvested thalli; however, both the density and the surface area of harvested thalli were different among the sites. The possible causes of these differences are discussed in the context of potential use of P umbilicalis in IMTA. (C) 2007 Elsevier B.V. All rights reserved.

Isolation, antimicrobial activity, and metabolites of fungus Cladosporium sp associated with red alga Porphyra yezoensis

Cladosporium sp. isolate N5 was isolated as a dominant fungus from the healthy conchocelis of Porphyra yezoensis. In the re-infection test, it did not cause any pathogenic symptoms in the alga. Twenty-one cultural conditions were chosen to test its antimicrobial activity in order to obtain the best condition for large-scale fermentation. Phenylacetic acid, p-hydroxyphenylethyl alcohol, and L-beta-phenyllactic acid were isolated from the crude extract as strong antimicrobial compounds and they are the first reported secondary metabolites for the genus Cladosporium. In addition, the Cladosporium sp. produced the reported Porphyra yezoensis growth regulators phenylacetic acid and p-hydroxyphenylacetic acid. No cytotoxicity was found in the brine shrimp lethality test, which indicated that the environmental-friendly Cladosporium sp. could be used as a potential biocontrol agent to protect the alga from pathogens.

Purification of High-quality Plastid DNA from the Brown Alga Laminaria japonica Sporophytes

Extracting DNA from a variety of algae is rather difficult because of high levels of polysaccharides, tannins, and phenolics as these interfere with DNA isolation and downstream applications. High-quality plastid DNA (ptDNA) purification is particularly difficult because of its small proportion in total genomic DNA. This report describes an improved protocol for ptDNA purification that efficiently produces high-quality ptDNA from sporophytes of Laminaria japonica and several other algae. This improved protocol simplifies procedures for ptDNA purification and improves yield to 150-200 mu g of ptDNA per 100 g of frozen algal tissue. Polymerase chain reaction (PCR) amplification of conserved sequences has been used to verify purity of the ptDNA product.

Molecular phylogenetic analysis of attached Ulvaceae species and free-floating Enteromorpha from Qingdao coasts in 2007

Based on the sequence data of the nuclear ribosomal DNA internal transcribed spacer (ITS) 1, 5.8 S, and ITS 2, the molecular phylogeny was analyzed on Ulvaceae species collected from Qingdao coasts in summer of 2007, including 15 attached Ulva and Enteromorpha samples from 10 locations and 10 free-floating Enteromorpha samples from seven locations. The result supported the monophyly of all free-floating Enteromorpha samples, implying the unialgal composition of the free-floating Enteromorpha, and the attached Ulvaceae species from Qingdao coasts were grouped into other five clades, suggesting that they were not the biogeographic origin of the free-floating Enteromorpha in that season.

Induction and characterization of green pigmentation mutant in Porphyra yezoensis Ueda

The free living conchocelis of Porphyra yezoensis Ueda was treated with N-methyl-N-nitro-N-nitrosoguanidine to induce pigmentation mutants. The artificial green pigmentation mutant of P. yezoensis conchocelis, which was composed entirely of green cells, was isolated through visualization with the unaided eye. The acquired green conchocelis was further developed into a green gametophytic blade. This mutant was relatively stable in color in both gametophytic blade and conchocelis phases. The gametophytic blade mutant was successively cultivated for commerce at some Porphyra farms in Rudong, China, and few wild type or sectorially variegated gametophytic blade occurred, indicating that the green mutant has commercial value. The green mutant was characterized as having lower phycoerythrin and higher phycocyanin content, and SDS-PAGE suggested that phycoerythrin was missing the gamma-subunit in comparison to the wild type. The wild type and the green mutant showed a clear difference in 02 evolution rates in white, green, yellow, and red light, which might be due to the qualitative and quantitative changes of phycoerythrin, and the quantitative difference of phycocyanin. (C) 2008 Elsevier B.V. All rights reserved.

Natural bromophenols from the marine red alga Polysiphonia urceolata (Rhodomelaceae): Structural elucidation and DPPH radical-scavenging activity

Three new natural occurring bromophenols, 3-(3-bromo-4,5-dihydroxyphenyl)-2-(3,5-dibromo-4-hydroxyphenyl)propionic acid (1), (E)-4-(3-bromo-4,5-dihydroxyphenyl)-but-3-en-2-one (2), and (3,5-dibromo-4-hydroxyphenyl) acetic acid butyl ester (3), together with one known bromophenol, 1,2-bis(3-bromo-4,5-dihydroxyphenyl)ethane (4), were isolated and identified from the marine red alga Polysiphonia urceolata. The structures of these compounds were elucidated by extensive analysis of ID and 2D NMR and IR spectra and MS data. Each of the isolated compounds was evaluated for scavenging alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical activity and all of them exhibited significant activity with IC50 values ranging from 9.67 to 21.90 mu M, compared to the positive control, a well-known antioxidant butylated hydroxytoluene (BHT), with IC50 83.84 mu M. (C) 2007 Elsevier Ltd. All rights reserved.

Cultivation of the brown alga Hizikia fusiformis (Harvey) Okamura: stress resistance of artificially raised young seedlings revealed by chlorophyll fluorescence measurement

Commercial farming of the intertidal brown alga Hizikia fusiformis (Harvey) Okamura in China and South Korea in the sea depends on three sources of seedlings: holdfast-derived regenerated seedlings, young plants from wild population and zygote-derived seedlings. Like many successfully farmed seaweed species, the sustainable development of Hizikia farming will rely on a stable supply of artificial seedlings via sexual reproduction under controlled conditions. However, the high rate of detachment of seedlings after transfer to open sea is one of the main obstacles, and has limited large-scale application of zygote-derived seedlings. To seek the optimal condition for growing seedlings on substratum in land-based tanks for avoidance of detachment in this investigation, young seedlings were grown in both outdoor tanks exposed directly to sunlight and in indoor raceway tanks in reduced, filtered sunlight. Results showed that young seedlings, immediately after fertilization, could withstand a daily fluctuation of direct solar irradiance up to a level of 1800 mu mol photons m(-1)s(-1), and maintained a faster growth rate than seedlings grown in indoor tanks. Detailed experiments by use of chlorophyll fluorescence measurements further demonstrated that the overnight (12 h) recovery of optimal fluorescence quantum yield (F-v/F-m) of seedlings after 1 h treatment at 40 degrees C was 98%, and the 48 h recovery of F-v/F-m of seedlings after 1 h exposure to 1800 mu mol m(-2)s(-1) was 92%. Forty-one-day-old seedlings showed no significant decrease of optimal fluorescence quantum yield at salinity ranging from 30 to 5 ppt for a treatment up to 17 h. Six-hour desiccation treatment did not have any influence on the optimal fluorescence quantum yield. Exposure to 18 mmol L-1 sodium hypochlorite for 10 min did not damage the PSII efficiency, and thus could be used to remove epiphytic algae. The strong tolerance of young seedlings to high temperature, high irradiance, low salinity and desiccation found in this investigation supports the view that mass production of Hizikia seedlings should be performed in ambient light and temperature instead of in shaded greenhouse tanks.

Circadian rhythms in the growth and reproduction of the brown alga Undaria pinnatifida and gametogenesis under different photoperiods

Circadian growth rhythm of the juvenile sporophyte of the brown alga Undaria pirznatifida was measured with the computer-aided image analysis system in constant florescent white light under constant temperature (10 degrees C). The growth rhythm persisted for 4 d in constant light with a free-running period of 25. 6 h. Egg release from filamentous gametophytes pre-cultured in the light - dark regime was evaluated for six consecutive days at fixed time intervals in constant white light and 12 h light per day. Egg release rhythm persisted for 3 d in both regimes, indicating the endogenous nature. Temporal scale of egg release and gametogenesis in 18, 16, 12 and 8 h light per day were evaluated respectively using vegetatively propagated filamentous gametophytes. Egg release occurred 2 h after the onset of dark phase and peaked at midnight. Evaluation of the rates of oogonium formation, egg release or fertilization revealed no significant differences in four light-dark regimes, indicating; the great plasticity of sexual reproduction. No photoperiodic effect in gametogenesis in terms of oogonium formation and egg release was found, but fertilization in short days was significantly higher than in long days. Results of this investigation further confirmed the general occurrence of circadian rhythms in inter-tidal seaweed species.

Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata

Antioxidant activity (AA), total phenolic content, and reducing power of the crude extract, fractions, and subfractions derived from a red alga, Polysiphonia urceolata, were evaluated and determined. The antioxidative activity was measured using the alpha,alpha-diphenyl-beta-pierylhydrazyl (DPPH) radical scavenging assay and the P-carotene-linoleate assay systems, and compared with that of butylated hydroxytoluene (BHT), gallic acid (GA), and ascorbic acid (AscA). The results showed that the crude extract and the ethyl acetate-soluble fraction exhibited higher AA than BHT in the DPPH assay model, at all of four concentration levels tested (from 0.4 to 50 mu g/ml), while, in the beta-carotene-linoleate assay system, the crude extract and the ethyl acetate-soluble fraction exhibited similar or, in most cases, higher AA than GA and AscA at the same concentrations (from 10 to 200 mu g/ml). The ethyl acetate-soluble fraction was further fractionated into seven subfractions F1-F7 by silica gel vacuum liquid chromatography. F1 was found to be the most effective subfraction in both assay systems. The total phenolic content and reducing power were determined using the Folin-Ciocalteu and the potassium ferricyanide reduction methods, respectively. Statistical analysis indicated a significant association between the antioxidant potency and total phenolic content as well as between the antioxidant potency and reducing power. (c) 2005 Elsevier Ltd. All rights reserved.

Isolation and characterization of the oxygen-evolving photosystem II complex from the economical red alga Bangia fusco-purpurea

The highly pure and active photosystem II (PSII) complex was isolated from Bangia fusco-purpurea (Dillw) Lyngb., an important economic red alga in China, through two steps of sucrose density gradient ultracentrifugation and characterized by the room absorption and fluorescence emission spectra, DCIP (2,6-dichloroindophenol) reduction, and oxygen evolution rates. The PSII complex from B. fusco-purpurea had the characteristic absorption peaks of chlorophyll (Chl) a (436 and 676 nm) and typical fluorescence emission peak at 685 nm (Ex = 436 nm). Moreover, the acquired PSII complex displayed high oxygen evolution (139 mu mol O-2/(mg Chl h) in the presence of 2.5 mM 2,6-dimethybenzoqinone as an artificial acceptor and was active in photoreduction of DCIP (2,6-dichloroindophenol) by DPC (1,5-diphenylcarbazide) at 163 U/(mg Chl a h). SDS-PAGE also suggested that the purified PSII complex contained four intrinsic proteins (D1, D2, CP43, and CP47) and four extrinsic proteins (33-kD protein, 20-kD protein, cyt c-550, and 14-kD protein).

Laurenidificin, a new brominated C-15-acetogenin from the marine red alga Laurencia nidifica

new brominated C-15-acetogenin, namely, laurenidificin, was isolated from the marine red alga Laurencia nidifica. Its structure was determined on the basis of spectroscopic methods. (C) 2010 Bin Gui Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Method for large-scale isolation and purification of R-phycoerythrin from red alga Polysiphonia urceolata Grev

R-phycoerythrin was isolated and purified from a red alga, Polysiphonia urceolata Grev, using Streamline column combined with ion-exchange chromatography or hydroxyapatite chromatography. The purity of R-phycoerythrin isolated by Streamline column was up to 1.66 and the yield of R-phycoerythrin could be as high as 0.68 mg/g frozen P. urceolata. All the eluates from Streamline column were divided into two equivalent parts, respectively. One part was pumped into the ion-exchange column loaded with Q-Sepharose and the other was applied to the adsorption column loaded with hydroxyapatite. The purities of R-phycoerythrin purified using these two methods were both up to 3.26, more than 3.2 the commonly accepted criterion. The yield of purified R-phycoerythrin from the ion-exchange chromatography was 0.40 mg/g frozen P. urceolata and that from the hydroxyapatite chromatography could reach 0.34 mg/g frozen P. urceolata. The purified protein had three absorption peaks at 498, 535, and 565 nm and displayed a fluorescence maximum at 580 nm, which was consistent with the typical spectrum of R-phycoerythrin. The purified R-PE was also identified with electrophoresis. Only one single protein band appeared on native-PAGE with silver staining. SDS-PAGE demonstrated the presence of one 20 kDa major subunit, and one low intensity band corresponding to 33 kDa subunit. The results indicate that using the expanded bed adsorption combined with ion-exchange chromatography or hydroxyapatite chromatography, R-phycoerythrin can be purified from frozen P. urceolata on large scale. (c) 2006 Elsevier Inc. All rights reserved.

Cultivation of the brown alga Hizikia fusiformis (Harvey) Okamura: controlled fertilization and early development of seedlings in raceway tanks in ambient light and temperature

Commercial cultivation of the dioecious brown macroalga Hizikia fusiformis (Harvey) Okamura in East Asia depends on the supply of young seedlings from regenerated holdfasts or from wild population. Recent development of synchronized release of male and female gametes in tumble culture provides a possibility of mass production of young seedlings via sexual reproduction. In this paper, we demonstrate that controlled fertilization can be efficiently realized in ambient light and temperature in a specially designed raceway tank in which the sperm-containing water has been recirculated. The effective fertilization time of eggs by sperm was found to be within six hours. Fast growth and development of the young seedlings relied on the presence of water currents. Velocity tests demonstrated that young seedlings of 2-3 mm in length could withstand a water current of 190 cm s(-1) stop without detachment. Culture experiments at 24 h postfertilization showed that elongation of both the seedlings and their rhizoids were not hampered by high irradiance up to 600 mu mol photons m(-2) stop s(-1) stop. However, growth was slightly retarded if cultured at a temperature of 16 degrees C compared to other culture temperatures of 22, 25 and 29 degrees C. No seedling detachment was observed after transfer of the young seedlings to raft cultivation in the sea after one and 1.5 months post-fertilization, indicating the feasibility of obtaining large quantity of seedlings in such a system.