The agar component of the red seaweed Gelidium purpurascens

Chemical composition and rheological properties of agar isolated from Gelidium purpurascens, the agar after alkaline treatment, and a commercial agar are presented. The agar and alkali-treated agar gave weaker gels, as measured with an Instron 1122, than those of commercial agar. Xylose, glucose and glucuronic acid in the agar were removed together with 86% of the nitrogen content on alkaline treatment, indicating occurrence of these residues as carbohydrate-protein complexes. Sequential extraction of the alga accounted for low yields of agar as losses incurred on ethanol precipitation. Acid treatment of the residue from exhaustive aqueous extraction of the alga liberated a further 10% agar with increased gel strengths despite increased glucose inclusion, suggesting a lack of involvement of these ‘contaminant’ carbohydrate-protein residues in helix coil formation during gelling.     

Optimization and scale-up of a new photobleaching agar extraction process from Gracilaria lemaneiformis

An eco-friendly photobleaching extraction process for agar extraction from the red alga Gracilaria lemaneiformis was developed for the benefit of workers’ health and environmental safety. Here we report the optimization of key process parameters (alkali modification concentration, photobleaching duration, algal length and screen filter opening size) in order to scale up this new technique. The optimal conditions were found to be modification by 3–5% NaOH, photobleaching for 5 h, using algal fragments 2 –4 cm in length, and a filter screen with a 6 μm opening. A 20-L agar extraction reactor was thus constructed, and the scale-up of the agar extraction process was tested in six batch experiments. The resulting agar quality was similar to that of the laboratory-scale extraction. In addition, batch-to-batch reproducibility was excellent. The results demonstrate the excellent scale-up ability and potential application of this new photobleaching agar extraction process on a commercial scale. The agar yield and gel strength for 5% NaOH modified agar were 26.8% and 1,897 g cm⁻², while those for 3% NaOH modified agar were 28.2% and 1,287 g cm⁻², respectively. It is clear that the agar yield and quality can be manipulated via alkali modification in this new eco-friendly extraction to meet market demands.     

Relationship between gene expression of UDP-glucose pyrophosphorylase and agar yield in Gracilariopsis lemaneiformis (Rhodophyta)

UDP-glucose pyrophosphorylase (UGPase) is an enzyme involved in the biosynthesis of UDP-D-galactose, a subunit of agar in red seaweeds. The relationship between agar content and expression levels of the UGPase encoding gene (glugp) was studied in thalli under different treatment conditions using a quantitative real-time PCR-based method (qPCR). Moreover, this qPCR method for the measurement of glugp expression was also applied to commercial varieties of Gracilariopsis lemaneiformis, a red macroalga, in order to examine its reliability on material obtained from field cultivation. Both the agar content and glugp gene expression in G. lemaneiformis grown under low salinity (17?‰) conditions for 1 week showed a slight increase in comparison with the control group (33?‰ salinity, natural salinity of seawater), but the difference was not statistically significant (P?>?0.05). However, when the culture time was extended to 2 weeks, the increase in both the agar yield and glugp expression became significant (P?glugp expression (P?>?0.05). Our results suggest that glugp gene expression and agar content are highly positively correlated and that the measurement of glugp expression, using only a small amount of thalli material, may be an efficient approach to evaluate agar content. In addition, both the agar content and glugp expression in cultivars 981, 07-2, and ZC differed significantly from those of MT-18. The findings of this study suggest that UGPase may be involved in agar biosynthesis and indicate that glugp gene expression could be a fairly reliable molecular marker to reflect the agar content of strains during breeding and selection of G. lemaneiformis.     

Growth,pigments, UV-absorbing compounds and agar yield of the economic red seaweed <Emphasis Type="Italic">Gracilaria lemaneiformis</Emphasis> (Rhodophyta) grown at different depths in the coastal waters of the South China Sea

The economic red alga, Gracilaria lemaneiformis Bory, was grown at different depths in the coastal waters of the South China Sea, and its growth, pigments, ultra-violet (UV)-absorbing compounds and agar yield were investigated in order to see the impacts of depth change. Gracilaria lemaneiformis grew slower at greater depths in March, while the highest relative growth rate (RGR) was found at about 1.0 m depth in April, about 9% higher than that at surface water (0.5 m below the surface). The RGR increased with the increasing daily photosynthetically active radiation (PAR) dose received by the thalli at different depths. The contents of phycoerythrin and chlorophyll a increased, while that of UV-absorbing compounds (UVAC, absorption peak at 325 nm) decreased with increased depth. The highest levels of the UVAC in the thalli grown in surface seawater played a protective role against solar UV radiation (280–400 nm). The content of UVAC declined at deeper depths and under indoor low PAR. The agar yield of the thalli increased with the increasing depths, with the highest content found at 3.5 m depth.     

两种抗生素对龙须菜的光合生理效应

探讨了不同浓度的两种抗生素(氯霉素和青霉素G钠)对龙须菜(Gracilaria lemaneiformis)生长、光合作用、呼吸作用、色素含量以及可溶性蛋白含量等生理特性的影响。结果表明:龙须菜的生长受到两种抗生素的影响,但是氯霉素的影响要比青霉素G钠的影响大的多。在氯霉素处理的过程中,光合作用、有效光化学效率(Yield)、藻红藻蓝蛋白以及可溶性蛋白含量都随着氯霉素浓度的升高而显著下降,但是呼吸作用速率由于氯霉素的处理而升高;此外光合色素含量不受氯霉素的影响。在青霉素G钠的处理中,光合作用、有效光化学效率随着青霉素G钠的升高而下降,龙须菜叶绿素a与类胡萝卜素含量随着青霉素G钠浓度的升高而具有升高的趋势,但藻红蛋白、藻蓝蛋白以及可溶性蛋白在各处理组之间均没有表现出一定趋势。这些结果说明,氯霉素对生长的影响主要是光合作用速率的下降,以及有关蛋白合成下降引起,而青霉素G钠对生长的影响可能原因是呼吸作用速率的增加引起。由于龙须菜对氯霉素的敏感性比对青霉素G钠的敏感性更强,氯霉素在基因工程的育种中可能更适合作为选择压力。     

Chemical composition of agars from a newly reported Japanese agarophyte,Gracilariopsis lemaneiformis

The chemical composition of agars from Gracilariopsis lemaneiformis, newly reported from Japan, was investigated. Native agars were isolated by a sequential extraction of plants in water at 22 °C and 100 °C, and in boiling 20, 40 and 60° ethanol. Agars in each extract were analyzed by chemical methods, ¹H, ¹³C NMR; and IR spectroscopy. The highest yield of agar (total carbohydrate) was obtained from the 40° ethanol extract (55°). Highest sulfate content was attained in non-alkali treated agars extracted with hot water (4.81°, DS 0.2). The 3,6-anhydrogalactose content was highest in the 40° ethanol extract (36.1° in non-alkali treatment, 40.3° in alkali treatment). The highest methoxyl content (6.51°, DS 0.66) was obtained in the 60° ethanol extract. The G. lemaneiformis agar is composed of the biological precursor to agarobiose repeating units and agarobiose containing 6-O-methyl agarobiose and a small amount of 2-O-methyl-α-l-galactopyranose residues. Alkali treatment improved the chemical quality of the agar fractions, which was comparable with Japanese commercial agar and agarose.     

Physiological effects of nitrogen deficiency and recovery on the macroalga Gracilariopsis lemaneiformis (Rhodophyta)

Algal metabolites are the most promising feedstocks for bio‐energy production. Gracilariopsis lemaneiformis seems to be a good candidate red alga for polysaccharide production, especially relating to the agar production industry. Nitrogen deficiency is an efficient environmental pressure used to increase the accumulation of metabolites in algae. However, there are no studies on the physiological effects of G. lemaneiformis in response to nitrogen deficiency and its subsequent recovery. Here we integrated physiological data with molecular studies to explore the response strategy of G. lemaneiformis under nitrogen deficiency and recovery. Physiological measurements indicated that amino acids and protein biosynthesis were decreased, while endogenous NH₄⁺ and soluble polysaccharides levels were increased under nitrogen stress. The expression of key genes involved in these pathways further suggested that G. lemaneiformis responded to nitrogen stress through up‐regulation or down‐regulation of genes related to nitrogen metabolism, and increased levels of endogenous NH₄⁺ to complement the deficiency of exogenous nitrogen. Consistent with the highest accumulation of soluble polysaccharides, the gene encoding UDP‐glucose pyrophosphorylase, a molecular marker used to evaluate agar content, was dramatically up‐regulated more than 4‐fold compared to the relative expression of actin after 4 d of nitrogen recovery. The present data provide important information on the mechanisms of nutrient balance in macroalgae.     

The metabolism of 8-heptadecene in Pyropia (Bangiaceae,Rhodophyta)

The metabolic pathway of 8-heptadecene in red algae was investigated. The results showed that the amounts of 8-heptadecene in the primitive bangiophycidean red algae Pyropia and Bangia were 30–50 % of the volatile compounds, much higher than that in the green alga Ulva pertusa, the brown alga Sargassum thunbergii and the florideophycidean red alga Gracilaria lemaneiformis. Studies on the metabolism of 8-heptadecene in Pyropia found that its enzymatic system has no significant catalytic activity on palmitic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid. However, the isolated enzymatic solution showed activity in the presence of eicosapentaenoic acid. This activity produced about four times the amount of 8-heptadecene compared with other substrates and the control, indicating the solution had a specific catalytic function for eicosapentaenoic acid. Furthermore, the enzyme solution was strongly inhibited by NaN₃ but not by phenidone and phenanthroline suggesting that the enzyme is structurally related to heme protein. Thus, it is believed that a constant amount of 8-heptadecene is maintained in the primitive red alga Pyropia and the 8-heptadecene is a metabolite of eicosapentaenoic acid and may catalyzed by enzymes including a heme lipoxygenase-like enzyme.     

Application of SSR and AFLP to the analysis of genetic diversity in <Emphasis Type="Italic">Gracilariopsis lemaneiformis</Emphasis> (Rhodophyta)

The agarophyte Gracilariopsis lemaneiformis is both important for biological research and of significant economic value. However, the genetic diversity of wild populations of the alga has not been studied. We used amplified fragment length polymorphism (AFLP) PCR and simple sequence repeat (SSR) analysis to investigate diversity in four field populations, three from the coast of Qingdao and one from Weihai, China. Forty G. lemaneiformis isolates collected from the four different geographical groups were analyzed using 16 pairs of SSR primers for PCR amplification. However, no polymorphisms were detected, indicative of a degree of genetic homogeneity. A total of 347 reproducible bands were then amplified using eight AFLP primer pairs, and genetic indices of diversity within and between populations were calculated. This analysis revealed only low levels of genetic diversity both within and between the four geographical groups of G. lemaneiformis. The Weihai population showed a higher within-population genetic diversity than any of the Qingdao populations. The data suggest that there is only limited gene flow between populations. An UPGMA dendrogram revealed two main clusters, and one of these included all the Qingdao isolates. The order of clustering was in accordance with their geographical distribution. These results suggest that the wild G. lemaneiformis populations are closely related and that there is little genetic diversity within wild germplasm in the regions sampled.     

Nutritional Composition and Assessment of Gracilaria lemaneiformis Bory

The chemical composition, mineral elements, vitamins, free fatty acids and amino acid content of the edible red alga Gracilaria lemaneiformis Bory, grown in the sea near Nan＇ao island, Guangdong Province, were analyzed in the present study. Gracilaria lemaneiformis Bory showed a total sugar content of 14.65%. The protein content was 21%, of which approximately 41% was determined to be essential amino acids （EAA）. The major amino acid components were glutamic acid, leucine, arginine, and alanine. Of the EAA assayed, methionine and cysteine appeared to be the most limiting amino acids compared with the EAA pattern provided by Food and Agricultural Organization of the United Nations. The total lipids content was 0.87% and comprised a high composition of unsaturated fatty acids （61%）, mainly as linoleic acid and oleic acid, and a little amount of polyunsaturated fatty acid; palmitic acid was the main component （39%） of saturated acids. Relatively high levels of vitamin C, iodine, phosphorus, and zinc were also present in G. lemaneiformis. The nutritional composition between G. lemaneiformis and Nostoc flagelliforme, a rare alga that is widely eaten in Chinese society, was compared. The results suggest that N. flagelliforme can be substituted for by G. lemaneiformis, not only because of their similar shape, but also because of their approximate nutritional composition. Gracilaria lemaneiformis may possibly serve as a potential healthy food in human diets in the future.     

Bioaccumulation of trace metals by the live macroalga Gracilaria lemaneiformis

The macroalga Gracilaria lemaneiformis is an important and commercially valuable renewable resource. It is distributed widely in shallow marine waters but grows mostly on tropical or subtropical coasts. We investigated the accumulation of Cd, Cu, and Pb by live G. lemaneiformis under low concentrations. There was a positive correlation between the organisms’ metal concentrations and exposure concentrations. When exposed to both Cu and Cd, the concentrations of Cu and Cd in G. lemaneiformis were higher than those exposed to solutions of Cu and Cd alone. However, the concentrations of heavy metals in G. lemaneiformis were not markedly different (p?<?0.05) between the treatment groups and the control groups. We analyzed the results with nonlinear curve fitting and employed a two-compartment model to study the accumulation kinetics of heavy metals by G. lemaneiformis. The uptake rate constants and bioconcentration factors (BCFs) of the metals decreased with increased exposure concentration. The theoretical equilibrium concentrations increased significantly with the exposure concentrations. Our results suggested that G. lemaneiformis obviously accumulated heavy metals from seawater. As an important resource for food and pharmaceuticals, G. lemaneiformis should be cultivated in clear seawater.     

MIPS: A Calmodulin-Binding Protein of Gracilaria Lemaneiformis Under Heat Shock

To study the Ca²⁺/Calmodulin (CaM) signal transduction pathway of Gracilaria lemaneiformis under heat stress, myo-inositol-1-phosphate synthase (MIPS), a calmodulin-binding protein, was isolated using the yeast two-hybrid system. cDNA and DNA sequences of mips were cloned from G. lemaneiformis by using 5′RACE and genome walking procedures. The MIPS DNA sequence was 2,067 nucleotides long, containing an open reading frame (ORF) of 1,623 nucleotides with no intron. The mips ORF was predicted to encode 540 amino acids, which included the conserved MIPS domain and was 61–67 % similar to that of other species. After analyzing the amino acid sequence of MIPS, the CaM-Binding Domain (CaMBD) was inferred to be at a site spanning from amino acid 212 to amino acid 236. The yeast two-hybrid results proved that MIPS can interact with CaM and that MIPS is a type of calmodulin-binding protein. Next, the expression of CaM and MIPS in wild-type G. lemaneiformis and a heat-tolerant G. lemaneiformis cultivar, "981," were analyzed using real-time PCR under a heat shock of 32 °C. The expression level displayed a cyclical upward trend. Compared with wild type, the CaM expression levels of cultivar 981 were higher, which might directly relate to its resistance to high temperatures. This paper indicates that MIPS and CaM may play important roles in the high-temperature resistance of G. lemaneiformis.     

Algicidal effect of 2,4-dichlorophenoxy acetic acid on blue-green alga Cylindrospermum sp.

The effect of the herbicide 2,4-Dichlorophenoxy acetic acid generally used in agriculture was studied on the nitrogen fixing blue-green alga Cylindrospermum sp. The alga could tolerate up to 150 μg per ml in liquid culture and 100 μg per ml on agar plates without any inhibitory effect on growth and survival. The maximum tolerance was up to 800 μg per ml and higher concentrations were lethal.     

The green tide-forming macroalga Ulva linza outcompetes the red macroalga Gracilaria lemaneiformis via allelopathy and fast nutrients uptake

The objective of this study was to evaluate the interactions between green tide-forming macroalgae Ulva linza and red macroalgae Gracilaria lemaneiformis in the laboratory. The results demonstrated that the presence of U. linza can restrict growth (9–31 %) and photosynthesis (25–85 %) of G. lemaneiformis. In contrast, G. lemaneiformis had little apparent effect on the growth of U. linza. Culture medium experiments confirmed that allelochemicals may be released by both the tested macroalgae. The causative mechanism for the growth and photosynthesis inhibition of G. lemaneiformis was not light limitation nor increase of pH, but a combination of allelopathic effects of U. linza and nutrient competition between the two macroalgae. Moreover, the “green tide” macroalga U. linza was a stronger competitor for nutrient than G. lemaneiformis. The results from this study provide evidence for the mechanisms of “green tide” formation by U. linza: potent allelopathic effects on G. lemaneiformis and faster nutrients uptake than its competitors.     

Effects of macroalgae Ulva pertusa (Chlorophyta) and Gracilaria lemaneiformis (Rhodophyta) on growth of four species of bloom-forming dinoflagellates

The effects of fresh thalli and culture medium filtrates from two species of marine macroalgae, Ulva pertusa Kjellm (Chlorophyta) and Gracilaria lemaneiformis (Bory) Dawson (Rhodophyta), on growth of marine microalgae were investigated in co-culture under controlled laboratory conditions. A selection of microalgal species were used, all being identified as bloom-forming dinoflagellates: Prorocentrum donghaiense Lu sp., Alexandrium tamarense (Lebour) Balech, Amphidinium carterae Hulburt and Scrippsiella trochoide (Stein) Loeblich III. Results showed that the fresh thalli of either U. pertusa or G. lemaneiformis significantly inhibited the microalgal growth, or caused mortality at the end of the experiment. However, the overall effects of the macroalgal culture filtrates on the growth of the dinoflagellates were species-specific (inhibitory, stimulatory or none) for different microalgal species. Results indicated an allelopathic effect of macroalga on the co-cultured dinoflagellate. We then took P. donghaiense as an example to further assess this hypothesis. The present study was carried out under controlled conditions, thereby excluded the fluctuation in light and temperature. Nutrient assays showed that nitrate and phosphate were almost exhausted in G. lemaneiformis co-culture, but remained at enough high levels in U. pertusa co-culture, which were well above the nutrient limitation for the microalgal growth, when all cells of P. donghaiense were killed in the co-culture. Daily f/2 medium enrichment greatly alleviated the growth inhibition on P. donghaiense in G. lemaneiformis co-culture, but could not eliminate it. Other environmental factors, such as carbonate limitation, bacterial presence and the change of pH were also not necessary for the results. We thus concluded that allelopathy was the most possible reason leading to the negative effect of U. pertusa on P. donghaiense, and the combined roles of allelopathy and nutrient competition were essential for the effect of G. lemaneiformis on P. donghaiense.     

The agar-type polysaccharide from the red alga Ceramium rubrum

Aqueous extraction of the red alga C. rubrum gave a galactan sulphate and, possibly, a separate glucan and xylan. The galactan sulphate has an alternating structure of the agar-type with D-galactose or 6-O-methyl-D-galactose as one alternating unit, and L-galactose, 3,6-anhydro-L-galactose; and their respective 2-methyl ethers as the other unit. Sulphate hemi-ester groups are present on position 6 of both D- and L-galactose residues, with smaller amounts on positions 2 and 4 of, probably, D-galactose residues. The polysaccharide differs from others previously examined in that most of the L-galactose residues are non-sulphated.     

Isolation and identification of two strains of pathogenic bacteria and their effects on the volatile metabolites of Gracilariopsis lemaneiformis (Rhodophyta)

Apical necrosis is a widely distributed disease in the culture of the marine agar-producing alga, Gracilariopsis lemaneiformis. In this study, 16 strains of epiphytic bacteria were isolated from the tip bleaching parts of G. lemaneiformis; of the 16 strains, two strains could induce healthy algal tips to become necrotic. They were identified as Thalassospira sp. and Vibrio parahaemolyticus by 16S rDNA sequence analysis and biochemical characterization. Using solid phase microextraction and gas chromatography–mass spectrometry, the variation of volatile metabolites of G. lemaneiformis infected by these two strains of pathogenic bacteria was analyzed. The results showed that E-2-nonenal and 1-octen-3-one differed very significantly (P < 0.01) between the Thalassospira infected group and the control group, while trichloromethane, 3-methyl butanal, benzaldehyde, and E-2-decenal differed significantly (P < 0.05). The difference of 1-octen-3-one, benzaldehyde, and E-2-nonenal between the V. parahaemolyticus infected group and the control group was very significant (P < 0.01), while 3-methyl butanal and octanal were significantly different (P < 0.05). In conclusion, 1-octen-3-one, E-2-nonenal, and benzaldehyde might be the characteristic metabolites for the pathogenic infection and could be used as biomarkers for the disease prevention of G. lemaneiformis.     

Cloning and characterization of two types of ubiquitin genes from Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta)

Two types of ubiquitin genes were isolated from the marine red alga Gracilaria lemaneiformis: a ubiquitin-52 amino acid fusion protein gene, and a 6-unit polyubiquitin gene. Alignment of polyubiquitins among three red algae (Gracilaria lemaneiformis, Gracilaria verrucosa, Aglaothamnion neglectum) and other species revealed that there were six ubiquitin repeats in all three red algae polyubiquitins, and that glutamine was the final amino acid residue in the terminal repeat of the polyprotein in the two Gracilaria sequences. Southern blot analysis revealed that both genes were encoded by low-copy number genes. Semi-quantitative RT-PCR was performed to investigate the expression of these two genes in two phases of G. lemaneiformis. The result revealed that the monoubiquitin was phase-relative, and upregulated in tetrasporophytes compared with female gametophytes. The polyubiquitin gene was expressed at similar levels in both phases.