Alginate from the macroalgae <Emphasis Type="Bold">Sargassum sinicola</Emphasis> as a novel source for microbial immobilization material in wastewater treatment and plant growth promotion

Alginate extracted from the macroalgae Sargassum sinicola was used as the raw material for co-immobilization of the microalgae Chlorella sorokiniana and growth-promoting bacterium Azospirillum brasilense for wastewater treatment and as an inoculant carrier of A. brasilense for plant growth promotion. The composition, structure, viscosity, color, and phenolic compound content of the alginate were analyzed and compared with commercially available alginate produced from the macroalgae Macrocystis pyrifera. From ¹H NMR analysis of alginate, S. sinicola was found to have more guluronic acid (F _G=0.64) than it had mannuronic acid (F _M=0.38) and had a viscosity of 13.5 m Pa s compared to 50 m Pa s for M. pyrifera. The S. sinicola alginate had dark brown color, reducing light penetration, with more phenolic compounds than M. pyrifera alginate. Nonetheless, growth of C. sorokiniana and A. brasilense in S. sinicola alginate was not significantly different than the growth in M. pyrifera alginate beads. Nutrient removal from wastewater by the co-immobilized microorganisms was similar for both types of alginate beads, and so was the growth enhancement of tomato plants inoculated with microbeads containing A. brasilense. This study shows the potential use of S. sinicola alginate as a raw material for cell immobilization for wastewater treatment and plant growth promotion.     

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.     

Monomer composition and sequence of sodium alginate extracted at pilot plant scale from three commercially important seaweeds from Mexico

The marine waters of the Baja California peninsula (Mexico) are a rich source of brown seaweeds with a great potential for exploitation. For that reason, Sargassum sinicola, Eisenia arborea, and Macrocystis pyrifera collected from different locations were subjected to extraction of sodium alginate using a pilot-plant scale process developed in our facilities. The composition and sequence parameters of the recovered alginate were studied by infrared and nuclear magnetic resonance spectroscopy. The spectral analysis of the products revealed that sodium alginate from S. sinicola contains a greater proportion of guluronate monomers (64%) than that from E. arborea (48%), and M. pyrifera (38%). Computation of the frequencies of diads and triads indicated that the alginate from S. sinicola was constructed by intercalated guluronate-blocks of 14 residues in length. In contrast, the length of the G-block in the alginates from E. arborea and M. pyrifera were 7 and 4 residues, respectively. The results show that S. sinicola, E. arborea, and M. pyrifera are sources of sodium alginate with different mannuronate/guluronate ratios, as well as a varied building-block length. In consequence, aqueous dispersions of sodium alginate from the three studied species are expected to exhibit different physical properties.     

Pilot plant scale extraction of alginates from Macrocystis pyrifera. 2. Studies on extraction conditions andmethods of separating the alkaline-insoluble residue

The effect of temperature (70, 80, 90 ^°C) and time (1–9 h) during the alkaline extraction step on alginate yield and quality were studied. The alginate yield increased with time and maximum yield was obtained after 3.5 h treatment, ranging from19.4% at 70 ^°C to 21.9% at 90 ^°C. The viscosity of the alginate produced was inversely correlated with the temperature and time. At70 ^°C the slope of the curve was almost zero(753 to 923 mPa s); at 90 ^°C the viscosity loss was 154 mPa s per hour during the first two hours, reducing from 523 to 86 mPa s after 5 h; 80 ^°C yielded values between those for 70 ^°C and90 ^°C. The best conditions for alkaline extraction were using pH 10 at 80 ^°C for two hours. The curves obtained gave useful information for controlling the viscosity of the alginate during production. It was found that viscosity of the paste formed during alkaline extraction (`process viscosity') was the best parameter to determine there action rate during extraction. Alginate yield increased during filtration time from 17.6% to 23.7%after 55 min at 70 ^°C. In this step the viscosity of the alginate obtained remained almost constant (522–610 mPa s), indicating no degradation of the products during filtration. The best dilution to filter the alginate extract was obtained at 45 mPa s. Diatomaceous earth (Celite) and expanded lava(Perlite) were tested as filter aids. Expanded lava was the best filter aid, using 1 kg per kilogram of alginate produced. Three methods were studied to separate the alkaline-insoluble residues after extraction: filtration, centrifugation, flocculation, and combinations of them. The best system found was filtration with flocculant in a rotary vacuum filter, with a knife advance of 0.1 mm every 3.5 seconds and drum rotation of 2 rpm, yielding an average filtration flow rate of 10.5 L min^-1. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Production of bioflavor by regeneration from protoplasts of Ulva pertusa (Ulvales,Chlorophyta)

Protoplasts were isolated from thalli of Ulva pertusa using a mixed enzyme solution of 2.0% Cellulase Onozuka R-10, 2.0% Macerozyme R-10, and 2.0% Driselase. Isolated protoplasts regenerated cell walls, developed into thalli, and propagated in large numbers under aeration in the preparative scale-culture system. Typical bioflavor compounds produced from the regenerated plants, as well as from field-collected plants, were found to be long chain aldehydes, which gave a typical seaweed odor. The long chain aldehydes were formed enzymatically from unsaturated fatty acids and released into the culture fluid. A Percoll/mannitol discontinuous density gradient separation of the heterogeneous protoplasts led to a selection of cell lines with high production of bioflavor. The cells that regenerated from protoplasts were immobilized by polymer matrices such as alginate, -carrageenan, agarose, and agar. Living cells entrapped in alginate beads in aerated cultures survived best. However, the beads started to breakdown after two months. The immobilized cells demonstrated a higher bioflavor production than did the cultured cells.     

NEW INSIGHTS IN THE TAXONOMY OF THE CERAMIUM SINICOLA COMPLEX: RESURRECTION OF CERAMIUM INTERRUPTUM (CERAMIACEAE,RHODOPHYTA)1

Phylogenetic relationships of the Ceramium sinicola complex (C. interruptum and C. sinicola) including C. codicola were studied using nucleotide sequences of rbcL and small subunit rDNA, and the RUBISCO spacer was used for sequence comparison of each species. A reassessment of the taxonomic rank and the evolutionary trend within the complex was inferred from a comparative morphological study and molecular data sets based on 11 samples from eight populations from the Pacific coast of the United States and Mexico. Intraspecific relationships were poorly resolved, but the resurrection of C. interruptum as a distinct species was strongly supported by both morphological and molecular data. Ceramium interruptum is distinguished by the combination of the following features: thalli uncorticated at the first internode above the dichotomy, presence of four corticating filaments, 7–11 segments between branching points, rhizoids digitate, and epiphytic on a variety of hosts. Our molecular analyses show that C. sinicola is the sister group to C. codicola, and C. interruptum is basal to them. These phylogenetic relationships allowed for an assessment of the trend in the evolution of cortication pattern and attachment mode to the host.     

Carbohydrate degradation and methane production during fermentation ofLaminaria saccharina (Laminariales,Phaeophyceae)

Anaerobic digestion of the brown algaLaminaria saccharina (L.) Lamour. harvested in spring and autumn was carried out at controlled laboratory conditions in stirred fermentor systems. Due to the normal seasonal variations, the autumn material had a much higher content of carbohydrates such as mannitol and laminaran. Both batch and semi-continuous feeding conditions were investigated for periods up to 800 h, with inoculum provided from previous kelp fermentations. In batch cultures, the methane yield from the autumn material was doubled compared to that of the spring material. Semi-continuous conditions gave more similar methane yields for both raw materials, 0.22 and 0.27 l CH₄ per g VS for spring and autumn material, respectively. In all experiments, mannitol and laminaran were reduced to less than 5 of the initial values within 24–48 hours after inoculation, whereas 30 of the alginate content was detectable even after 30 days. Viscometry revealed that this material was severely depolymerized, and alginate lyase activity was found to develop rapidly in all cultures. Although mannitol and laminaran were fermented much faster than alginate, the total accumulated methane yields seemed to be determined by the total carbohydrate content of the raw material during extended semi-continuous feeding.(*author for correspondence)     

Pilot plant scale extraction of alginates from Macrocystis pyrifera 4. Conversion of alginic acid to sodium alginate, drying and milling

The last three steps of the alginate production process were studied:conversion of alginic acid to sodium alginate, drying, and milling. Threemethods were used to follow the conversion reaction: measuring the pH (a) intheethanol-water liquid of the reaction mixture, (b) after dissolving a sample ofthe fiber taken from the reaction mixture, (c) after dissolving the driedsodiumalginate obtained from the reaction. To obtain a neutral dried sodium alginate,in the first method the pH should be adjusted to 9, and in the second the pHshould be adjusted to 8. The best method to control the reaction was todissolvea sample of the fiber and adjust the pH to 8. The best proportion to reach thecritical point, where pH just begins to rise, was 0.25 parts of sodiumcarbonateto 1 part of alginate in the initial dry algae. A pH above 7 may produce abreakdown of the molecule, reducing significantly the viscosity of the finalalginate. Four different temperatures were used to dry the alginate: 50, 60,70,and 80 °C. Drying time to reach 12% moisture ranged from 1.5h at 80 °C to 3 h at 50°C. The best drying temperature was 60 °C for2.5 h. The effect of drying temperature on alginate viscosity wasdependent on the alginate type. Low and medium viscosity alginates were notsignificantly affected, but alginate with high viscosity was reduced by 40 to54% using the temperature range of 60 to 80 °C. A fixed hammermill was used to reduce the particle size of the dried sodium alginate.Particlesize measurements showed that after a first milling the product contained 76%large particles (20–60 mesh) and 24% fine particles (80–120 mesh).After a third milling the product still contained 42.9% large particles. Nosignificant effect was found on alginate viscosity because of the millingsteps.     

Changes in alginate molecular mass distributions, broth viscosity and morphology of Azotobacter vinelandii cultured in shake flasks

The effect of different aeration conditions during the culture of Azotobacter vinelandii on the production and molecular mass of alginate was evaluated in shake flasks. In baffled flasks, the bacteria grew faster and produced less alginate (1.5 g/l) than in conventional (unbaffled) flasks (4.5 g/l). The viscosity of the culture broth was also influenced by the type of flask. Higher final viscosities were attained in unbaffled flasks [520 cP (520 mPa s)] as compared to baffled flasks (30 cP). This latter phenomenon was closely related to the changes in the molecular mass distribution. In either cases, the mean molecular mass increased with culture age; however, at the end of the fermentation, the mean molecular mass of the alginate obtained in unbaffled flasks was fivefold higher than that obtained in baffled flasks. As the culture proceeded, the cells of Azotobacter grown in unbaffled flasks increased in diameter, whereas those cultured in baffled flasks decreased in size. Received: 13 December 1996 / Received revision: 10 April 1997 / Accepted: 27 April 1997     

Pilot plant scale extraction of alginates from Macrocystis pyrifera 3. Precipitation, bleaching and conversion of calcium alginate to alginic acid

Three steps of the alginate production process were studied at pilot plantlevel. The effect of the amount of calcium chloride used during theprecipitation was measured in terms of filtration time of the precipitatedcalcium alginate. Three different proportions of calcium chloride per gramof alginate were tested. The best proportion used was 2.2 parts ofcalcium chloride per one part of alginate, yielding a filtration rate of 97.9L min^-1 on a screen area of 1.32 m². The method ofadding the solutions and the degree of mixing are discussed as other factorsaffecting the precipitation step. The effect of bleaching the calciumalginate with sodium hypochlorite (5%) was studied. Seven proportions,ranging from 0 to 0.77 mL of sodium hypochlorite per gram of sodiumalginate were tested. The effect of hypochlorite was compared foralginates with three different viscosities. Using alginates with mediumviscosity (300–500 mPa s), the best proportion was 0.4 mL hypochloriteper gram of alginate, yielding an alginate of light cream color with 20%less viscosity than the control. Alginates with lower viscosity showed asmaller loss of viscosity. The effect of pH during conversion of calciumalginate to alginic acid was determined using four combinations of pH,ranging from 2.2 to 1.6, in three acid washings. The extent of conversionwas determined by measuring the percent reduction of the alginate viscosity(RV) in 1% solution before and after adding a sequestrant of calcium. When a pH 1.8 or 1.6 was used for each washing, only two washings werenecessary to produce a RV lower than 40% (maximum recommended). The use of pH 2 required three acid washings to produce the same effect. The pH 2.2 did not remove enough calcium, even with three washings,the RV of the resulting sodium alginate being greater that 40%. Theresults of these experiments provide the information that producers needwhen deciding the best parameters to obtain a product with the desiredcharacteristics.     

CLASSIFICATION OF THE GENUS ISHIGE (ISHIGEALES,PHAEOPHYCEAE) IN THE NORTH PACIFIC OCEAN WITH RECOGNITION OF ISHIGE FOLIACEA BASED ON PLASTID rbcl AND MITOCHONDRIAL cox3 GENE SEQUENCES1

The taxonomy and biogeography of a genus with species that occur in geographically isolated regions is interesting. The brown algal genus Ishige Yendo is a good example, with species that apparently inhabit warm regions of both the northwestern and northeastern Pacific Ocean. We determined the sequences of mitochondrial cox3 and plastid rbcL genes from specimens of the genus collected over its distributional range. Analyses of the 86 cox3 and 97 rbcL sequences resulted in congruent trees in which Ishige sinicola (Setch. et N. L. Gardner) Chihara consisted of two distinct clades: one comprising samples from Korea and Japan, and the other comprising samples from the Gulf of California. Additional observations of the morphology and anatomy of the specimens agree with the molecular data. On the basis of results, we reinstated Ishige foliacea S. Okamura (considered a synonym of I. sinicola from the Gulf of California) for plants from the northwest Pacific region and designated a specimen in the Yendo Herbarium (SAP) as the lectotype. I. foliacea is distinguished by large (up to 20 cm) and wide (up to 20 mm) thalli, with a cortex of 4–7 cells, and a medulla composed of long, tangled hyphal cells. Both cox3 and rbcL sequence data strongly support the sister‐area relationship between the northwest Pacific region and the Gulf of California. A likely explanation for this pattern would be the presence of a species ancestral to contemporary species of Ishige in both regions during the paleogeological period, with descendants later isolated by distance.     

Induction of callus in the liverwort Marchantia palmata Nees

Abstract

In Marchantia palmata Nees, normal growth of thalli occurs on Nitsch's basal medium with 1% sucrose. With 4% sucrose, dark green callus is initiated by light intensities of 150–4,500 lux after 13–15 days of growth. In the dark only rhizoids develop from callus and at low light intensity (150 lux) rhizoids as well as knob-like green outgrowths differentiate. Increased light intensities induce differentiation of thalli and rhizoids from callus. The callus inducing effect of increased sucrose level can be replaced by higher concentrations of mannitol. Histological studies reveal that callus initially arises from the lower epidermis.     

Two new species of Gelidium (Rhodophyta, Gelidiales), Gelidium tenuifolium and Gelidium koshikianum, from Japan

Two new marine red algae, Gelidium tenuifolium sp. nov. and Gelidium koshikianum sp. nov. (Gelidiales, Gelidiaceae) are described from Japan. Gelidium tenuifolium with large‐sized thalli (up to 30 cm tall) is distinguished from other species with such thalIi by the production of wide, flattened and thin branches (up to 2 mm wide and 60–80 urn thick), the presence of an apical depression and simple determinate branches. Gelidium koshikianum with middle‐sized thalli (5–8 cm tall) is distinguished from other species with such thalli by having wide axes (up to 2.5 mm wide) and short (2.0–3.2 mm), unbranched, second‐ and third‐order branches issuing at short intervals (0.8–1.4 mm). In phylogenetic analyses of rbcL sequences, four Gelidium species that are chiefly distributed in Japan including G. tenuifolium were clustered together with 99% bootstrap value (Japanese Gelidium‐complex clade). Gelidium linoides Kützing came to the position of the sister group to G. tenuifolium with 99% bootstrap value. There were four substitutions (0.3% divergence) between G. linoides and G. tenuifolium sequences. Gelidium koshikianum and Gelidium allanii Chapman were clustered together with 100% bootstrap value and they came to the position of the sister group to the Japanese GeIidium‐complex clade with 83% bootstrap value. There were six substitutions (0.4% divergence) between G. koshikianum and G. allanii sequences.     

Effect of carbon dioxide on the rheological behavior of submerged cultures of Chlorella minutissima in stirred tank reactors

The objective of this study was to quantify the effect of algal biomass concentration on the rheology of the algal culture broth. Batch cultivations of Chlorella minutissima were carried out with air and carbon dioxide in a stirred tank bioreactor with a working volume of 1.8 L. The apparent viscosity of the culture broth was significantly affected by the cell mass concentrations in the bioreactor. Culture broth containing 50 g/L cell mass from air fed was found to exhibit an apparent viscosity of 1.52 mPa.s. The apparent viscosity of the carbon‐dioxide‐fed cultivations was found to increase by 20% at a shear rate of 100 s^?1. The flow behavior of the system was adequately described by the Herschel–Bulkley model with a small yield stress.     

Regeneration from Laminaria japonica Areschoug (Laminariales, Phaeophyceae) protoplasts isolated with bacterial alginase

Summary The conditions for effective isolation of viable protoplasts from Laminaria japonica with an alginase produced by marine bacterium Alteromonas sp. and a commercially available cellulase were investigated. The highest yields of viable protoplasts (7.910.4x10⁶ cells g^–1 FW) were obtained with a hypertonic solution containing 50 % seawater, 25 mM MgCl₂, 5 mM HEPES buffer system, and 0.5 M mannitol. Protoplasts were not obtained from thalli of L. japonica when an abalone alginase (abalone acetone powder; AAP: Sigma) was used instead of the bacterial alginase. The isolated protoplasts were cultured in an PESI medium at 5 °C. Complete cell wall formation was observed within 7 days, and dividing cells were first observed in a 9-day-old culture. Some protoplasts regenerated into sheet-shaped thalli and rhizoid structures were also observed on some thalli after 30 to 40 days in culture. This is the first report of protoplast regeneration into plantlets of L. japonica Areschoug (Laminariales, Phaeophyceae).Abbreviations FW Flesh weight - AAP Abalone acetone powder - HEPES N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid - Tris Tris(hyrdoxymethyl)aminomethane - PESI Provasoli's enriched seawater with iodine     

Plant regeneration from protoplasts of Laminaria japonica Areschoug (Laminariales, Phaeophyceae) in a continuous-flow culture system

A continuous-flow culture system was developed for culturing Laminaria japonica protoplasts. Protoplasts were settled on 5-μm pore size nylon mesh fixed inside a 50-ml plastic syringe, and cultured in Provasoli's enriched seawater with iodine medium with a gentle upward flow generated by a peristaltic pump. In the culture system, 50% of the protoplasts regenerated their cell wall within 24 hours and almost all protoplasts regenerated a cell wall after 3 days culture. After cell wall regeneration, a number of cells divided and regenerated into sheet-shaped thalli. The thalli transferred to a tissue culture flask developed into sporophyte-like plantlets within 1 month. Plantlets then differentiated into blade, stipe, and holdfast, with a proper mucilage canal. Received: 21 April 1997 / Revision received: 27 June 1997 / Accepted: 5 July 1997     

Viscosity has dichotomous effects on Bdellovibrio bacteriovorus HD100 predation

Bdellovibrio bacteriovorus HD100 is a highly motile predatory bacterium that consumes other Gram-negative bacteria for its sustenance. Here, we describe the impacts the media viscosity has both on the motility of predator and its attack rates. Experiments performed in polyethylene glycol (PEG) solutions, a linear polymer, found a viscosity of 10 mPa s (5% PEG) negatively impacted predation over a 24-h period. When the viscosity was increased to 27 mPa s (10% PEG), predation was nearly abolished. Tests with three other B. bacteriovorus strains, i.e., 109J and two natural isolates, found identical results. Short-term (2-h) experiments, however, found attack rates were improved in 1% PEG, which had a viscosity of 5.4 mPa s, using bioluminescent prey and their viabilities. In contrast, when experiments were performed in dextran, a branched polymer, no increase in predation was seen even though the viscosity was a comparable 5.1 mPa s. The enhanced attack rates in this solution coincided with a 31% increase in B. bacteriovorus HD100 swimming speeds (62 μm s⁻¹ in 1% PEG vs. 47.5 μm s⁻¹ in HEPES-salt).     

DNA encapsulation by an air-agitated, liquid-liquid mixer

Smooth and spherical alginate microspheres and nylon-membrane bound microcapsules were formed in an air-agitated, liquid-liquid mixer by emulsification/internal gelation and interfacial polymerization respectively. The mean diameter of the alginate microspheres ranged from 100 to 800 mum, and was controlled by process modifications. Increase in emulsifier concentration, gas flowrate, and emulsification time resulted in smaller microsphere size as did a decrease in liquid height. Increase in the dispersed phase viscosity resulted in a longer emulsification time required for approaching a minimum microsphere size. Microspheres could be formed with the proportion of dispersed phase approaching 30%. The yield of alginate microspheres was 70%, with losses attributed to incomplete recovery during washing and filtration operations. The yield of DNA encapsulation within the fraction of recovered microspheres, was 94%. The small loss was thought to occur by surface release during the washing of the microspheres. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 464-470, 1997.     

A rapid,sensitive, simple plate assay for detection of microbial alginate lyase activity

Screening of microorganisms capable of producing alginate lyase enzyme is commonly carried out by investigating their abilities to grow on alginate-containing solid media plates and occurrence of a clearance zone after flooding the plates with agents such as 10% (w/v) cetyl pyridinium chloride (CPC), which can form complexes with alginate. Although the CPC method is good, advantageous, and routinely used, the agar in the media interferes with the action of CPC, which makes judgment about clearance zones very difficult. In addition, this method takes a minimum of 30 min to obtain the zone of hydrolysis after flooding and the hydrolyzed area is not sharply discernible. An improved plate assay is reported herein for the detection of extracellular alginate lyase production by microorganisms. In this method, alginate-containing agar plates are flooded with Gram's iodine instead of CPC. Gram's iodine forms a bluish black complex with alginate but not with hydrolyzed alginate, giving sharp, distinct zones around the alginate lyase producing microbial colonies within 2–3 min. Gram's iodine method was found to be more effective than the CPC method in terms of visualization and measurement of zone size. The alginate-lyase-activity area indicated using the Gram's iodine method was found to be larger than that indicated by the CPC method. Both methods (CPC and Gram's iodine) showed the largest alginate lyase activity area for Saccharophagus degradans (ATCC 43961) followed by Microbulbifer mangrovi (KCTC 23483), Bacillus cereus (KF801505) and Paracoccus sp. LL1 (KP288668) grown on minimal sea salt medium. The rate of growth and metabolite production in alginate-containing minimal sea salt liquid medium, followed trends similar to that of the zone activity areas for the four bacteria under study. These results suggested that the assay developed in this study of Gram's iodine could be useful to predict the potential of microorganisms to produce alginate lyase. The method also worked well for screening and identification of alginate lyase producers and non-producers from environmental samples on common laboratory media. They did this by clearly showing the presence or absence of clearance zones around the microbial colonies grown. This new method is rapid, efficient, and could easily be performed for screening a large number of microbial cultures. This is the first report on the use of Gram's iodine for the detection of alginate lyase production by microorganisms using plate assay.