Non-selective microbiota reduction after the elicitation of a seaweed's immune response.

Pattern-triggered immunity (PTI) is an integral part of the innate immune system of many eukaryotic hosts, assisting in the defence against pathogen invasions. In plants and animals, PTI exerts a selective pressure on the microbiota that can alter community composition. However, the effect of PTI on the microbiota for non-model hosts, including seaweeds, remains unknown. Using quantitative polymerase chain reaction complemented with 16S rRNA gene and transcript amplicon sequencing, this study profiled the impact that PTI of the red seaweed Gracilaria gracilis has on its microbiota. PTI elicitation with agar oligosaccharides resulted in a significant reduction in the number of bacteria (by >75% within 72 h after treatment). However, the PTI elicitation did not cause any significant difference in the community diversity or structure. These findings demonstrated that PTI can be non-selective, and this might help to maintain a stable microbiota by uniformly reducing bacterial loads.

Marinomonas agarivorans sp. nov., an agar-degrading marine bacterium isolated from red algae.

A Gram-stain-negative, aerobic and curved-rod-shaped bacterium, designated QM202T, was isolated from red algae (Gracilaria blodgettii). Cells of strain QM202T were 0.2-0.3 µm wide and 1.0-2.5 µm long, catalase-negative and oxidase-positive. The strain exhibited an agar-degrading activity. It was motile by means of a single polar flagellum. Optimal growth occurred at 28-30 °C, pH 7.0-7.5 and in the presence of 2.0-3.0 % (w/v) NaCl. The DNA G+C content was 41.4 mol%. The isoprenoid quinone was identified as Q-8. Phophatidylethanolamine and phosphatidylglycerol were the predominant phospholipids. The dominant fatty acids were C18:1ω7c, C16:0 and C16:1ω7c and/or iso-C15:0 2-OH. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain QM202T belonged to the genus Marinomonas. The closest described neighbour in terms of 16S rRNA gene sequence identity was Marinomonas blandensis MED121T (95.5 %). The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness indicated that strain QM202T can be considered to represent a novel species, Marinomonas agarivorans sp. nov. The type strain is QM202T (=KCTC 52475T=MCCC 1H00145T).

Cyclonerane Derivatives from the Algicolous Endophytic Fungus Trichoderma asperellum A-YMD-9-2.

Seven previously unreported cyclonerane derivatives, namely, 3,7,11-trihydroxycycloneran-10-one, cycloneran-3,7,10,11-tetraol, cycloneran-3,7,11-triol, 11,12,15-trinorcycloneran-3,7,10-triol, 7,10S-epoxycycloneran-3,15-diol, 7,10R-epoxycycloneran-3,15-diol, and (10Z)-15-acetoxy-10-cycloneren-3,7-diol, were isolated in addition to the known (10Z)-cyclonerotriol, (10E)-cyclonerotriol, catenioblin C, and chokol E from the culture of Trichoderma asperellum A-YMD-9-2, an endophytic fungus obtained from the marine red alga Gracilaria verrucosa. The structures of previously unreported compounds were established by spectroscopic techniques, including 1D/2D NMR, MS, and IR. The isolation of these new cyclonerane derivatives greatly adds to the structural diversity of unusual cyclonerane sesquiterpenes, and several isolates exhibit potent inhibition against some marine phytoplankton species.

Seven chromanoid norbisabolane derivatives from the marine-alga-endophytic fungus Trichoderma asperellum A-YMD-9-2.

An examination of the endophytic fungus Trichoderma asperellum A-YMD-9-2 obtained from the marine red alga Gracilaria verrucosa led to the isolation of seven new chromanoid norbisabolane derivatives, trichobisabolins I-L (1-4) and trichaspsides C-E (5-7). Their structures and relative configurations were established on the basis of spectroscopic techniques, mainly including 1D/2D NMR and MS, and the absolute configuration of 1 was assigned by X-ray crystallographic analysis using Cu Kα radiation. All of these isolates feature a 1,9-epoxy ring system, and 5-7 represent the second occurrence of norbisabolane aminoglycosides. Compounds 1-7 exhibited potent inhibition of several marine phytoplankton species.

Agaribacter flavus sp. nov., isolated from red algae.

A Gram-stain-negative, non-motile, aerobic, curved-rod bacterium, designated as strain 2p52T, was isolated from the marine algae Gracilaria blodgettii collected off the coast of Lingshui county, in Hainan province, China. Strain 2p52T grew at 15-42 °C (optimally at 30-33 °C), at pH 6.0-10.0 (7.5-8.0) and in the presence of 1.0-8.0 % (w/v) NaCl (2.0-3.0 %). The most closely related species was Agaribacter marinus (96.5 % 16S rRNA gene sequence similarity). Phylogenetic analysis based on the sequence of the 16S rRNA gene revealed that strain 2p52T belonged to the genus Agaribacter. The novel strain contained phophatidylethanolamine and phosphatidylglycerol as the major polar lipids. The predominant isoprenoid quinine was Q-8, and the DNA G+C content was 43.2 mol%. The major cellular fatty acids were C16 : 1ω7c and/or iso-C15 : 0 2-OH, C16 : 0, and C18 : 1ω7c. The phenotypic and systematic comparative analyses indicated that the isolate is representative of a novel species of the genus Agaribacter, and the name Agaribacter flavus sp. nov. is proposed. The type strain is 2p52T (=KCTC 52473T=MCCC 1H00151T).

Seonamhaeicola marinus sp. nov., isolated from marine algae.

A Gram-stain-negative, facultatively anaerobic, rod-shaped, non-flagellated and non-gliding bacterium, designated strain B011T, was isolated from marine algae Gracilaria blodgettii collected from the coast of Lingshui county, Hainan, China. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain B011T, Seonamhaeicola algicola (96.8 % 16S rRNA gene sequence similarity) and Seonamhaeicola aphaedonensis (95.0 %) belonged to the same clade. Optimal growth occurred in the presence of 3.0 % (w/v) NaCl, at pH 6.5-7.5 and at 28 °C. Menaquinone-6 (MK-6) was the predominant respiratory quinone. The major fatty acids (>10 %) were iso-C15 : 1 G (23.4 %) and iso-C15 : 0 (22.8 %). The major polar lipids were phosphatidylethanolamine, one aminolipid and three unknown lipids. The DNA G+C content of strain B011T was 33.9 mol%. On the basis of polyphasic analysis, strain B011T is considered to represent a novel species in the genus Seonamhaeicola, for which the name Seonamhaeicolamarinus sp. nov. is proposed. The type strain is B011T (=MCCC 1H00146T=NBRC 112333T).

Agarilytica rhodophyticola gen. nov., sp. nov., isolated from Gracilaria blodgettii.

A novel Gram-stain-negative, rod-shaped, non-spore-forming, aerobic, agarolytic bacterium, designated 017T, was isolated from Gracilaria blodgettii collected at the coast of Lingshui county, Hainan province, China. Optimal growth occurred at 28-33 °C (range 15-40 °C), with 3 % (w/v) NaCl (range 2-4 %) and at pH 8.0 (range pH 6.5-8.5). Cells of strain 017T were motile and formed yellow colonies on marine agar 2216. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 017T shared the highest similarity with Teredinibacter turnerae T7902T (94.4 %). The predominant polar lipids of the novel isolate consisted of phosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and some other unknown lipids. Major cellular fatty acids (>10 %) were C16 : 0, C18 : 1ω7c and summed feature 3 (C16 : 1ω7c/iso-C15 : 0 2-OH), and the sole respiratory lipoquinone was Q-8. The DNA G+C content of strain 017T was 40.2 mol%. Comparative analysis of 16S rRNA gene sequences and phenotypic characterization indicated that strain 017T represents a novel species in a new genus of the family Cellvibrionaceae, order Cellvibrionales, for which the name Agarilytica rhodophyticola gen. nov., sp. nov. is proposed. The type strain of Agarilytica rhodophyticola is 017T (=KCTC 42584T=MCCC 1H00123T).

Crocinitomix algicola sp. nov., isolated from Gracilaria blodgettii.

A Gram-stain-negative, strictly aerobic, non-flagellated, yellow-pigmented, rod-shaped bacterial strain, designated 0182T, was isolated from Gracilaria blodgettii, an algae of phylum Rhodophyta collected from coast of Lingshui county, Hainan, China (110° 03' 44.2'' E, 18° 24' 29.8'' N). The strain grew optimally at 30 °C, pH 7.0-7.5 and in the presence of 2.0-3.0 % (w/v) NaCl. Cells of strain 0182T were approximately 0.9-2.5 µm in length and 0.2-0.4 µm in width. The major respiratory quinone was MK-7. The predominant cellular fatty acids were iso-C15 : 0, C15 : 0 and iso-C17 : 0 3-OH. The major polar lipids were phosphoaminolipid, glycolipid, two unknown aminolipids and four unknown lipids. The DNA G+C content was approximately 35.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 0182T was phylogenetically related to members of the genus Crocinitomix and was closely related to Crocinitomix catalasitica with 94.6 % sequence similarity. On the basis of genotypic and phenotypic characteristics and phylogenetic evidence, strain 0182T is thought to represent a novel species of the genus Crocinitomix, for which the name Crocinitomix algicola sp. nov. is proposed. The type strain is 0182T (=KCTC 42868T =MCCC 1H00128T).

Comparison of dairy desserts produced with a potentially probiotic mixed culture and dispersions obtained from Gracilaria birdiae and Gracilaria domingensis seaweeds used as thickening agents.

Dairy desserts have emerged as interesting options for the incorporation of probiotics, bioactive ingredients and alternative sources of thickeners. This shows an opportunity to investigate the use of Gracilaria seaweeds in the formulation of potentially probiotic dairy desserts. This study aimed to compare the effects of dispersions obtained from Gracilaria domingensis and Gracilaria birdiae used as thickening agents on texture properties of dairy desserts fermented with SAB 440-A, composed of the starter Streptococcus thermophilus and the potential probiotics Bifidobacterium animalis and Lactobacillus acidophilus, and also to study their physicochemical characteristics, microbial viability and sensory acceptability. No significant differences between desserts with G. birdiae or G. domingensis dispersions regarding total solids, ash and fat content, as well as pH, titratable acidity, the viability of the microorganisms of the mixed culture and sensory acceptability were verified (P > 0.05). Nonetheless, the dessert with G. domingensis dispersion showed higher dietary fibre content and significantly increased firmness than the one produced with G. birdiae (P < 0.05). Moreover, B. animalis was able to maintain higher populations, above 7 log cfu g-1 during 21 days of storage of desserts, in the presence of either G. birdiae or G. domingensis dispersions, despite the fact that L. acidophilus has shown low viability in the final products. Therefore, the G. domingensis dispersion is suitable to be used as a thickening agent to produce dairy desserts with enhanced firmness and good sensory acceptability, it being also advisable to use only B. animalis as a probiotic for this product.

Comparison of microbial communities during the anaerobic digestion of Gracilaria under mesophilic and thermophilic conditions.

Mesophilic and thermophilic anaerobic digesters (MD and TD, respectively) utilizing Gracilaria and marine sediment as the substrate and inoculum, respectively, were compared by analyzing their performances and microbial community changes. During three successive transfers, the average cumulative methane yields in the MD and TD were 222.6 ± 17.3 mL CH4/g volatile solids (VS) and 246.1 ± 11 mL CH4/g VS, respectively. The higher hydrolysis rate and acidogenesis in the TD resulted in a several fold greater accumulation of volatile fatty acids (acetate, propionate, and butyrate) followed by a larger pH drop with a prolonged recovery than in the MD. However, the operational stability between both digesters remained comparable. Pyrosequencing analyses revealed that the MD had more complex microbial diversity indices and microbial community changes than the TD. Interestingly, Methanomassiliicoccales, the seventh methanogen order was the predominant archaeal order in the MD along with bacterial orders of Clostridiales, Bacteriodales, and Synergistales. Meanwhile, Coprothermobacter and Methanobacteriales dominated the bacterial and archaeal community in the TD, respectively. Although the methane yield is comparable, both MD and TD show a different profile of pH, VFA and the microbial communities.

Seonamhaeicola algicola sp. nov., a complex-polysaccharide-degrading bacterium isolated from Gracilaria blodgettii, and emended description of the genus Seonamhaeicola.

A novel Gram-stain-negative, yellow, rod-shaped, facultatively anaerobic, gliding bacterial strain, designated Gy8T, was isolated from the surface of Gracilaria blodgettii. This bacterium was able to degrade various polysaccharides, especially agar and alginate. The major cellular fatty acids (>10 % of the total fatty acids) were C15 : 0, iso-C15 : 0, C15 : 0 3-OH and iso-C15 : 1. The major menaquinone was MK-6. The DNA G+C content was 35.3 mol%. The major polar lipids consisted of phosphatidylethanolamine and two unknown polar lipids. Strain Gy8T showed highest 16S rRNA gene sequence similarity to Seonamhaeicola aphaedonensis AH-M5T (95.6 %), and these two strains formed a distinct branch in phylogenetic trees generated with the neighbour-joining, maximum-likelihood and maximum-parsimony algorithms. The novel strain and the reference type strain of the single species described to date in the genus Seonamhaeicola contained MK-6 as the major menaquinone, iso-C15 : 0 and iso-C15 : 1 as the major fatty acids, and phosphatidylethanolamine and an unknown lipid as the major polar lipids. Based on phenotypic, chemotaxonomic and phylogenetic analysis, strain Gy8T is considered to represent a novel species within the genus Seonamhaeicola in the family Flavobacteriaceae, phylum Bacteroidetes, for which the name Seonamhaeicola algicola sp. nov. is proposed. The type strain is Gy8T ( = KCTC 42396T = CICC 23816T).

Wenyingzhuangia gracilariae sp. nov., a novel marine bacterium of the phylum Bacteroidetes isolated from the red alga Gracilaria vermiculophylla.

A Gram-negative, strictly aerobic, beige-pigmented, non-motile, rod-shaped bacterial strain designated N5DB13-4(T) was isolated from the red alga Gracilaria vermiculophylla (Rhodophyta) collected at Sodegaura Beach, Chiba, Japan. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the novel isolate is affiliated with the family Flavobacteriaceae within the phylum Bacteroidetes and that it showed highest sequence similarity (97.3 %) to Wenyingzhuangia heitensis H-MN17(T). The hybridization values for DNA-DNA relatedness between the strains N5DB13-4(T) and W. heitensis H-MN17(T) were 34.1 ± 3.5 %, which is below the threshold accepted for the phylogenetic definition of a novel prokaryotic species. The DNA G+C content of strain N5DB13-4(T) was determined to be 31.8 mol%; MK-6 was identified as the major menaquinone; and the presence of iso-C15:0, iso-C15:0 3-OH and iso-C17:0 3-OH as the major (>10 %) cellular fatty acids. A complex polar lipid profile was present consisting of phosphatidylethanolamine, two unidentified glycolipids and four unidentified lipids. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel species of the genus Wenyingzhuangia for which the name Wenyingzhuangia gracilariae sp. nov. is proposed. The type strain of W. gracilariae sp. nov. is N5DB13-4(T) (=KCTC 42246 (T)=NBRC 110602(T)).

The ecological perspective of microbial communities in two pairs of competitive Hawaiian native and invasive macroalgae.

Marine macroalgae are known to harbor large populations of microbial symbionts, and yet, microbe symbiosis in invasive macroalgae remains largely unknown. In this study, we applied molecular methods to study microbial communities associated with two invasive algae Acanthophora spicifera and Gracilaria salicornia and the two native algae Gracilaria coronopifolia and Laurencia nidifica at spatial and temporal scales in Hawaiian coral reef ecosystems. Bacterial communities of both the invasive and native macroalgae displayed little spatial and temporal variations, suggesting consistent and stable bacterial associations with these macroalgae. Results of this study identified three types of bacterial populations: nonspecific (present in both algal and water samples); algae-specific (found in all algal species); and species-specific (only found in individual species). The bacterial diversity of invasive algae was lower than that of their native counterparts at phylum and species levels. Notably, the vast majority (71 %) of bacterial communities associated with the invasive algae G. salicornia were representatives of Cyanobacteria, suggesting a potential ecological significance of symbiotic Cyanobacteria.

The lipidic extract of the seaweed Gracilariopsis longissima (Rhodophyta, Gracilariales): a potential resource for biotechnological purposes?

In recent years seaweeds increasingly attracted interest in the search for new drugs and have been shown to be a primary source of bioactive natural products including antibiotics. In the present investigation the antimicrobial activity of Gracilariopsis longissima lipidic extract was assayed and its chemical characterization was carried out by means of advanced analytical techniques such as gas-chromatography and multinuclear and multidimensional NMR spectroscopy. G. longissima lipidic extract showed an antibacterial activity against several Vibrio species. These results are interesting considering both the resistance against antibiotics developed by vibrios and the need to control fish and shellfish diseases due to vibriosis. Analysis of fatty acid methyl esters performed by gas-chromatography showed that palmitic acid methyl ester (16:0) was the predominant saturated fatty acid (42%), while, among monounsaturated fatty acids, oleic acid methyl ester (18:1) prevailed (8.5%). Because the palmitic acid represents the main component of fatty acids we hypothesized its involvement in the antibacterial activity observed. However, a pure sample of palmitic acid did not show an antibacterial activity. The fatty acid profile of G. longissima revealed also an interesting composition in polyunsaturated fatty acids and in particular the ratio of ω-3 to ω-6 fatty acids was >1 thus suggesting that this macroalga may be used as a natural source of ω3. Moreover, the (1)H NMR spectrum in CDCl(3) of algal lipid fraction shows the characteristic signals of saturated and unsaturated fatty acids as well as other metabolites. Interestingly, in the lipid extract the presence of polyhydroxybutyrate, a linear biodegradable and biocompatible polyester, was clearly identified by NMR spectroscopy. In conclusion, the lipidic extract of G. longissima on account of its antimicrobial activity, nutritional value and content in biodegradable and biocompatible polyester represents an interesting potential biotechnological resource.

Role of bacterial isolates in enhancing the bud induction in the industrially important red alga Gracilaria dura.

Plant growth depends on the integration of environmental cues, nitrogen fixation and phytohormone-signaling pathways. The growth and development of Gracilaria dura was significantly influenced by the association of bacterial isolates. The putative bud-inducing epiphytic Exiguobacterium homiense and endophytic Bacillus pumilus, Bacillus licheniformis were examined for their ability to fix nitrogen and produce indole-3-acetic acid (IAA). These bacterial isolates were identified to the species level by biochemical tests, fatty acid and partial 16S rRNA gene sequence analysis. The B. pumilus, B. licheniformis and E. homiense produced 445.5, 335 and 184.1 µg mL(-1) IAA and 12.51, 10.14 and 6.9 mM mL(-1) ammonium, respectively, as determined using HPLC and spectroscopy. New bud regeneration observed after the addition of total protein of the bacterial isolates suggests that IAA is conjugated with protein. The epi- and endophytic bacterial isolates were able to induce five and 10 new buds per frond, respectively, in comparison to the control, where one to two buds were observed. The combination of 25 °C and 30‰ showed the optimum condition for bud induction in G. dura when incubated with the total protein of B. pumilus. Our finding revealed for the first time that IAA coupled with nitrogen fixation induce and regenerate new buds in G. dura.

Characterization of Microbulbifer strain CMC-5, a new biochemical variant of Microbulbifer elongatus type strain DSM6810T isolated from decomposing seaweeds.

A Gram-negative, rod-shaped, non-spore forming, non-motile and moderate halophilic bacteria designated as strain CMC-5 was isolated from decomposing seaweeds by enrichment culture. The growth of strain CMC-5 was assessed in synthetic seawater-based medium containing polysaccharide. The bacterium degraded and utilized agar, alginate, carrageenan, xylan, carboxymethyl cellulose and chitin. The strain was characterized using a polyphasic approach for taxonomic identification. Cellular fatty acid analysis showed the presence of iso-C(15:0) as major fatty acid and significant amounts of iso-C(17:1x9c) and C(18:1x7c). Phylogenetic analysis based on 16S rDNA sequence indicated that strain CMC-5 is phylogenetically related to Microbulbifer genus and 99% similar to type strain Microbulbifer elongatus DSM6810T. However in contrast to Microbulbifer elongatus DSM6810T, strain CMC-5 is non-motile, utilizes glucose, galactose, inositol and xylan, does not utilize fructose and succinate nor does it produce H2S. Further growth of bacterial strain CMC-5 was observed when inoculated in seawater-based medium containing sterile pieces of Gracilaria corticata thalli. The bacterial growth was associated with release of reducing sugar in the broth suggesting its role in carbon recycling of polysaccharides from seaweeds in marine ecosystem.

Selection of lactic acid bacteria from fermented plant beverages to use as inoculants for improving the quality of the finished product.

Lactic Acid Bacteria (LAB) from fermented plant beverages were selected based on their antibacterial actions against potential food borne pathogenic bacteria (Staphylococcus aureus PSSCMI 0004, Escherichia coli PSSCMI 0001, Salmonella typhimurium PSSCMI 0034 and Vibrio parahaemolyticus VP 4). Antibacterial activities were measured using an agar spot method. The Lactobacillus plantarum W90A strain isolated from a wild forest noni (Morinda coreia Ham) beverage was used as an inoculant. Three different inoculation procedures were conducted with the fruit of wild forest noni fermentations to establish which one was the best for controlling the numbers of yeast in the finished product. A 5% inoculum of L. plantarum W90A (LAB set), initial cell density 8.6 log cfu mL(-1), produced a better product and inhibitory properties against the test organisms, particularly E. coli PSSCMI 0001 than one with no inoculum or with a 5% inoculum from a previous natural fermented product. An LAB inoculum resulted in a reduced total bacterial count and no yeast throughout fermentation period (90 days). The lower yeast resulted in a reduction of the ethanol content to 2.9 g L(-1) compared to 12.2 g L(-1) inthe culture with no inoculum. The highest acidity (1.3-1.4%) with the same pH (3.3) was observed in both sets of inoculated fermentations, whereas the uninoculated set gave a pH value of 3.7 (1.2% acidity).

Investigation of the role of a beta(1-4) agarase produced by Pseudoalteromonas gracilis B9 in eliciting disease symptoms in the red alga Gracilaria gracilis.

Gracilaria species are an important source of agar. The South African Gracilaria industry has experienced a number of setbacks over the last decade in the form of complete or partial die-offs of the agarophyte growing in Saldanha Bay, which may be attributed to bacterial infection. Since a positive correlation was observed between the presence of agarolytic epiphytes and bacterial pathogenicity, we investigated the role of an agarase in the virulence mechanism employed by a bacterium that elicits disease in Gracilaria gracilis. The recombinant plasmid pDA1, isolated from a Pseudoalteromonas gracilis B9 genomic library, was responsible for the agarolytic activity exhibited by Escherichia coli transformants when grown on solid medium. A BLAST search of the GenBank database showed that an 873 bp ORF (aagA) located on pDA1 had 85 % identity to the beta-agarase (dagA) from Pseudoalteromonas atlantica ATCC 19262(T) (or IAM 12927(T)) at the amino acid level. AagA was purified from the extracellular medium of an E. coli transformant harbouring pDA1 by using a combination of gel filtration and ion-exchange chromatography. AagA has an M(r) of 30 000 on SDS-PAGE. TLC of the digestion products of AagA showed that the enzyme cleaves the beta-(1,4) linkages of agarose to yield predominately neoagarotetraose. Western hybridization confirmed that the cloned agarase was in fact the extracellular beta-agarase of P. gracilis B9. The observed relationship between disease symptoms of G. gracilis and the agarolytic phenotype of P. gracilis B9 was confirmed. Transmission electron microscope examination of cross sections of both healthy G. gracilis and G. gracilis infected with P. gracilis, revealed a weakening of the cell structure in the latter plants. Immunogold-labelled antibodies localized the agarase in situ to the cell walls of bleached G. gracilis. Thus, the weakening observed in the cell structure of G. gracilis infected with P. gracilis can be attributed to degradation of the mucilaginous component of the cell wall of the bleached thalli.