Using agar extraction waste of Gracilaria lemaneiformis in the papermaking industry

In China, fiber supply lags behind the growing demand for paper and paperboard products. The increasing consumption of paper products necessitated the need for new fiber sources. The red alga Gracilariopsis lemaneiformis is economically cultivated on a large scale in China for industrial agar extraction. During the extraction processes, considerable amounts of solid residues are produced as extraction wastes. In this study, we explored the potential of using the agar extraction residues as raw materials for pulping and papermaking. The results show that the extraction wastes of G. lemaneiformis could indeed be utilized for papermaking. Evaluation of the paper handsheets showed that a higher content of algal material resulted in paper that had lower strength and permeability but higher waterproof and greaseproof characteristics, as well as better antimicrobial effects. The results indicated that alga extraction residues could be employed as functional fillers to produce paper products that are potentially useful in the food-packaging industry.     

Agars from three species of Gracilaria (Rhodophyta) from Yucatán Peninsula

Gracilaria cervicornis, Gracilaria blodgettii and Gracilaria crassissima growing along the coasts of Yucatán were investigated for their agar content. The effect of different concentrations of NaOH in the alkali treatment was evaluated. The three species of Gracilaria produced agars, both native and alkali treated, with different properties confirming the heterogeneity of the agar polymers in this genera. G. cervicornis produces agar polymers with an occurrence of methoxylation and sulphation at the C-6 of the beta-D-galactose residues, and with an extra methylation due to the presence of the 4-O-methyl-alpha-L-galactose residue. The presences of these residues is responsible for the extremely poor gelling ability of its agarocolloids, whose commercial value seems to be quite low. Agar extracts from G. blodgettii showed the typical pattern of unsubstituted agar with a very low degree of methylation on both galactose residues. The discrepancy found between sulphate content and NMR data of agar from this species requires a more detailed structural investigation. G. crassissima produces a good quality agar after sulphate precursor sequences have been removed by alkaline treatment, and it may be considered for exploitation as a source of commercial grade agar. Alkali treatment was effective both in removing alkali-labile sulphate and increasing the gel strength in G. crassissima but not in G. cervicornis and G. blodgettii.     

Agar Processed from Gracilaria foliifera of Florida

Agar producing a firm gel with specific rheological properties as well as a good commercial value was processed from Gracilaria foliifera (Forsskal) Børgesen, a red alga harvested along the Florida west coast. The alkali pretreatment of the seaweed based on the Funaki-Kojima’s method was applied before the process of agar extraction. The chemical and physical properties of the processed agar were compared with other typical agar experimentally processed.     

Structural studies of water-soluble glycoproteins from Cannabis sativa L

Two carbohydrate-protein fractions, isolated from Cannabis sativa L. by extraction with water and chromatography on DEAE-cellulose, contained arabinose, galactose, glucose, mannose, galacturonic acid, 2-acetamido-2-deoxyglucose, and 2-acetamido-2-deoxygalactose. The structure of the carbohydrate moieties was investigated by methylation analysis and Smith degradation. A high percentage of end-groups indicates a large degree of branching, glucose and galactose being the main branch-points, linked at C-3 and C-6. The hexoses are also present as unbranched residues in the chain, largely as (1→3)- and (1→4)-linked units and as end-groups. Arabinofuranosyl units constitute the main part of the non-reducing end-groups, and are also present as part of the chain. The polysaccharide chains are probably linked to protein through the hydroxyl group of hydroxyproline.     

Effects of seasons on the chemical structure and gel strength of Gracilaria pseudoverrucosa agar (Gracilariaceae, rhodophyta)

The seasonal effects on the chemical structure and rheological properties of Gracilaria pseudoverrucosa agar have been investigated using a sequential solvent extraction, ¹³C NMR and infrared spectroscopy, and gel strength measurements. The results showed that agar enriched in precursor to the agarobiose repeat unit were obtained from algae collected in summer. In contrast, algae collected in winter contained agar molecules richer in alkali-stable sulfate groups attributed in part to -galactose-4-sulfate. A similar total concentration of 6-O-methylated agarobiose repeat units was present in the agar from both algal samples but the distribution of the methylated disaccharide varied in the fractions. Agar fractions from the summer-collected sample had higher gel strength than those of the winter ones. Alkali treatment markedly improved the gel strength of the agar from the summer harvested seaweed. Different gel strengths were observed for the native and alkali-treated agar fractions extracted from the same algal sample and a gel strength comparable to that obtained for a commercial bacteriological grade agar was obtained from the alkali-treated 40% ethanol extract agar from the summer collected alga. The chemical and rheological variations due to seasonal changes are interpreted as reflecting the ratio of actively-growing (young) to resting (old) tissue in the alga and are proposed to represent a type of ‘secondarization’ of the algal cell-wall.     

Production and properties of agar from the invasive marine alga, Gracilaria vermiculophylla (Gracilariales, Rhodophyta)

The utilization potential, in terms of agar production, of the invasive alga, Gracilaria vermiculophylla, collected at Ria de Aveiro, northwestern Portugal was investigated. The agar yield ranged from 15% to 33%, with pre-extraction treatment with alkali generally increasing the yield. The gel quality (gel strength and apparent Young’s modulus) was best (>600 g cm^?2 and >1,000 kPa, respectively) when alkali treatment with 6% NaOH for 3.5 h was performed. At these pretreatment conditions, the effect of extraction time was also investigated and highest yield and best gel quality were obtained with a 2 h extraction time. By employing these extraction conditions, G. vermiculophylla can be a source of industrial food-grade agar. The structure of agar from G. vermiculophylla was determined through chemical techniques and FTIR and NMR spectrometry. It is mainly composed of alternating 3-linked d-galactose and 4-linked 3,6-anhydro-l-galactose, with methyl substitution occurring at 16–19 mol% of C-6 in 3-linked units and 2–3 mol% of C-2 in 4-linked units. A minor sulfation on C-4 of 3-linked units was also detected; while precursor units (6-sulfated 4-linked galactosyl moieties) were found in the native extract.     

Comparative hydrolysis and fermentation of sugarcane and agave bagasse

Sugarcane and agave bagasse samples were hydrolyzed with either mineral acids (HCl), commercial glucanases or a combined treatment consisting of alkaline delignification followed by enzymatic hydrolysis. Acid hydrolysis of sugar cane bagasse yielded a higher level of reducing sugars (37.21% for depithed bagasse and 35.37% for pith bagasse), when compared to metzal or metzontete (agave pinecone and leaves, 5.02% and 9.91%, respectively). An optimized enzyme formulation was used to process sugar cane bagasse, which contained Celluclast, Novozyme and Viscozyme L. From alkaline–enzymatic hydrolysis of sugarcane bagasse samples, a reduced level of reducing sugar yield was obtained (11–20%) compared to agave bagasse (12–58%). Selected hydrolyzates were fermented with a non-recombinant strain of Saccharomyces cerevisiae. Maximum alcohol yield by fermentation (32.6%) was obtained from the hydrolyzate of sugarcane depithed bagasse. Hydrolyzed agave waste residues provide an increased glucose decreased xylose product useful for biotechnological conversion.     

Hydrothermal fractionation of Sargassum muticum biomass

Sargassum muticum is an invasive brown alga which could be regarded as a renewable resource susceptible to fractionation and integral valorization. S. muticum biomass was processed according to a scheme consisting of conventional alkaline extraction of alginate, ultrafiltration to concentrate antioxidant compounds lost in the waste streams and autohydrolysis of the solid residue remaining after alginate extraction. The effect of temperature during non isothermal autohydrolysis was optimized to maximize yields and antioxidant activity of the solubilized fraction. The overall solubilization yield reached 88% of the initial material and the solubilization yield from the alginate-exhausted solid up to 82%. The waste fraction concentrated by membrane technology and that solubilized during autohydrolysis showed radical scavenging and reducing activities comparable to commercial antioxidants. The solid insoluble by-product from the autohydrolysis stage could be potentially useful for agricultural purposes.     

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.     

Optimization of agar extraction from local seaweed species,Gracilaria salicornia in Tanzania

This study aimed to develop agar extraction protocols for Gracilaria salicornia from Tanzania and investigate its physico‐chemical characteristics. A 3³ factorial experimental design was used in the extraction of agar whereby three independent variables of NaOH concentration (10, 20 and 30% w/v), alkali pre‐treatment duration (0.5, 1 and 2 h) and extraction temperatures (115, 120 and 125°C) were used to determine the optimum conditions for production of high‐quality agar. Agar yield, gel strength, sulfate content, gelling and melting temperatures were evaluated as dependent variables. The optimal condition was observed at 30% NaOH concentration, 2 h alkali pre‐treatment duration and 120°C extraction temperature. The yield, gel strength, sulfate content, gelling and melting temperatures of the agar obtained under these conditions were 26.9 ± 0.7%, 510.3 ± 16.2 g cm^?2, 0.29 ± 0.04%, 39.3°C and 88.4°C, respectively. These properties are very close to that of imported commercial agar. It was concluded that the local agar is capable of replacing imported agar for most general purposes. This offers a new possibility of using quality local agar in place of commercial agar.     

Constitutive mutants for dextransucrase from Leuconostoc mesenteroides NRRL B-512F

Constitutive mutants for dextransucrase were isolated from cells of Leuconostoc mesenteroides NRRL B-512F by treatment with N-methyl-N′-nitro-N-nitrosoguanidine, growing on an agar plate containing glucose as a carbon source and overlaying a soft agar with sucrose and tetracycline. These mutants were able to produce the enzyme in a liquid media containing sugars other than sucrose, such as glucose, fructose and maltose, without simultaneous synthesis of dextran. The enzyme activity of one mutant strain, SH 3002, was 2- to 3-fold higher than that of the wild strain grown on sucrose. When the concentration of glucose in the medium was increased from 2 to 4%, a 1.7-fold increase of enzyme activity was obtained for the mutant, whereas only a slight increase of the activity was observed on sucrose for both the wild strain and the mutant.     

Carbohydrates of the antarctic brown seaweed Ascoseira mirabilis

Mannitol, sucrose and four monosaccharides were obtained from an ethanolic extract of Ascoseira mirabilis. Sequential extraction with aqueous calcium chloride, dilute acid and dilute alkali gave mixtures of laminaran, ‘fucan’ and alginic acid. Laminarans fractionated from the extracts contained different proportions of uniformly (1 → 3) and (1 → 6) linked chains of β-D-glucose residues. The ‘fucan’ contained varying proportions of fucose, galactose and glucuronic acid, small amounts of xylose, mannose, glucose, half ester sulphate and protein. Extraction of the weed under mild alkaline conditions gave a yield of 13.4% of low molecular weight calcium alginate with a mannuronate to guluronate ratio of 30:70 and only a small proportion of sequences of alternating residues. Selective extraction and fractionation gave alginate fractions rich (> 80%) in mannuronate or guluronate.     

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.     

Optimization of native agar extraction from Hydropuntia cornea from Yucatán, México

Response surface methodology (RSM) was used to determine optimum extraction conditions for yield, gelling temperature, melting temperature and apparent viscosity of native agar from the red alga Hydropuntia cornea. Two independent variables were selected during extraction, temperature (80, 90 and 100 degrees C) and time (2, 3 and 4 h). The extraction procedure using 100 degrees C and 3 h produced the greatest yield (43.3%), the greatest gelling temperature (32 degrees C) and the greatest melting temperature (78 degrees C). The extraction using 65 degrees C and 4 h produced the optimum statistical apparent viscosity (80.73 cPs), though agar yield under these conditions had low quality for industrial purposes. Temperature and time do not affect melting temperature, but do play an important role in gelling temperature. From the independent variables studied, extraction temperature is most important in producing an increase in yield, and time is the only variable that contributes significantly to increasing viscosity. Most of the physicochemical properties found in the native agar from H. cornea extracted at 100 degrees C/3 h were similar to commercial samples. These findings suggest the feasibility of using this colloid in spreads and soft-texture food products.     

Pilot plant scale extraction of alginate from Macrocystis pyrifera. 1. Effect of pre-extraction treatments on yield and quality of alginate

In the extraction of alginate from brown seaweeds, the acid pre-extraction treatment has been considered by many authors as an essential step because it makes the alginate more readily soluble in an alkaline solution. At pilot plant level, extractions were made (i) using formalin treatment prior to the acid pre-extraction treatment (ii) using different acid treatments so the calcium ions exchanged varied from 83% to 4%. The use of formalin treatment gave a product with less color. During the acid pre-extraction treatment, it was possible to reduce the calcium exchanged from 33.4% to almost zero with a maximum reduction in alginate yield of 7%. The degree of acid treatment was positively correlated to calcium exchanged and yield but negatively correlated with alginate viscosity. Using strong acid conditions the viscosity was 168 mPa s, while mild acid conditions produced an alginate with 623 mPa s. The direct extraction from calcium alginate to sodium alginate is possible because strong alkaline conditions were used, pH 10 at 80 °C for two hours and with a low water volume. The best pre-extraction treatment to obtain an alginate with high viscoity is to hydrate the alga with 0.1% formalin overnight, then wash the alga once with hydrochloric acid at pH 4 using a batch system with continuous agitation during 15 min. This revised version was published online in August 2006 with corrections to the Cover Date.     

Agars fromGracilaria chilensis (Gracilariales)

The chemical composition of the agars extracted with water at 95 °C fromGracilaria chilensis of Estero de Lenga (43.2% of yield) and of Bahia de Coquimbo (43.4% of yield) were determined. The gel strength, gel melting and gelation temperature of the agars were also measured. Polysaccharides obtained by extraction with cold water and by extraction of the alkali pre-treatedG. chilensis of Bahia de Coquimbo were also analyzed. The physical constants and the contents of sulfate and 3,6-anhydrogalactose of the alkali-modified agar fromG. chilensis of Estero de Lenga were comparable to those of commercial agar. This algae represents an excellent potential commercial resource of agar.G. chilensis of Bahia de Coquimbo gave hydrocolloids with low gel strength values more suitable for use in the food industry. Anion exchange chromatography of the agars obtained by extraction with water at 95 °C indicated that the whole polysaccharides and all of the acidic fractions were not homogeneous. Neutral fractions comprised only about 25% of the agars.     

The structure of the low molecular-weight glucans isolated from the siphonous green alga caulerpa simpliciuscula

A β-d-glucan of low molecular weight isolated from the marine alga Caulerpa simpliciuscula has been shown to contain 30 glucose residues. At least 27 of these are β-d-(1→3) linked. There are 1-2β-(1→6) branches per molecule, with a maximum of 4 d-glucose residues per side chain. As normally isolated, this glucan is associated with a soluble (1→4)-α-d-glucan (soluble starch) of the same molecular weight, in the ratio of 3 molecules of β-d-glucan per molecule of α-d-linked glucan.     

Agar from the red seaweed,Laurencia flexilis (Ceramiales,Rhodophyta) from northern Philippines

The worldwide production of the gelling agent agar mainly rely on the red algae of the order Gracilariales and Gelidiales for raw material. We investigate here the potential of a species from another red algal order, Ceramiales as an agar source. The agar from Laurencia flexilis collected in northern Philippines was extracted using native and alkali treatment procedures and the properties of the extracts were determined using chemical, spectroscopic and physical methods. The native agar, 26% dry weight basis, forms a gel with moderate gel strength (200 g cm^?2). Alkali‐treatment did not enhance the gel strength, indicating insignificant amounts of galactose‐6‐sulfate residue, the precursor of the gel‐forming 3,6‐anhydrogalactose (3,6‐AG) moieties. Furthermore, the Fourier transform infrared and chemical analysis showed low sulfate and high 3,6‐AG levels, not affected significantly by the alkali treatment. Nuclear magnetic resonance spectroscopic analysis revealed 3‐linked 6‐O‐methyl‐D‐galactose and 4‐linked 3,6‐anhydro‐L‐galactose as the major repeating unit of the native extract, with minor sulfation at 4‐position of the 3‐linked galactose residues. The native and alkali treated agars have comparably high gelling and melting temperatures, whereas the former exhibits higher gel syneresis. Laurencia flexilis could be a good source of agar that possesses physico‐chemical and rheological qualities appropriate for food applications. Due to the inability of alkali treatment to enhance the key gel qualities of the native extract, it is recommended that commercial agar extraction from this seaweed would be done without pursuing this widely‐used industrial procedure.     

Purification of Synechococcus lividus by equilibrium centrifugation and its synchronization by differential centrifugation

A culture of the thermophilic, unicellular, blue-green alga Synechococcus lividus was freed from two thermophilic bacteria by use of equilibrium (isopycnic) centrifugation in Ficoll density gradients. After removal of the bacteria, the alga would grow only on agar plates in a high carbon dioxide atmosphere. Intermittent illumination, equilibrium centrifugation, and differential centrifugation were tested as techniques for obtaining synchronized cultures of the alga. Daughter cells selected by differential centrifugation in Ficoll density gradients yielded the best synchrony, and a cross-wall index of 85% was observed during the period of cell division.     

Biosynthesis of dolichyl phosphate: characterization and site of synthesis in algae

This is the first report not only on the presence of polyprenyl phosphates and their site of synthesis in algae, but also on the formation of their sugar derivatives in this system.

A glucose acceptor lipid was isolated from the nonphotosynthetic alga Prototheca zopfii. The lipid was acidic and resistant to mild acid and alkaline treatments. The glucosylated lipid was labile to mild acid hydrolysis and resistant to phenol treatment and catalytic hydrogenation, as dolichyl phosphate glucose is. These results are consistent with the properties of an α-saturated polyprenyl phosphate.

The polyprenylic nature of the lipid was confirmed by biosynthesis from radioactive mevalonate. The [¹⁴C]lipid had the same chromatographic properties as dolichyl phosphate in DEAE-cellulose and Sephadex LH-20. Strong alkaline treatment and enzymic hydrolysis liberated free alcohols with chain lengths ranging from C₉₀ to C₁₀₅, C₉₅ and C₁₀₀ being the most abundant molecular forms. The glucose acceptor activity of the biosynthesized polyprenyl phosphate was confirmed.

The ability of different subcellular fractions to synthesize dolichyl phosphate was studied. Mitochondria and the Golgi apparatus were the sites of dolichyl phosphate synthesis from mevalonate.