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.

The past, present and future of phycology in China

Algae have been part of Chinese life for thousands of years. They are widely used as food and have been cited in Chinese literature as early as 2500 years ago. However, formal taxonomic studies on Chinese algae were initiated by foreign scientists only about 200 years ago, and by Chinese phycologists only about 90 years ago. This paper summarizes the history of modern phycological studies on Chinese algae and provides an overview of the achievements of phycological studies by Chinese scientists, especially on algal taxonomy, morphology, genetics, ecology and environmental research, physiology, biotechnology, algal culture, applied phycology and space phycology, in the last century. Recent development in phycological research focuses on algal floristic and molecular systematics, algal molecular biotechnology, applied phycology including micro and macroalgal cultivation and algal product development, and the roles of algae in environmental pollution control. These areas will also be the main focuses of Chinese phycological research in the foreseeable future.

Review of genetic engineering of Laminaria japonica (Laminariales, Phaeophyta) in China

Progress has been made in establishing a genetic transformation model for Laminaria japonica (Phaeophyta, Laminariales). The model includes introduction of foreign genes by biolistic bombardment, use of promoter SV40 to drive gene expression, algal regeneration by parthenogenesis and selection by chloramphenicol or hygromycin.

Integrated cultivation of the red alga Kappaphycus alvarezii and the pearl oyster Pinctada martensi

The effect of simultaneously cultivating the pearl oyster Pinctada martensi and the red alga Kappaphycus alvarezii on growth rates of both species was investigated in laboratory and field studies conducted from December 1993 to June 1995. The two study sites were in subtidal areas 100 km apart off the east coast of Hainan Island, China. Pearl oysters were cultivated in the center of an algal farm and red alga was cultivated in the center of the pearl oyster farm. These field experiments showed higher growth rates of both P. martensi and K. alvarezii in a co-culture system than in a monospecies culture system. Laboratory studies showed that the algae removed nitrogenous wastes released by pearl oysters. Algae treated with pearl oyster wastes grew much faster than those without oyster wastes. Algae treated with the seawater to which NH4Cl, NaNO3 and NaNO2 were added grew at the same rate as those treated with natural seawater containing oyster nitrogenous wastes, suggesting that enhanced growth of algae in the co-culture system was largely due to nitrogenous metabolites of the pearl oysters. In the co-culture, growth of pearl oysters was positively influenced by the presence of rapidly growing algae but when seawater temperature decreased below 20 degrees C, the algae grew slowly and there was no measurable benefit of mixed culture to either algae or pearl oyster.

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.

Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)

Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo. (C) 2008 Elsevier B.V. All rights reserved.

Morphological and phylogenetic analysis of a Gymnodinium-like species from the Chinese Coast

A Gymnodinium-like species was studied with light microscopy (LM) and scanning electron microscopy (SEM). Also, the internal transcribed spacers (containing 5.8S rDNA) and large ribosomal subunit DNA (D1-D2) sequences were obtained by PCR amplification, and then sequenced to explore the relationships within our isolate, Gymnodinium and other Gymnodinium-like species, including Karenia, Gyrodinium, Karlodinium and Symbiodinium. The LM observation showed that the species was characterized by moving in a levorotatory direction, visible hypocone, epicone and transverse groove, all of which are typical for Gymnodinium. In addition, two flagella could be found under SEM. The phylogenetic analysis revealed that the isolate grouped with Symbiodium, rather than other relevant dinoflagellates. All results showed our isolate belongs to Symbiodium. The strain was isolated from a red tide water sample, denoting that Symbiodium may be causative species for algal bloom.

Effect of temperature and irradiance on the growth and reproduction of Enteromorpha prolifera J. Ag. (Chlorophycophyta, Chlorophyceae)

Effect of temperature and irradiance on growth and reproduction of Enteromorpha prolifera that bloomed offshore along the Qingdao coast in summer 2008, was studied. It was showed that E. prolifera propagated mainly asexually with specific growth rate (SGR) of 10.47 at 25A degrees C/40 mu mol m(-2)s(-1). Under this condition, gametes with two flagellate formed and released in 5 days. At the beginning of the development, the unicell gamete divided into two cells with heteropolarity, and then the apical cell developed into thalli primordial cells, whereas the basal cell developed into rhizoid primordial cells. In 8-day culture, the monoplast gamete developed into juvenile germling of 240 mu m in length. Unreleased gametes can develop directly within the alga body. E. prolifera could either reproduce through lateral branching or fragmenting except apomixis revealed by Microscopic observation. On aged tissue of E. prolifera, although the degraded pigments partially remained in faded algal filaments, numerous vegetative cells could still divide actively in the algal tissues.

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.

Effect of iron on growth and lipid accumulation in Chlorella vulgaris

The economic feasibility of algal mass culture for biodiesel production is enhanced by the increase in biomass productivity and storage lipids. Effect of iron on growth and lipid accumulation in marine microalgae Chlorella vulgaris were investigated. In experiment I, supplementing the growth media with chelated FeCl3 in the late growth phase increased the final cell density but did not induce lipid accumulation in cells. In experiment II, cells in the late-exponential growth phase were collected by centrifugation and re-inoculated into new media supplemented with five levels of Fe3+ concentration. Total lipid content in cultures supplemented with 1.2 x 10(-5) mol L-1 FeCl3 was up to 56.6% biomass by dry weight and was 3-7-fold that in other media supplemented with lower iron concentration. Moreover, a simple and rapid method determining the lipid accumulation in C. vulgaris with spectrofluorimetry was developed. (c) 2007 Elsevier Ltd. 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.

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.

Investigations of the characteristics and mode of action of an algalytic bacterium isolated from Tai Lake

An algalytic bacterium provisionally designated as TL1 was isolated from Tai Lake, a large freshwater lake in the Yangtze Delta plain on the border of the Jiangsu and Zhejiang provinces and close to Wuxi city in the People's Republic of China. Strain TL1 was identified as Achromobacter sp. based on its biophysical and biochemical properties and the analysis of its 16S rRNA sequence. Microcystis aeruginosa, which is the most common toxic cyanobacterium in eutrophic freshwater, could be decomposed by strain TL1. The results showed that after inoculation with the algalytic bacterium, the content of chlorophyll-a, maximum PSII quantum yield, and maximum electron transport rates of the alga decreased sharply. At first, the algal cells enhanced the activities of some antioxidative enzymes, but subsequently, the activities of antioxidative enzymes fell sharply once damage of the algal cells was achieved. The filtrate from strain TL1 culture suspension, after autoclaving and treatments with proteinase K, strongly inhibited algal growth, indicating that the lytic metabolites were extracellular and thermostable, not a protein.

Molecular cloning, characterization and evolutionary analysis of phytoene desaturase (PDS) gene from Haematococcus pluvialis

Phytoene desaturase is one of the most important enzymes necessary for the biosynthesis of carotenoids in some cyanobacteria, green algae and plants. In this study, genomic DNA and cDNA of pds were cloned from unicellular green alga Haematococcus pluvialis strain323 using PCR and RT-PCR methods. The cDNA was cloned into plasmid pET-28a and efficiently expressed in Escherichia coli BL21. The complete genomic PDS gene of H. pluvialis, 3.3 kb in size, included eight exons and seven introns. To locate transcriptional regulation elements, an approximate 1 kb of 5'-flanking region was isolated by genome-walking method. Results of bioinformatic analysis showed several putative cis-elements e.g. the ABRE motif (abscisic acid responsive element), the C-repeat/DRE (dehydration responsive element) motif and the GCN4 motif were located in 5'-flanking region of pds. Results of phylogenetic analyses reveal that different sources of PDS genes form a separate clade, respectively, with 100% bootstrap support. Moreover, a maximum likelihood approach was employed to detect evidence of positive selection in the evolution of PDS genes. Results of branch-site model analysis suggest that 7.9% of sites along the green algal branch are under positive selection, and the PDS gene in green algae exhibits a different evolutionary pattern from its counterparts in cyanobacteria and plants.

Dynamic changes of total bacteria and Vibrio in an integrated seaweed-abalone culture system

Polyculture of seaweeds alongside fed animal aquaculture is an environmentally friendly means of avoiding eutrophication problem both in land-based and sea-based monoculture systems. Many aspects of such polyculture systems have been described, but little attention has been given to the impact of live seaweeds on the microbiological properties of the water that connects the algae and animals. In this investigation, the Pacific red alga Gracilaria textorii was cultured in a recirculated dual tank system (150 L) with the juvenile abalone Haliotis discus hannai. Dynamic changes of total bacteria (TB) and total Vibrio (TV) in the water of polyculture and monoculture systems were evaluated. Results revealed that (1) level of TB in the polyculture was constantly higher than in the monoculture over a 6.5-day period. While levels of TV in the polyculture was detected to be constantly lower than in the monoculture, (2) integration of G. textorii in the abalone culture changed the Vibrio compositions in the water as judged by the changes of bacteria colony types; (3) application of artificial diet led to dramatic increase of the levels in TB and TV in both systems at 12 h after application in the 24-h test and resulted in selective propagation of Vibrio in the water in the monoculture system; (4) polyculture of G. textorii with juvenile abalone in combination with feeding with live algal diet helped to maintain low levels of TV and the balance of the Vibrio composition; (5) living biomass of G. textorii was effective in preventing propagation of two purified Vibrio strains (V alginolaticus and V logei) in the water. These results provide a general basis of the dynamic changes of levels in TB and TV in a seaweed-abalone polyculture system with or without artificial diet in tanks. (c) 2005 Elsevier B.V All rights reserved.