An efficient method for DNA isolation from red algae

A simple, inexpensive and efficient method was developed for rapid isolation of total genomic DNA from 15 red algal species. It resulted in 0.1 mug high quality DNA from 1 mg fresh algal material, with an A(260)/A(280) ratio of 1.68 - 1.90. Using this rapidly isolated DNA, the 18S ribosomal RNA genes ( rDNA) and the nuclear ribosomal DNA of the internal transcribed spacer (ITS) regions were amplified. The tested DNA was suitable for restriction endonuclease digestion, genetic marker analysis and polymerase chain reaction (PCR) amplification, and may be valid for other genetic manipulation.

Molecular biotechnology of marine algae in China

Molecular biotechnology of marine algae is referred to as the biotechnology on the identification, modification, production and utilization of marine algal molecules. It involves not only the manipulation of macromolecules such as DNA, RNA and proteins, but also deals with low molecular weight compounds such as secondary metabolites. In the last decade, molecular systematic researches to investigate the relationship and to examine the evolutionary divergence among Chinese marine algae have been carried out by Chinese scientists. For example, RAPD has been widely used in several laboratories to elucidate genetic variations of the reds, such as Porphyra, Gracilaria, Grateloupia and the greens such as Ulva and Enteromorpha. Some important data have been obtained. The study on molecular genetic markers for strain improvement is now in progress. In 1990s, genetic engineering of economic seaweeds such as Laminaria, Undaria, Porphyra, Gracilaria and Grateloupia has been studied in China. For Laminaria japonica, the successfully cultivated kelp in China, a model transformation system has been set up based on the application of plant genetic techniques and knowledge of the algal life history. Progress has been made recently in incorporating a vaccine gene into kelp genome. Evidence has been provided showing the expression of gene products as detectable vaccines. In the present paper, the progress of molecular biotechnological studies of marine algae in China, especially researches on elucidating and manipulating nucleic acids of marine algae, are reviewed.

Structural analysis of peptides of PSII light-harvesting complexes in siphonous green algae, Codium fragile

peptide composition and arrangement of 4 major light-harvesting complexes LHCP1-3 and LHCP3, isolated from siphonous green algae (Codium fragile (Sur.) Hariot.) were investigated. LHCP1 showed five main peptides, 34.4, 31.5, 29.5, 28.2 and 26.5 kD in SDS-PAGE, the 34.4 and 31.5 kD peptides were never found in higher plants. LHCP3 contained the other four kinds of LHCP1 peptides except 34.4 kD, while LHCP3, consisted of only 28.2 and 26.5 kD peptides. We found that 34.4, 28.2 and 26.5 kD peptides were easy to decompose from LHCP1 when subjected to SDS-PACE without pretreatment. They might be located at the exterior of LHCP1, while the 31.5 and 29.5 kD peptides were at the central part. The 28.2 and 26.5 kD peptides often occurred in CPa, the center complex of PS II. They are possibly the LHC II peptides tightly associated with CC II. According to the results described above, a peptide map of LHCP1 was sketched.

Characteristics and comparative study of chlorophyll-protein complexes from siphonous green algae

By mild PAGE method, 11, 11, 7 and 9 chlorophyll-protein complexes were isolated from two species of siphonous green algae ( Codium fragile (Sur.) Harlot and Bryopsis corticulans Setch.), green alga (Ulothrix flacca (Dillw.) Thur.), and spinach (Spinacia oleracea Mill.), respectively. Apparent molecular weights, Chi a/b ratios, distribution of chlorophyll, absorption spectra, low temperature fluorescence spectra of these complexes were determined, and compared with one another. PS I complexes of two siphonous green algae are larger in apparent molecular weight because of the attachment of relative highly aggregated LHC I. Four isolated light-harvesting complexes of PSII are all siphonaxanthin-Chl a/b-protein complexes, and they are not monomers and oligomers like those in higher plants. Especially, the absence of 730 nn fluorescence in PS I complexes indicates a distinct structure and energy transfer pattern.

Preparation of chloroplasts from brown algae

Thalli from a brown alga Undaria pinnatifida were soaked by CaCl2 solution with different concentration and time at 4 degreesC, the effect of CaCl2 solution on efficiency and influorescence emission spectra of chloroplasts were examined. The results show that the efficiency of collected chloroplasts is increased markedly after soaking in CaCl2 solution. According to the results of collected efficiency and characteristic of influorescence emission spectra at room temperature of chloroplasts, it was suggested that soaking in the 0.2 mol/L CaCl2 solution for 10 min is optimum. Under this condition, the efficiency of collected chloroplasts is as 5 fold as control group, and the characteristics of chloroplasts obtained by CaCl2 soaking are similar to that of traditional method.

Study on the concentration and seasonal variation of inorganic elements in 35 species of marine algae

The concentrations of five major and 28 trace elements in 35 marine algae collected along the coast of China were determined by instrumental neutron activation analysis. The concentrations of halogens, rare earth elements and many transition metal elements in marine algae are remarkably higher than those in terrestrial plants. The concentration factors for 31 elements in all collected algae were calculated, those for tri- and tetra-valent elements were higher than those of the mono- and di-valent elements in marine algae. The biogeochemical characteristics of inorganic elements in marine algae were investigated. In addition, the seasonal variation of inorganic elements in Sargassum kjellmanianum was also studied. (C) 1998 Elsevier Science B.V. All rights reserved.

Temperature and light tolerance of representative brown, green and red algae in tumble culture revealed by chlorophyll fluorescence measurements

Laminaria japonica, Undaria pinnatifida, Ulva lactuca, Grateloupia turuturu and Palmaria palmata are Suitable species that fit the requirements of a seaweed-animal integrated aquaculture system in terms of their viable biomass, rapid growth and promising nutrient uptake rates. fit this investigation, the responses of the optimal chlorophyll fluorescence yield of the five algal species in tumble Culture were assessed at a temperature range of 10 similar to 30 degrees C. The results revealed that Ulva lactuca was the most resistant species to high temperature, withstanding 30 degrees C for 4 h without apparent decline in the optimal chlorophyll fluorescence yield. While the arctic alga Palmaria palmata was the most vulnerable one, showing significant decline in the optimal chlorophyll fluorescence yield at 25 degrees C for 2 h. The cold-water species Laminaria japonica, however, demonstrated strong ability to cope with higher temperature (24 similar to 26 degrees C) for shorter time (within 24 h) without significant decline in the optimal chlorophyll fluorescence yield. Grateloupia turuturu showed a general decrease in the optimal chlorophyll fluorescence yield with the rising temperature from 23 to 30 degrees C, similar to the temperate kelp Undaria pinnatifida. Changes of chlorophyll fluorescence yields of these algae were characterized differently indicating the existence of species-unique strategy to cope with high light. Measurements of the optimal chlorophyll fluorescence yield after short exposure to direct solar irradiance revealed how long these exposures could be without significant photoinhibition or with promising recovery in photosynthetic activities. Seasonal pattern of alternation of algal species in tank culture in the Northern Hemisphere at the latitude of 36 degrees N was proposed according to these basic measurements.

Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column

A gradient reversed-phase high-performance liquid chromatography (HPLC) method using a C30 column was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus pluvialis and the mutant E1, which was obtained from the mutagenesis of H. pluvialis by exposure to UV-irradiation and ethyl methanesulphonate (EMS) with subsequent screening using nicotine. The results showed that the contents of total astaxanthins including free astaxanthin and astaxanthin esters ranged from 1.4 to 30.9 mg/g dry biomass in these green algae. The lower total astaxanthin levels (< 2 mg/g dry biomass) were detected in the green algae Chlorococcum sp. and C. zofingiensis. The higher total astaxanthin levels (> 16 mg/g dry biomass) were found in the green alga H. pluvialis and its mutant E1. It is notable that the mutant E1 is found to have considerably higher amounts of total astaxanthin (30.9 mg/g) as compared to the wild strain of H. pluvialis (16.1 mg/g). This indicates that UV-irradiation and EMS compound mutagenesis with subsequent screening using nicotine is an effective method for breeding of a high-producing astaxanthin strain of H. pluvialis. In addition, the green alga C. zofingiensis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a remarkably lower percentage of astaxanthin monoesters (18.0% of total astaxanthins) in comparison with H. pluvialis (35.5% for diesters and 60.9% for monoesters), the mutant E1 (49.1% and 48.1%) and Chlorococcum sp. (18.0% and 58.6%).

Evaluation of 28 marine algae from the Qingdao coast for antioxidative capacity and determination of antioxidant efficiency and total phenolic content of fractions and subfractions derived from Symphyocladia latiuscula (Rhodomelaceae)

The extracts obtained from 28 species of marine algae were evaluated for their antioxidant activity (AA) versus the positive controls butylated hydroxytoluene (BHT), gallic acid (GA), and ascorbic acid (AscA). Most of the tested samples displayed antioxidant activity to various degrees. Among them, the extract of Symphyocladia latiuscula exhibited the strongest AA, which was comparable to BHT, GA, and AscA in radical scavenging activity, as shown in the DPPH (alpha,alpha-diphenyl-beta-picrylhydrazyl) assay, and higher than those of the positive controls in beta-carotene-linoleate assay system. In addition, the ethyl acetate-soluble fraction isolated from the crude extract of S. latiuscula exhibited the highest antioxidant activity in both assay systems. This fraction was further fractionated into seven subfractions (F1-F7) by vacuum liquid chromatography (VLC). F1 and F4 were found to be the most effective subfractions in scavenging DPPH radical assay and in the beta-carotene-linoleate assay, respectively. The total phenolic content (TPC) and reducing power (RP) for all of the extracts, fractions, and subfractions (F1-F7) were also determined. The TPC of the 28 extracts ranged from 0.10 to 8.00 gallic acid equivalents (mg/g seaweed dry weight) while the RP ranged from 0.07 to 11.60 ascorbic acid equivalents (mg center dot g(-1) seaweed dry weight). Highly positive relationships between AA and TPC as well as between AA and RP were found for the extracts and fractions, while for the subfractions F1-F7 only weak or no such relations were found. The results obtained from this study indicate that further analysis is needed of those marine algal species that contain the most antioxidant activity in order to identify the active principles.

Phylogenetic studies on two strains of Antarctic ice algae based on morphological and molecular characteristics

The taxonomic characterization of two strains of Antarctic ice algae, Chlamydomonas sp. ICE-L and Chlamydomonas sp. ICE-W, were analyzed on the basis of morphological and molecular traits. The results indicate that they are the same species and belong to Chlamydomonas (Chlorophyta). According to I SS rDNA and ITS-I sequences they are very close relatives of Chlamydomonas sp. Antarctic 2E9, if not identified as such. They belong to the 'monadina clade', Cd. monadina and Cm. subdivisa as the sister group, on the basis of 18S rDNA sequence. They occur in 'Chlamydomonas clade' according to rbcL sequencing and are close relatives of Cd. kuwadae. The ITS sequences of ICE-L and ICE-W are 1302 base pairs and 1300 base pairs in length, the longest Volvocales ITS sequences ever reported.

Application of rotifer Brachionus plicatilis in detecting the toxicity of harmful algae

The toxicity of seven major HAB (harmful algal bloom) species/strains, Prorocentrum donghaiense, Phaeocystis globosa, Prorocentrum micans, Alexandrium tamarense (AT-6, non-PSP producer), Alexandrium lusitanicum, Alexandrum tamarense (ATHK) and Heterosigma akashiwo were studied against rotifer Brachionus plicatilis under laboratory conditions. The results show that P. donghaiense, P. globosa, P. micans, A. tamarense (AT-6), or A. lusitanicum could maintain the individual survival and reproduction, as well as the population increase of the rotifer, but the individual reproduction would decrease when exposed to these five algae at higher densities for nine days; H. akashiwo could decrease the individual survival and reproduction, as well as population increase of the rotifer, which is similar to that of the starvation group, indicating that starvation might be its one lethal factor except for the algal toxins; A. tamarense (ATHK) has strong lethal effect on the rotifer with 48h LC50 at 800 cells/mL. The experiment on ingestion ability indicated by gut pigment change shows that P. donghaiense, P. globosa, P. micans, A. tamarense (AT-6) and A. lusitanicum can be taken by the rotifers as food, but A. tamarense (ATHK) or H. akashiwo can be ingested by the rotifers. The results indicate that all the indexes of individual survival and reproduction, population increase, gut pigment change of the rotifers are good and convenient to be used to reflect the toxicities of HAB species. Therefore, rotifer is suggested as one of the toxicity testing organisms in detecting the toxicity of harmful algae.

A modified HPLC method for analysis of PSP toxins in algae and shellfish from China

Improvements to an established HPLC method are introduced. The modified method is more efficient for separation and detection of the toxins responsible for paralytic shellfish poisoning (PSP). The PSP toxin content of two strains of Alexandrium tamarense and approximately forty shellfish samples collected from different locations in China have been analyzed with this HPLC method. Only one shellfish sample, collected from Lianyungang, China, contained PSP toxins.

The influence of diets containing dried bivalve feces and/or powdered algae on growth and energy distribution in sea cucumber Apostichopus japonicus (Selenka) (Echinodermata : Holothuroidea)

In recent years, bivalve feces and powdered algae have been used as the food sources of holothurians in China. In this study, growth and energy budget for sea cucumber Apostichopus japonicus (Selenka) with initial wet body,veights of 32.5 1.0 g (mean +/- SE, n=45) when fed with five different granule diets containing dried bivalve feces and/or powdered algae in water temperature 13.2-19.8 degrees C and salinity 30-32ppt were quantified in order to investigate how diets influence growth and energy distribution and to find out the proper diet for land-based intensive culture of this species. Results showed that diets affected the food ingestion, feces production, food conversion efficiency and apparent digestive ratios, hence the growth and energy budget. Sea cucumbers fed with dried feces of bivalve showed poorer energy absorption, assimilation and growth than individuals fed with other four diets; this could be because feces-drying process removed much of the benefits. Dried bivalve feces alone, therefore, were not a suitable diet for sea cucumbers in intensive cultivation. The mixed diets of feces and powered algae showed promising results for cultivation of sub-adult Apostichopus japonicus, while animals fed with powdered algae alone, could not obtain the best growth. According to SGR of tested animals, a formula of 75% feces and 25% powdered algae is the best diet for culture of this species. Extruded diets were used in the present experiment to overcome shortcomings of the traditional powdered feeds, however, it seems a conflict exists between drying bivalve feces to form extruded diets and feeding sea cucumbers with fresh feces which contain beneficial bacteria. Compared with other echinoderms, in holothurians the energy deposited in growth is lower and the energy loss in feces accounts for the majority of the ingested energy. Such detailed information could be helpful in further development of more appropriate diets for culture of holothurians. (c) 2006 Elsevier B.V. All rights reserved.

The effect of the causative algae of large-scale HAB in the East China Sea on egg hatching of Argopecten irradians, and population growth of Brachionus plicatilis and Moina mongolica

The impacts of Prorocentrum donghaiense Lu and Alexandrium catenella Balech, causative species of the large-scale HAB in the East China Sea, were studied under laboratory conditions. According to bloom densities, the effects of monoculture and mixture of the two species were examined on the egg-hatching success of Argopecten irradians Lamarck, and the population growth of Brachionus plicatilis Muller and Moina mongolica Daday. The results showed that monoculture of A. catenella had a significant inhibition on the egg hatching success of A. irradians, and the population growth of B. plicatilis and M. mongolica. The median effective densities ( EDSo) inhibiting the egg hatching success of A. irradians for 24 h and the population growth of B. plicatilis and M. mongolica for 96 h were 800, 630, and 2 400 cells/cm(3), respectively. Monoculture of P. donghaiense has no such inhibitory effect on the egg hatching success of A. irradians; P. donghaiense at lower suitable densities could sustain the population growth of B. plicatilis (1 x 10(4) similar to 3 x 10(4)cells/cm(3)) and M. mongolica (2 x 10(4) similar to 5 x 10(4) cells/cm(3)); P. doaghaiense at higher densities had significantly adverse effect on the population growth of B. plicatilis (4 x 10(4) similar to 10 x 10(4) cells/cm(3)) and M. mongolica (10 x 10(4) cells/cm(3)). When the two algae were mixed according to bloom densities, P. donghaiense at suitable densities to some extent could decrease the toxicity of A. catenella to B. plicatilis and M. mongolica. The results indicated that the large-scale HAB in the East China Sea could have adverse effect on zooplankton, and might further influence the marine ecosystem, especially when there was also Alexandrium bloom.

Biosorption of Cr(VI) from aqueous solutions by nonliving green algae Cladophora albida

Biosorption of Cr(VI) from aqueous solutions by nonliving green algae Cladophora albida was investigated in batch experiments. The influence of pH, algal dosage, initial Cr(VI) concentration, temperature and coexisting anions on removal efficiencies of C. albida was studied. Cr(VI) removal process was influenced significantly by the variation of pH, and the optimum pH was chosen at a range of 1.0-3.0. The optimum algal dosage 2 g/L was used in the experiment. The removal rate of Cr(VI) was relatively rapid in the first 60 min, but then the rate decreased gradually. Removal mechanism was studied by analyzing Cr(VI) and total Cr in the solution. Biosorption and bioreduction were involved in the Cr(VI) removal. Biosorption of Cr(VI) was the first step. followed by Cr(VI) bioreduction and Cr(III) biosorption on the algal biomass. Actual industrial wastewater was used to evaluate the practicality of the biomass C. albida. From a practical viewpoint, the abundant and economic biomass C. albida could be used for removal of Cr(VI) from wastewater by the reduction of toxic Cr(VI) to less toxic Cr(III). (C) 2008 Elsevier Ltd. All rights reserved.