Biological degradation of Ascophyllum nodosum

The alginate forms the major structural component of the cell wall and the intercellular matrix of the brown alga Ascophyllum nodosum. Successful biological degradation of A. nodosum would largely depend on the dissolution of the alginate, but reactive compounds in the alga such as polyphenols may also have toxic effects on the microbial population involved. Aerobic and anaerobic batch reactors, operated at 35°C and pH 7, were fed milled A. nodosum, nutrients and inocula adapted to seaweed degradation. The dominant factor for conversion of organic matter during anaerobic digestion was the inhibitory effect of the polyphenols on alginate lyases and methane production. Probably, the relative large fraction of high molecular weight polyphenols (>10 kDa) in this alga gave efficient binding of proteins during digestion. The anaerobic degradation was greatly stimulated when the polyphenols were fixed with low amounts of formaldehyde. An accumulated content of guluronate in the remaining alginate indicated that Ca-crosslinking also limited the guluronate lyase access to the polymer. In contrast, the aerobic digestion of alga gave no increase in the guluronate content of the residual alginate. Compared to anaerobic conditions, the phenols had a much lower influence on the hydrolytic rate of organic matter during aerobic conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Alginate degradation during anaerobic digestion of Laminaria hyperborea stipes

Polyphenols and divalent metal ions present in the tissue may seriously affect the degradation of alginate during anaerobic digestion of brown seaweeds. Laminaria hyperborea stipes, harvested at 59 °N off the Norwegian coast in the autumn, were degraded at different concentrations of polyphenols in anaerobic batch reactors at 35 °C and pH 7. This was done by removing or adding the mechanically peeled outer phenolic layer of the algae, and using methanogenic and alginate degrading inocula already adapted to L. hyperborea degradation. Initial alginate released from the algal particles was affected by NaOH titrations because the Ca/Na-ratio was reduced. After a rapid consumption of the mannitol, alginate lyases were induced, and guluronate lyases showed the highest extracellular activity. Then the microbes digested 0.12–0.23 g Na-alginate L⁻¹ h⁻¹. Later the degradation rate of alginates declined almost to zero, and 13–50% of the alginate remained insoluble. The total solubilisation of alginates was apparently limited by both Ca-crosslinked guluronate residues and complexation with compounds such as polyphenols. The methane production had a lag phase that increased at higher amounts of soluble polyphenols, and the total fermentation probably also became product inhibited if soluble compounds such as acetate, ethanol and butyrate were accumulated. This revised version was published online in September 2006 with corrections to the Cover Date.     

Extraction and characterisation of alginate from brown seaweeds (Fucales,Phaeophyceae) collected from Port Dickson,Peninsular Malaysia

Four species of brown seaweeds, namely Sargassum baccularia, Sargassum binderi, Sargassum siliquosum and Turbinaria conoides, harvested from Port Dickson, Negeri Sembilan, Malaysia were analysed for ash content, alginate yield and alginate properties. Seaweeds calcined at 450°C were found to have low amount of non-combustible residue as these were not contaminated by calcareous animals. Alginate was extracted from these seaweeds by two methods: hot and cold. In the hot method, the storing time was 3 h and the processing temperature was 50°C, whilst in the cold method, the sample was stored overnight at room temperature. Higher yield of alginate was obtained by the hot method compared to the cold method, but alginate extracted by the cold method gave higher molecular weight. In the hot method, 49.9% of alginate was extracted from S. siliquosum, followed by T. conoides (41.4%), S. binderi (38.9%) and S. baccularia (26.7%). Alginate extracted from T. conoides has an average molecular weight, M _w, of 8.06 × 10⁵ g mol⁻¹, whereas alginate from S. siliquosum was the lowest in M _w (4.81 × 10⁵ g mol⁻¹) when the extraction was done at room temperature. Alginate extracted from S. baccularia was found to be very heat-sensitive. Its M _w has dropped more than 83%, from 7.52 × 10⁵ to 1.23 × 10⁵ g mol⁻¹, when the extraction temperature was raised. The effect of heat on the extent of depolymerisation of the alginate molecule of the other three brown seaweed species was less significant, with decrease in molecular weight ranging between 13% and 16%.     

Alginate,mannitol, phenolic compounds and biological activities of two range-extending brown algae, <Emphasis Type="Italic">Sargassum mangarevense</Emphasis> and <Emphasis Type="Italic">Turbinaria ornata</Emphasis> (Phaeophyta: Fucales), from Tahiti (French Polynesia)

This study deals with two range-extending brown algae from Tahitian coral reefs, Sargassum mangarevense and Turbinaria ornata; their alginate properties, mannitol and phenolic contents, antioxidant and antimicrobial activities were determined. Turbinaria ornata showed the richest alginate content with the highest extraction yield (19.2 ± 1.3% dw). Their alginates also exhibited the highest viscosity (50 ± 18 mPa.s), but the M:G ratios (mannuronic acid to glucuronic acid) of alginates (1.25–1.42) were similar in both species. Alginate yield displayed spatial variations, but no significant seasonal changes. The highest mannitol content was found in S. mangarevense (12.2 ± 2.1% dw) during the austral winter. With respect to other tropical Fucales, both algae exhibited also a high phenolic content (2.45–2.85% dw) with significant spatio-temporal variations. Furthermore, high antioxidant activity and activity against Staphylococcus aureus were also detected in extracts. According to these preliminary results, these two range-extending algae are of key interest in numerous industrial areas.     

18S rDNA gene metabarcoding of microeukaryotes and epi-endophytes in the holobiome of seven species of large brown algae

Brown algae (Phaeophyceae) are habitat-forming species in coastal ecosystems and include kelp forests and seaweed beds that support a wide diversity of marine life. Host-associated microbial communities are an integral part of phaeophyte biology, and whereas the bacterial microbial partners have received considerable attention, the microbial eukaryotes associated with brown algae have hardly been studied. Here, we used broadly targeted “pan-eukaryotic” primers (metabarcoding) to investigate brown algal-associated eukaryotes (the eukaryome). Using this approach, we aimed to investigate the eukaryome of seven large brown algae that are important and common species in coastal ecosystems. We also aimed to assess whether these macroalgae harbor novel eukaryotic diversity and to ascribe putative functional roles to the host-associated eukaryome based on taxonomic affiliation and phylogenetic placement. We detected a significant diversity of microeukaryotic and algal lineages associated with the brown algal species investigated. The operational taxonomic units (OTUs) were taxonomically assigned to 10 of the eukaryotic major supergroups, including taxonomic groups known to be associated with seaweeds as epibionts, endobionts, parasites, and commensals. Additionally, we revealed previously unrecorded sequence types, including novel phaeophyte OTUs, particularly in the Fucus spp. samples, that may represent fucoid genomic variants, sequencing artifacts, or undescribed epi-/endophytes. Our results provide baseline data and technical insights that will be useful for more comprehensive seaweed eukaryome studies investigating the evidently lineage-rich and functionally diverse symbionts of brown algae.     

Aerobic microbial degradation of alginate in Laminaria hyperborea stipes containing different levels of polyphenols

The polyphenols present in brown seaweed tissue may seriously affect aerobic microbial degradation, particularly the alginate present. Laminaria hyperborea stipes, harvested at 59 °N off the Norwegian coast in autumn, were degraded at different levels of polyphenols in aerated batch reactors at 35 °C and pH 7. This was achieved by manipulating the relative amounts of peripheral tissue, by removing or adding the mechanically peeled outer phenolic layer, using standardized inocula already adapted to L. hyperborea degradation. The degradation of organic matter was clearly depressed by increasing the amount of peripheral tissue. Alginate lyase activity was also negatively correlated to the amount of peripheral tissue loaded, presumably due to the release of reactive polyphenols. The total digestion rates of alginate were reduced by more than a factor of two at enhanced amounts of peripheral tissue. The guluronic content of extracted Na-alginate increased during the degradation, despite the presence of significant amounts of guluronate specific alginate lyase activity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Composition of phytobenthos in “lab-lab”, a periphyton-based extensive aquaculture technology for milkfish in brackishwater ponds during dry and wet seasons

The study compared the species composition of phytoperiphyton (“lab-lab”) present in ponds when gradually filled with water weekly to depths of 5, 10, 15 and 30 cm between the wet and dry seasons, for one month before the stocking of fish was studied. This was done during the dry season (March–April, 2003) and wet season (June–July, 2002). Periphyton was allowed to grow on 24 artificial substrates set at equal distances in a 1000 m² pond. “Lab-lab” that colonized the artificial substrates and that on the pond surrounding the substrates were scraped off from a measured surface area. Simultaneously, water was collected for the analysis of physical, chemical and biological parameters. Sampling was done bi-weekly coinciding with 2 and 7 days submergence at a desired depth before adjusting the water level. The major algae consisted of the diatoms (Bacilliarophyta), the blue green algae (Cyanobacteria), and the green algae (Chlorophyta). The diatoms were dominant during the dry season while the cyanobacteria dominated during the wet season. Twenty eight genera were observed during the dry season and 25 genera were noted in the wet season. Variation in genera and density that were observed every sampling period, was influenced by environmental conditions and the incoming water. The total algal density ranged from 100.7 × 10⁸ – 855.1 × 10⁸ and to 24.7 × 10⁸ – 83.9 × 10⁸ organisms.m⁻² during the dry and wet seasons, respectively. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Effect of Processing on the Microflora of Norwegian Seaweed Meal,with Observations on Sporendonema minutum (H?ye) Frank and Hess

Meal from the brown seaweed Ascophyllum nodosum (L.) Le Jol. is mainly used as an animal feed supplement. Since moist weed often develops a marked mold growth and since little was known about the microflora of seaweed meal, a cultural procedure was developed to enumerate the populations of bacteria, yeasts, and molds of seaweed meals manufactured by different drying processes. The microflora could be supported by a variety of media varying in levels of nutrition and in the source and concentration of salts. Fresh weed contained less than 10³ bacteria and less than 10² yeasts and molds per g (dry weight). The type and extent of microbial populations in seaweed meal appeared to be dependent upon the method of seaweed drying. Rotary drum-drying at temperatures decreasing from 800 to 80 C maintained or reduced the microbial populations to 10³ organisms per g (dry weight). Although meals with high nutritional quality can be obtained with warm air- or rock-dried weed, these conditions can also permit bacterial and mold development. Extended rock-drying in variable weather conditions and prolonged storage of moist weed, both of which decrease the nutritional quality, also lead to high bacterial numbers and to a marked development of the halophilic brown mold Sporendonema minutum which attained populations of 10⁸ viable spores per g of dried weed. A poultry diet containing 5% badly molded weed had no apparent toxic or growth-depressing effect when fed to chicks.     

The cellulose contents of Indian seaweeds

Cellulose content as well as alpha (α) and beta (β) celluloses were evaluated in 21 seaweed species belonging to different classes growing in Indian waters. The greatest yields of cellulose (crude) and β-cellulose were obtained from Caulerpa taxifolia (approx. 11.0% and 5.2%, respectively), whilst α-cellulose (approx. 8.2%) was the greatest in Padina tetrastromatica. The lowest cellulose (crude), α- and β- contents were recorded from the calcareous red alga Liagora ceranoides (approx. 0.85%, 0.62% and 0.18%, respectively). There was no variation in the yields of cellulose in the brown algae, whilst wide variations in the yields were found in the green and red algae.     

Viability of ectomycorrhizal fungus mycelium entrapped in calcium alginate gel

 We studied the viability of fragmented mycelium of Pisolithus tinctorius and Paxillus involutus entrapped in calcium alginate gel to determine the efficacy of this method of producing ectomycorrhizal fungus inoculum. Fungi were grown in MMN solution at 28  °C before being fragmented in a blender and subsequently entrapped in calcium alginate. We tested different ratios of alginate and mycelium suspension to 0.7 M CaCl₂. The ratio 8 : 10 resulted in well-formed beads of the highest viability for Paxillus involutus (99%) and for Pisolithus tinctorius (75%). Paxillus involutus mycelium was more than 90% viable when entrapped mycelium was 10 to 50 days old, and Pisolithus tinctorius attained its highest viability (55%) for 20- to 40-day-old mycelium. Gel entrapped Paxillus involutus mycelium grew well at all temperatures after 30 days of storage, but viability significantly decreased after 60 days storage at 6  °C on dry filter paper. For gel-entrapped Pisolithus tinctorius mycelium, viability was highest when stored at 25  °C in 0.7 M CaCl₂. Entrapment of Paxillus involutus fragmented mycelium in calcium alginate beads under the conditions that we propose can be used successfully to produce inoculum. Accepted: 11 October 1998     

微生物来源褐藻胶(alginate)生物合成及应用前景

褐藻胶及其制品在医药、食品及化工等领域具有重要价值。除褐藻以外,微生物是褐藻胶的另一重要来源。虽然微生物来源的褐藻胶未能够实现规模化生产,但是由于微生物合成褐藻胶具有发酵条件可控、产物单一、结构稳定并且易于纯化等优势,引起广泛的关注。并且利用生物工程技术已经实现了对微生物来源褐藻胶结构的调控和改造,促进了褐藻胶的高值化利用。此文综述了微生物褐藻胶生物合成的概况,并对利用基因工程改造褐藻胶的发展趋势和应用前景进行了论述。     

Effect of Processing on the Microflora of Norwegian Seaweed Meal, with Observations on Sporendonema minutum (Høye) Frank and Hess

Meal from the brown seaweed Ascophyllum nodosum (L.) Le Jol. is mainly used as an animal feed supplement. Since moist weed often develops a marked mold growth and since little was known about the microflora of seaweed meal, a cultural procedure was developed to enumerate the populations of bacteria, yeasts, and molds of seaweed meals manufactured by different drying processes. The microflora could be supported by a variety of media varying in levels of nutrition and in the source and concentration of salts. Fresh weed contained less than 10(3) bacteria and less than 10(2) yeasts and molds per g (dry weight). The type and extent of microbial populations in seaweed meal appeared to be dependent upon the method of seaweed drying. Rotary drum-drying at temperatures decreasing from 800 to 80 C maintained or reduced the microbial populations to 10(3) organisms per g (dry weight). Although meals with high nutritional quality can be obtained with warm air- or rock-dried weed, these conditions can also permit bacterial and mold development. Extended rock-drying in variable weather conditions and prolonged storage of moist weed, both of which decrease the nutritional quality, also lead to high bacterial numbers and to a marked development of the halophilic brown mold Sporendonema minutum which attained populations of 10(8) viable spores per g of dried weed. A poultry diet containing 5% badly molded weed had no apparent toxic or growth-depressing effect when fed to chicks.     

Biochemical characteristics of cellulose and a green alga degradation by Gilvimarinus japonicas 12-2T,and its application potential for seaweed saccharification

ABSTRACT

Cellulose is one of the major constituents of seaweeds, but reports of mechanisms in microbial seaweed degradation in marine environment are limited, in contrast to the multitude of reports for lignocellulose degradation in terrestrial environment. We studied the biochemical characteristics for marine cellulolytic bacterium Gilvimarinus japonicas 12-2^T in seaweed degradation. The bacterial strain was found to degrade green and red algae, but not brown algae. It was shown that the bacterial strain employs various polysaccharide hydrolases (endocellulase, agarase, carrageenanase, xylanase, and laminarinase) to degrade seaweed polysaccharides. Electrophoretic analysis and peptide sequencing showed that the major protein bands on the electrophoresis gel were homologous to known glucanases and glycoside hydrolases. A seaweed hydrolysate harvested from the bacterial culture was found useful as a substrate for yeasts to produce ethanol. These findings will provide insights into possible seaweed decomposition mechanisms of Gilvimarinus, and its biotechnological potential for ethanol production from inedible seaweeds.     

Accumulation of phlorotannins in the abalone Haliotis discus hannai after feeding the brown seaweed Ecklonia cava

Value-added abalone Haliotis discus hannai containing bioactive phlorotannins is produced by simply changing the feed to phlorotannin-rich brown seaweed Ecklonia cava 2 weeks prior to harvesting. We assessed the accumulation of phlorotannins by feeding with the seaweed after 4 days of starvation. Reverse-phase high-performance liquid chromatography afforded isolation of the major phlorotannins, which were identified by mass spectrometry and ¹H-nuclear magnetic resonance to be 7-phloroeckol and eckol. Throughout the E. cava feeding period of 20 days, 7-phloroeckolol accumulated in the flesh (foot muscle tissue), up to 0.85?±?0.21 mg g^?1 dry weight of tissue after 12 days. Eckol reached 0.31?±?0.08 mg g^?1 dry tissue after 14 days. Feeding Laminaria japonica as a control, we detected no phlorotannins in the abalone muscle tissue. Abalone seaweed consumption and growth rate were similar when fed with E. cava or L. japonica for 20 days. Reduction in phlorotannins to half-maximal accumulation took 1.0 and 2.7 days for 7-phloroeckol and eckol, respectively, after replacement of the feed with L. japonica.     

Characteristics of Aluminum Biosorption by Sargassum fluitans Biomass

Biomass of nonliving brown seaweed Sargassum fluitans pretreated by different methods is capable of taking up more than 10% (11 mEq/g) of its dry weight in aluminum at pH 4.5. There are indications that the biomass hydroxyl groups were involved in sequestering the aluminum in the form of polynuclear aluminum species. Aluminum-alginate complex (like cotton candy) was formed in the aluminum sorption solution as alginate was partially released from the biomass. Aluminum uptake of S. fluitans biomass was independent of residual alginate content in the biomass. Sodium ion added for pH adjustment was not adsorbed at all in the presence of aluminum ion. Received March 11, 1998; accepted October 9, 1998.     

Thermal ecotypes of amphi-Atlantic algae. I. Algae of Arctic to cold-temperate distribution (Chaetomorpha melagonium,Devaleraea ramentacea andPhycodrys rubens)

Three species of Arctic to cold-temperate amphi-Atlantic algae, all occurring also in the North Pacific, were tested for growth and/or survival at temperatures of −20 to 30°C. When isolates from both western and eastern Atlantic shores were tested side-by-side, it was found that thermal ecotypes may occur in such Arctic algae.Chaetomorpha melagonium was the most eurythermal of the 3 species. Isolates of this alga were alike in temperature tolerance and growth rate but Icelandic plants were more sensitive to the lethal temperature of 25°C than were more southerly isolates from both east and west. With regard toDevaleraea ramentacea, one Canadian isolate grew extraordinarily well at −2 and 0°C, and all tolerated temperatures 2–3°C higher than the lethal limit (18–20°C) of isolates from Europe. ConcerningPhycodrys rubens, both eastern and western isolates died at 20°C but European plants tolerated the lethal high temperature longer, were more sensitive to freezing, and attained more rapid growth at optimal temperatures. The intertidal species,C. melagonium andD. ramentacea, both survived freezing at −5 and −20°C, at least for short time periods.C. melagonium was more susceptible thanD. ramentacea to desiccation. Patterns of thermal tolerance may provide insight into the evolutionary history of seaweed species.     

Purification and characterization of a novel alginate lyase from the marine bacterium Cobetia sp. NAP1 isolated from brown algae

The application of marine resources, instead of fossil fuels, for biomass production is important for building a sustainable society. Seaweed is valuable as a source of marine biomass for producing biofuels such as ethanol, and can be used in various fields. Alginate is an anionic polysaccharide that forms the main component of brown algae. Various alginate lyases (e.g. exo- and endo-types and oligoalginate lyase) are generally used to degrade alginate. We herein describe a novel alginate lyase, AlgC-PL7, which belongs to the polysaccharide lyase 7 family. AlgC-PL7 was isolated from the halophilic Gram-negative bacterium Cobetia sp. NAP1 collected from the brown algae Padina arborescens Holmes. The optimal temperature and pH for AlgC-PL7 activity were 45 °C and 8, respectively. Additionally, AlgC-PL7 was thermostable and salt-tolerant, exhibited broad substrate specificity, and degraded alginate into monosaccharides. Therefore, AlgC-PL7 is a promising enzyme for the production of biofuels.     

褐藻降解菌群筛选及其复合酶固定化研究

以大连地区的褐藻为材料,筛选了褐藻降解菌群,其在30℃、pH7．5条件下培养74h时,酶活达1．883IU／mL。利用硅藻土吸附法经冷冻干燥制备了固定化复合酶,分析了复合酶系的酶学性质,其最适反应温度为45℃,并在40℃～55℃范围内具有良好的热稳定性;最适pH为7．5,并在pH7．0～8．5之间pH稳定性良好。利用固定化酶进行褐藻酸钠制备,提取率达48．3％,粘度为2．9Pa·s,与传统方法相比均有显著提高,为工程化生产褐藻酸钠提供一定基础。     

Sodium alginate from Sargassum wightii retards mortalities in Penaeus monodon postlarvae challenged with white spot syndrome virus

Sodium alginate extracted from brown seaweed Sargassum wightii (16.35 ± 1.42%, mean [±SD] yield from 5 extractions) was prepared as a powder or beads and used to enrich Artemia nauplii at concentrations of 100, 200, 300 and 400 mg l-1. The alginate-enriched nauplii were fed to Penaeus monodon shrimp postlarvae (PL) stage 15 (PL15, i.e. 15 d old) for 20 d. Mean weight gain and specific growth rate over this period were 0.24 g and 15.8%, respectively, in PL groups not fed alginate, and 0.20-0.28 g and 14.7-16.5%, respectively, in PL groups fed alginate. Amongst PL35 then challenged with white spot syndrome virus (WSSV) by immersion, all PL not fed alginate died within 9 d. However, amongst PL fed the 4 concentrations of alginate powder or beads, mortality rates reduced with increasing alginate concentration, and between 25 and 32% PL remained alive when the bioassay was terminated on Day 21. Amongst alginate-fed PL groups compared with the control group, mortality was reduced by 26.5 to 58.4%. Nested PCR detection of WSSV revealed sodium alginate concentration-dependent reductions in infection loads. The data indicate that sodium alginate extracted from brown seaweed and fed to P. monodon can retard progression of WSSV disease.     

Extraction and characterization of an alginate from the brown seaweed <Emphasis Type="Italic">Sargassum turbinarioides</Emphasis> Grunow

Matrix polysaccharide from the brown algae Sargassum turbinarioides collected in the coastal waters of Nosy Be (Madagascar) in the Indian Ocean was isolated and its structure was studied by ¹H-NMR spectroscopy, FT-IR, SEC-MALLS and HPAEC. An alginate with a molecular weight of 5.528 × 10⁵ g mol⁻¹ was identified as sole polysaccharide. Values of the M/G ratio, F _GG, F _MM and F _GM (or F _GM) blocks were measured at respectively 0.94, 0.39, 0.36 and 0.25 and compared with those of alginates from other Sargassum species. This sodium alginate appeared similar to some of the other Sargassum alginates with M/G < 1, high values of homopolymeric blocks (η < 1) and significant polyguluronic block content.