The extracellular polysaccharides of the red microalgae: Chemistry and rheology

Cells of red microalgae encapsulated within sulphated polysaccharides, are thought to have a wide range of potential industrial applications. Our group is thus carrying out a comprehensive research program aimed at bringing these biopolymers into industrial use. The program includes physiological studies on polysaccharide production, outdoor cultivation of the microalgae, and characterisation of the polysaccharides. Chemical composition and structure and physicochemical properties were investigated for the polysaccharides of three red microalgae, Porphyridium sp., P. aerugineum and Rhodella reticulata. Differences were found among the three species in the composition of the monosugars, half ester sulphate groups and glucuronic acid content, but a disaccharide isolated was identical in all the species examined. This disaccharide is thought to be the basic building block of these polysaccharides. In addition, monosugar sulphates were isolated and characterised. Fractionation by charge showed the polysaccharides to be heterogenous and composed of at least two fractions that differed in their composition. Although the polysaccharides differed in composition, their rheological characteristics were found to be similar. Aqueous solutions of the biopolymers were stable over a wide range of pH values and temperatures and were compatible with monovalent cations. Mixtures of the algal polysaccharides with locust bean gum exhibited synergism and syneresis. When the gel strength was compared with that of agar gel at the same concentration the polysaccharide gels were found to be weaker.     

Disentangling the role of extracellular polysaccharides in desiccation tolerance in lichen-forming microalgae. First evidence of sulfated polysaccharides and ancient sulfotransferase genes

Trebouxia sp. TR9 and Coccomyxa simplex are desiccation-tolerant microalgae with flexible cell walls, which undergo species-specific remodelling during dehydration–rehydration (D/R) due to their distinct ultrastructure and biochemical composition. Here, we tested the hypothesis that extracellular polysaccharides excreted by each microalga could be quantitatively and/or qualitatively modified by D/R. Extracellular polysaccharides were analysed by size exclusion and anion exchange chromatography, specific stains after gel electrophoresis and gas chromatography/mass spectrometry of trimethylsilyl derivatives (to determine their monosaccharide composition). The structure of a TR9-sulfated polymer was deduced from nuclear magnetic resonance (NMR) analyses. In addition, sugar-sulfotransferase encoding genes were identified in both microalgae, and their expression was measured by RT-qPCR. D/R did not alter the polydispersed profile of extracellular polysaccharides in either microalga but did induce quantitative changes in several peaks. Furthermore, medium-low-sized uronic acid-containing polysaccharides were almost completely substituted by higher molecular mass carbohydrates after D/R. Sulfated polysaccharide(s) were detected, for the first time, in the extracellular polymeric substances of both microalgae, but only increased significantly in TR9 after cyclic D/R, which induced a sugar-sulfotransferase gene and accumulated sulfated ß-D-galactofuranan(s). Biochemical remodelling of extracellular polysaccharides in aeroterrestrial desiccation-tolerant microalgae is species-specific and seems to play a role in the response to changes in environmental water availability.     

PARALYTIC SHELLFISH TOXINS ARE RESTRICTED TO FEW SPECIES AMONG AUSTRALIA'S TAXONOMIC DIVERSITY OF CULTURED MICROALGAE1

There are at least 40,000 species of microalgae in the aquatic environment. Fifteen species of marine dinoflagellates and freshwater cyanobacteria are known to produce paralytic shellfish toxins (PSTs) and represent a threat to human and/or livestock health. Although known toxic species are regularly monitored, the wider cross‐section of microalgae has not been systematically tested for PSTs. Advances in rapid screening techniques have resulted in the development of highly sensitive and specific methods to detect PSTs, including the sodium channel and saxiphilin binding assays. These assays were used in this study in 96‐well formats to screen 234 highly diverse isolates of Australian freshwater and marine microalgae for PSTs. The screening assays detected five toxic species, representing one freshwater cyanobacterium (Anabaena circinalis Rabenhorst) and four species of marine dinoflagellates (Alexandrium minutum Halim, A. catenella Balech, A. tamarense Balech, and Gymnodinium catenatum Graham). Liquid chromatography‐fluorescence detection was used to identify 14 saxitoxin analogues across the five species, and each species exhibited distinct toxin profiles. These results indicate that PST production is restricted to a narrow range of microalgal species found in Australian waters.     

The potential of production of sulfated polysaccharides from Porphyridium

Summary The environmental conditions prevailing in Israel make marine algae an attractive crop for the production of valuable chemicals. A marine species of Porphyridium seems to fit this purpose.The unicellular red alga Porphyridium is encapsulated by a polysaccharide envelope that is present in the gel state. This polysaccharide is an acidic heteropolymer composed of sulfated sugars. It forms ionic bridges through divalent cations, thus reaching a very high molecular weight. The thickness of the polysaccharide capsule varies according to the phase of growth and the growth conditions. Its outer part dissolves in the growth medium, which becomes progressively more viscous. Sulfated polysaccharides form theramlly reversible gels similar to agar and carrageenan, which are usually extracted from marine macroalgae. These gels have been finding increasing use in commercial applications as gelling agents, thickeners, stabilizers, and emulsifiers.We have done experiments on the cultivation of a marine species of Porphyridium for the production of polysaccharides. This unicellular alga has an advantage over the macroalgae due to its relatively faster growth rate and the possibility to regulate its growth. The potential for production of the polysaccharide, both that dissolved in the external medium and that attached to the cell (including an intracellular fraction), and the effects of growth conditions on productivity were suudied in the laboratory. Porphyridium was also cultivated outdoors in seawater in 1-m² ponds and its growth potential investigated.     

Drag reduction by Acinetobacter calcoaceticus BD4.

The encapsulated bacterium Acinetobacter calcoaceticus BD4 at a density of 3.6 X 10(9) cells per ml reduced the friction of turbulent water in a narrow pipe by 55%. This drag reduction was due to the tightly bound polysaccharide capsules (0.4 mg per ml) of culture. Capsule-deficient mutants of BD4 failed to reduce drag. The cell-bound polysaccharide demonstrated a threefold-higher drag-reducing activity than the polymer which was free in solution.     

Drag reduction by Acinetobacter calcoaceticus BD4

The encapsulated bacterium Acinetobacter calcoaceticus BD4 at a density of 3.6 X 10(9) cells per ml reduced the friction of turbulent water in a narrow pipe by 55%. This drag reduction was due to the tightly bound polysaccharide capsules (0.4 mg per ml) of culture. Capsule-deficient mutants of BD4 failed to reduce drag. The cell-bound polysaccharide demonstrated a threefold-higher drag-reducing activity than the polymer which was free in solution.     

微泡菌属菌株YPW1和YPW16多糖降解特性分析

【背景】海洋环境中分离到的微泡菌属菌株具有多糖降解能力,在环境中可以作为糖类代谢的重要执行者参与海洋碳循环过程。【目的】测定2株微泡菌属菌株的多糖降解活性,通过与微泡菌属其他菌株基因组比较分析2株菌的多糖降解基因特征。【方法】通过3,5-dinitrosalicylicacid(DNS)定糖法测定多糖降解活性,同时利用高通量测序技术对菌株基因组序列进行测定与组装,并与其他基因组注释结果进行比较分析。【结果】分离得到2株微泡菌属菌株YPW1和YPW16,二者均为潜在新种。结果表明,菌株YPW1能够降解琼胶、褐藻胶、果胶、几丁质、木聚糖、淀粉、普鲁兰等7种多糖,而菌株YPW16仅可降解淀粉和普鲁兰。基因组分析表明,YPW1具有上述7种多糖的降解酶基因,但菌株YPW16只具有淀粉酶与普鲁兰酶降解基因。相较于其他微泡菌属菌株,菌株YPW1多糖降解范围、多糖降解酶基因种类与丰度较高,但菌株YPW16多糖降解范围却较为狭窄。由此可知,多糖降解酶基因在微泡菌属基因组中的分布差异性较大。【结论】本研究为微泡菌属提供了2株潜在的新型菌株资源,为生物多糖降解提供了生化工具,也为研究微泡菌属菌株中多糖降解基...     

The effect of gradual increase in salinity on the biomass productivity and biochemical composition of several marine,halotolerant<Emphasis Type="Bold">,</Emphasis> and halophilic microalgae

Open ponds are the preferred cultivation system for large-scale microalgal biomass production. To be more sustainable, commercial scale biomass production should rely on seawater, as freshwater is a limiting resource, especially in places with high irradiance. If seawater is used for both pond fill and evaporative volume makeup, salinity of the growth media will rise over time. It is not possible for any species to achieve optimum growth over the whole saline spectrum (from seawater salinity level up to salt saturation state). In this study, we investigated the effects of gradual salinity increase (between 35 and 233 ppt) on biomass productivity and biochemical composition (lipid and carbohydrate) of six marine, two halotolerant, and a halophilic microalgae. A gradual and slow stepped salinity increase was found to expand the salinity tolerance range of tested species. A gradual reduction in biomass productivity and maximum photochemical efficiency was observed as a consequence of increased salinity in all tested species. Among the marine microalgae, Tetraselmis showed highest biomass productivity (32 mg L^?1 day^?1) with widest salinity tolerance range (35 to 109 ppt). Halotolerant Amphora and Navicula were able to grow from 35 ppt to 129 ppt salinity. Halophilic Dunaliella was the only species capable of growing between 35 and 233 ppt and showed highest lipid content (56.2%) among all tested species. This study showed that it should be possible to maintain high biomass in open outdoor cultivation utilizing seawater by growing Tetraselmis, Amphora, and Dunaliella one after another as salinity increases in the cultivation system.     

Bait worm packaging as a potential vector of invasive species

Invasive species have become an increasingly greater concern for the ecological health of coastal ecosystems, yet vectors of these introductions often are unclear. This project evaluated the potential for the brown seaweed Ascophyllum nodosum ecad scorpiodes (Hauck) Reinke, packaged with bait worms (Nereis virens) harvested from the coast of Maine (USA), as a vector of invasive marine fauna and flora. Often, the seaweed and contents of the bait boxes are discarded into the water by recreational fishermen after using the bait worms, and any included non-native species may then be introduced. Bait boxes were purchased from several commercial vendors in Connecticut and New York over a two-year period. Subsamples of the seaweed were placed in laboratory culture and the growth of associated macro- and microalgae was monitored. Marine invertebrate species present in the samples were also identified and quantified. Results indicated 13 species of macroalgae and 23 species of invertebrates were associated with baitboxes. Among the highly diverse microbial assemblage detected, two species of potentially toxic marine microalgae, Alexandrium fundyense Balech and Pseudonitzschia multiseries (Hasle) Hasle, were found both prior to and after incubation at various temperatures, indicating these harmful algae are brought to and can survive in receiving waters. These findings highlight the need to consider alternative choices of bait box packaging materials or appropriate disposal methods of the seaweed in order to minimize the transport of species which are not native to the receiving coastal waters.     

湛江沿海海洋微藻及其多糖、脂类和蛋白藻株多样性研究

从硇洲岛和徐闻珊瑚礁自然保护区潮间带采集的海水和沉积物样品中分离培养海洋微藻, 筛选其中富含多糖、脂类或蛋白质的藻株。采用形态学观察、18S rDNA序列比较及其系统发育分析法, 对分离培养的海洋微藻及其富含多糖、脂类或蛋白质的藻株进行分类鉴定和生物多样性分析。分离、培养、鉴定并储藏了189株海洋微藻, 归属于65个种, 分布于硅藻门(Bacillariophyta)、绿藻门(Chlorophyta)、定鞭藻门(Haptophyta)和红藻门(Rhodophyta)的9纲、25目、30个科、38个属; 其中多糖含量较高的46株海洋微藻, 分布于25个种, 20个属; 脂类含量较高的46株海洋微藻, 分布于32个种, 15个属; 蛋白含量较高的46株海洋微藻, 分布于28个种, 18个属。结果表明硇洲岛和徐闻珊瑚礁自然保护区潮间带可培养的海洋微藻及其富含多糖、脂类和蛋白质藻株的物种多样性丰富, 在新型药物、活性天然产物、功能食品和饲料及其添加剂的发掘等方面具有良好前景。     

Biological,chemical and physical factors influencing the seasonal change of polysaccharide concentration in irrigation-channel sediment

Variation of polysaccharide concentration in irrigation-channel sediment was determined concurrently with biological, chemical and physical factors influencing the benthic algal community. Phenol-sulphuric acid method was used to measure polysaccharide concentration. Polysaccharide concentration, biomass of benthic algae, and species composition changed spatially and temporally. Fluctuations of total suspended solid (TSS) concentration and exposure of channel bed to direct sunlight had major effects on algal growth and polysaccharide production. Polysaccharide concentration was correlated to chlorophyll a concentration (r=0.73, P<0.001) and algal biomass (r=0.57, P<0.001). Fragilaria construens and Aulacoseira (Melosira) italica were the most common diatoms in the benthic flora. Chlorophyll a concentration in the sediment showed a strong negative correlation (r=-0.99, P<0.001) with the seasonal variation of TSS concentration in channel water. The polysaccharides produced by benthic microorganisms play a major role in clogging channel bed and thereby reducing seepage from earthen irrigation channels. Correlations between polysaccharide concentration and chlorophyll a (and algal biomass) further indicate the importance of benthic algae for polysaccharide production. Since availability of light to the algal flora is critical for the production of polysaccharides, the effect of clogging can be maximized by exposing the channel bed to direct sunlight during non-irrigation period (winter).     

A GLYCOPROTEIN NONCOVALENTLY ASSOCIATED WITH CELL‐WALL POLYSACCHARIDE OF THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)1

The cells of the red microalga Porphyridium sp. (UTEX 637) are encapsulated in a cell wall of a negatively charged mucilaginous polysaccharide complex composed of 10 different sugars, sulfate, and proteins. In this work, we studied the proteins associated with the cell‐wall polysaccharide. A number of noncovalently associated proteins were resolved by SDS‐PAGE, but no covalently bound proteins were detected. The most prominent protein detected was a 66‐kDa glycoprotein consisting of a polypeptide of approximately 58 kDa and a glycan moiety of approximately 8 kDa containing N‐linked terminal mannose. In size‐exclusion chromatography, the 66‐kDa protein was coeluted with the polysaccharide and could be separated from the polysaccharide only after denaturation of the protein, indicating that the 66‐kDa protein was tightly bound to the polysaccharide. Western blot analysis revealed that the 66‐kDa protein was specific to Porphyridium sp. and P. cruentum, because it was not detected in the other species of red microalgae examined. Indirect immunofluorescence assay confirmed the location of the protein in the algal cell wall. The sequence of cDNA clone encoding the 66‐kDa glycoprotein, detected in our in‐house expressed sequence tag database of Porphyridium sp., revealed that this is a novel protein with no similarity to any protein in the public domain databases and our in‐house expressed sequence tag database of the red microalga Rhodella reticulata. The 66‐kDa protein bound polysaccharides from red algae but not from those of other origins tested. Possible roles of the 66‐kDa protein in the biosynthesis of the polysaccharide are discussed.     

Growth model for raceway pond cultivation of Desmodesmus sp. MCC34 isolated from a local water body

Biofuel production by microalgae has the advantage of higher biomass productivity over land crops. The selection of potential microalgae depends on the growth in outdoor mass cultivation during different seasons, which can be predicted by a mathematical model. Here, freshwater green algae were isolated from a local water body in Pilani, Rajasthan, India (geographical coordinates: 28°22′N 75°36′E) and characterized by microscopy and ribosomal RNA analysis. The strain was submitted to the Indian Agricultural Research Institute's microbial culture collection (IARI, India) and identified as Desmodesmus sp. MCC34. This strain, along with a fresh water green algae (Chlorella minutissima), two marine green algae species (Dunaliella salina and Dunaliella tertiolecta) and two nitrogen fixing cyanobacteria (Nostoc muscorum and Anabaena doliolum), were screened for lipid productivity and growth kinetics under culture room and raceway pond conditions. Desmodesmus sp. MCC34 showed the highest specific growth rate (0.26 day^?1), biomass production (1.9 g L^?1) and lipid productivity (103 mg L^?1 day^?1). The optimal temperature and saturating light intensity for maximal growth of Desmodesmus sp. MCC34 were 35 °C and 75 μmol m^?2 s^?1 with molar extinction coefficient of 0.22 m² g^?1, respectively. Desmodesmus sp. MCC34 was then subjected to outdoor cultivation in a 20‐m long raceway pond for 18 days during March and November 2013. The areal biomass productivity and volumetric biomass productivity were 13946.23 kg ha^?1 year^?1 and 56.94 mg L^? 1day^?1 during the month of March, decreasing to 6262.28 kg ha^?1 year^?1 and 25.57 mg L^? 1day^?1 during the month of November. A mathematical model was constructed to explain the relationship between biomass production and growth parameters such as temperature, light intensity and nutrient concentration. The productivity values predicted with the proposed model correspond well with the experimental data, suggesting the validity of the model.     

A novel polyculture growth model of native microalgal communities to estimate biomass productivity for biofuel production

Native polyculture microalgae is a promising scheme to produce microalgal biomass as biofuel feedstock in an open raceway pond. However, predicting biomass productivity of native polycultures microalgae is incredibly complicated. Therefore, developing polyculture growth model to forecast biomass yield is indispensable for commercial-scale production. This research aims to develop a polyculture growth model for native microalgal communities in the Minamisoma algae plant and to estimate biomass and biocrude oil productivity in a semicontinuous open raceway pond. The model was built based on monoculture growth of polyculture species and it is later formulated using species growth, polyculture factor (k_value), initial concentration, light intensity, and temperature. In order to calculate species growth, a simplified Monod model was applied. In the simulation, 115 samples of the 2014–2015 field dataset were used for model training, and 70 samples of the 2017 field dataset were used for model validation. The model simulation on biomass concentration showed that the polyculture growth model with k_value had a root-mean-square error of 0.12, whereas model validation provided a better result with a root-mean-square error of 0.08. Biomass productivity forecast showed maximum productivity of 18.87 g/m²/d in June with an annual average of 13.59 g/m²/d. Biocrude oil yield forecast indicated that hydrothermal liquefaction process was more suitable with a maximum productivity of 0.59 g/m²/d compared with solvent extraction which was only 0.19 g/m²/d. With satisfactory root-mean-square errors less than 0.3, this polyculture growth model can be applied to forecast the productivity of native microalgae.     

Effects of acidity on the insoluble polysaccharide content of germinating pollen of Pinus pinea L. and Pinus pinaster Aiton

The PAS reaction was used to stain total insoluble polysaccharides in pollen of two species of Pinus collected in two different years and germinated in vitro at different pH values. PAS-detectable polysaccharides are localized in the intine, cytoplasm and amyloplasts; PAS-undetectable (callose) in the middle of the intine, but only in the distal part of the grain. The total PAS stained polysaccharide content was quantified with a microdensitometer. Values are correlated with germination percentages and pollen tube length. The total polysaccharide content, as well as pollen tube length, varies in each species according to the year, probably due to environmental conditions. Also the various pHs deeply influence pollen germination and tube length, the lowest pH being the most unfavourable condition. At this pH (3.5) the total polysaccharide content is higher than in the control (pH 5.5): this is probably due to a lack of callose synthesis for tube wall or to a depolymerization of this polysaccharide, and a subsequent storage in a PAS-detectable form.     

Antiviral Activities of Marine Pseudomonas Polysaccharides and Their Oversulfated Derivatives

A marine Pseudomonas species WAK-1 strain simultaneously produces extracellular glycosaminoglycan and sulfated polysaccharide. Among the antiviral activities tested for these polysaccharides, the latter showed anti-HSV-1 activity in RPMI 8226 cells (50% effective concentration is 1.4 μg/ml). Oversulfated derivatives of these polysaccharides prepared by dicyclohexylcarbodiimide-mediated reaction for both polysaccharides showed antiviral activities against influenza virus type A (for glycosaminoglycan, 50% effective concentration is 11.0 μg/ml; for another, 2.9 μg/ml). Glycosaminoglycan, sulfated polysaccharide, and their chemically synthesized oversulfated derivatives did not show antiviral activities against influenza virus type B and human immunodeficiency virus type 1. No cytotoxicity of these products was noted against host cells at the 50% cytotoxic concentration of 100 μg/ml, except that naturally occurring sulfated polysaccharide had 50% cytotoxicity against MT-4 cells at 8–21 μg/ml. Received May 1, 1998; accepted July 24, 1998.     

Total Polysaccharide Content of Developing Pollen in Two Angiosperm Species

The PAS reaction was used to stain total insoluble polysaccharides in whole pollen grains of anthers of Borago officinalis and Lycopersicum peruvianum from early microspore to ripe pollen stage. Total polysaccharide content was quantified with a microdensitometer: in Borago officinalis values showed two peaks, one during the microspore and the other during the binucleate stage; in Lycopersicum, there was a single peak during the latter stage. In both species total polysaccharide content decreased markedly in the last stage of pollen development before anthesis, when starch was hydrolysed and the polysaccharide reserves of the pollen transferred to the cytoplasm. Pollen grain volume was also determined at the various stages. It was found to increase, though with a different pattern in the two species, and to decrease before dehiscence. The results are discussed in terms of cytoembryological data of pollen grain development.     

UV-B Radiation Suppresses the Growth and Antioxidant Systems of Two Marine Microalgae, Platymonas subcordiformis （Wille） Hazen and Nitzschia closterium （Ehrenb.） W. Sm

This study investigated whether increased solar UV-B radiation (280-315nm) could suppress the growth of marine microalgae through effects on their antioxidant systems. Two marine microalgae species, Platymonas subcordiformis (Wille) Hazen and Nitzschia closterium (Ehrenb.) W. Sm, were exposed to a range of UV-B radiation and both showed reductions in their growth rates, and the chlorophyll a(Chl a) and carotenoid (Car) contents when UV-B radiation dose increased. Superoxide anion radical (O2^ )production and the concentration of hydrogen peroxide (H2O2) and malodiadehyde (MDA) also increased with the increasing of UV-B radiation. Antioxidant systems, non-enzymic components (Car and glutathione content) and enzymic components (superoxide dismutase (SOD) and catalase (CAT) activity), decreased as a result of enhanced UV-B radiation. When the exogenous glutathione (GSH) was added, the effects of UV-B radiation on the growth of the two species were alleviated. These results suggest that enhanced UV-B radiation suppressed the antioxidant systems and caused some active oxygen species to accumulate, which in turns retarded the development of the marine microalgae.     

Effects of fertilizer-based culture media on the production of exocellular polysaccharides and cellular superoxide dismutase by <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> (Bohlin)

Microalgae cultures are receiving attention because of increasing biotechnological and biomedical production of active biomolecules. We evaluated various fertilizer-based culture media to scale up production of the marine microalga Phaeodactylum tricornutum for production of exocellular polysaccharides (EPS), soluble proteins, and cellular superoxide dismutase (SOD). The standard source of sodium nitrate was the same as that used in the synthetic f/2 culture medium and ammonium nitrate, urea, ammonium sulfate, and calcium nitrate as alternative sources of nitrogen. The maximum production of EPS was achieved in microalgae cells grown in the culture media containing 63 and 23% nitrogen from ammonium sulfate, and also in microalgae cells grown in the culture media containing 3% nitrogen from ammonium nitrate. The maximum production of cellular SOD was achieved in microalgae cells grown in the culture media containing 35 and 26% nitrogen from ammonium sulfate, and in the culture media containing 17% nitrogen from urea. The results suggest that it is possible to use a source of nitrogen, other than sodium nitrate, to scale up growth of P. tricornutum for production of EPS and SOD at reduced costs.     

Biomass productivity and productivity of fatty acids and amino acids of microalgae strains as key characteristics of suitability for biodiesel production

Microalgae are discussed as an alternative source for the production of biofuels. The lipid content compared to cultivation time of used species is the main reason for any choice of a special strain. This paper reviews more analytical data of 38 screened microalgae strains. After the cultivation period, total content of lipids was analysed. The extracted fatty acids were quantified as fatty acid methyl esters by GC analysis. The amino acids were analysed by HPLC. Chlorella sp., Chlorella saccharophila, Chlorella minutissima and Chlorella vulgaris were identified as species with the highest productivity of fatty acids relevant to transesterification reactions. The components were mainly linoleic acid, palmitic acid and oleic acid. To increase productivity of highly saturated fatty acids, cultivation parameters light intensity and temperature were varied. In this manner, the ideal conditions for biodiesel production were defined in this publication.