Sulfation of extracellular polysaccharides of red microalgae: preparation, characterization and properties

Polysaccharides are natural polymers with a variety of properties that may be translated into significant commercial applications. A program of chemical modifications of the extracellular polysaccharides of red microalgae, such as Porphyridium sp. and Rhodella reticulata, has been undertaken by our group in order to tailor new properties and hence to broaden the spectrum of potential applications. These algal biopolymers are anionic in nature due to the presence of uronic acids (about 10%) and sulfate half esters (about 7%). In the current study, the sulfate content of these biopolymers was increased to 35-40% by means of sulfation agents such as pyridine SO(3), DMF.SO(3) and ClSO(3)H. Reaction conditions were optimized in a model system based on potato starch as the model polysaccharide (type of reagent, temperature and time of reaction). After work-up procedures, the highest sulfate content was obtained by sulfation of the polysaccharide of Porphyridium sp. with a mixture of ClSO(3)H and pyridine at 70 degrees C for 1 h. The sulfated products were characterized by chemical and rheological analyses, IR spectroscopy, and GPC-HPLC chromatography. "Oversulfated" polymers (having sulfate contents exceeding 20%) with high molecular weights were found to inhibit mammalian cell growth when used at certain concentrations; for example, over 80% inhibition was obtained when oversulfated polymers at a concentration of 200 microg/ml were tested on T-cell lymphoma line 24-1. These preliminary results indicate that the modified polysaccharides do indeed exhibit potential therapeutic properties.     

Novel bacterial exopolysaccharides from deep-sea hydrothermal vents

Five bacterial strains recovered from deep-sea hydrothermal vents were studied for their ability to secrete extracellular polymers. A preliminary characterization displayed four different polysaccharides in terms of both chemical composition and rheological properties. One of them was secreted by Alteromonas macleodii subsp. fijiensis and exhibited similarities with xanthan, a commercial polysaccharide. Two of the three Pseudoalteromonas species were shown to produce the same polymer. The last polymer was secreted by a bacterium belonging to the Vibrio genus. They all contained glucose, galactose, mannose, glucuronic and galacturonic acids as the main sugars with the exception of the last one which was only constituted by uronic acids and hexosamines, in that similar to the structure of heparin, a glycosaminoglycan useful in pharmaceutical area. Applications for these polysaccharides could be expected in various biotechnological fields including the food industry, the wastewater treatment and pharmaceutical areas.     

Observations on parasitism in deep-sea hydrothermal vent and seep limpets

Parasite burdens of shallow-water molluscs have been well documented, but little is known about parasite burdens of molluscs from deep-sea chemosynthetic environments (e.g. hydrothermal vents and seeps). Chemosynthetic habitats are characterized by high concentrations of reduced sulfur and, in the case of vents, high heavy metal concentrations. These compounds are noxious and even stress-inducing in some environments, but are part of the natural chemical milieu of vents and seeps. To examine parasite types and infection intensities in limpets from vents and seeps we documented parasite burdens in 4 limpet species from 4 hydrothermal vent fields (3 on the East Pacific Rise, 1 on the Mid-Atlantic Ridge) and 1 seep site (Florida Escarpment). Approximately 50 % of all limpets examined were infected with 1 or more types of parasites. Limpet parasites were predominantly rickettsia-like inclusions in the digestive and gill epithelia. Limpets collected from the vent field on the Mid-Atlantic Ridge were free of parasites. We detected no histopathological effects that we could attribute to parasites.     

Colonization of nascent, deep-sea hydrothermal vents by a novel Archaeal and Nanoarchaeal assemblage

Active deep-sea hydrothermal vents are areas of intense mixing and severe thermal and chemical gradients, fostering a biotope rich in novel hyperthermophilic microorganisms and metabolic pathways. The goal of this study was to identify the earliest archaeal colonizers of nascent hydrothermal chimneys, organisms that may be previously uncharacterized as they are quickly replaced by a more stable climax community. During expeditions in 2001 and 2002 to the hydrothermal vents of the East Pacific Rise (EPR) (9 degrees 50'N, 104 degrees 17'W), we removed actively venting chimneys and in their place deployed mineral chambers and sampling units that promoted the growth of new, natural hydrothermal chimneys and allowed their collection within hours of formation. These samples were compared with those collected from established hydrothermal chimneys from EPR and Guaymas Basin vent sites. Using molecular and phylogenetic analysis of the 16S rDNA, we show here that at high temperatures, early colonization of a natural chimney is dominated by members of the archaeal genus Ignicoccus and its symbiont, Nanoarchaeum. We have identified 19 unique sequences closely related to the nanoarchaeal group, and five archaeal sequences that group closely with Ignicoccus. These organisms were found to colonize a natural, high temperature protochimney and vent-like mineral assemblages deployed over high temperature outflows within 92 h. When compared phylogenetically, several of these colonizing organisms form a unique clade independent of those found in mature chimneys and low-temperature mineral chamber samples. As a model ecosystem, the identification of pioneering consortia in deep-sea hydrothermal vents may help advance the understanding of how early microbial life forms gained a foothold in hydrothermal systems on early Earth and potentially on other planetary bodies.     

Capsular polysaccharides secreted by building façade colonisers: characterisation and adsorption to surfaces

Exopolymers secreted by algal and cyanobacterial strains isolated from building fa?ades were imaged by microscopy techniques. They were extracted and characterised to investigate their possible contribution to interactions with solid surfaces. The polymers were polysaccharides, with anionic and hydrophobic properties varying between the various strains. Capsular polysaccharides extracted from a strain of Klebsormidium flaccidum adsorbed in higher amounts on hydrophobic than on hydrophilic surfaces. These results tend to confirm the hypothesis that exopolymers are important in the colonisation process of microorganisms to surfaces.     

Sulfide ameliorates metal toxicity for deep-sea hydrothermal vent archaea

The chemical stress factors for microbial life at deep-sea hydrothermal vents include high concentrations of heavy metals and sulfide. Three hyperthermophilic vent archaea, the sulfur-reducing heterotrophs Thermococcus fumicolans and Pyrococcus strain GB-D and the chemolithoautotrophic methanogen Methanocaldococcus jannaschii, were tested for survival tolerance to heavy metals (Zn, Co, and Cu) and sulfide. The sulfide addition consistently ameliorated the high toxicity of free metal cations by the formation of dissolved metal-sulfide complexes as well as solid precipitates. Thus, chemical speciation of heavy metals with sulfide allows hydrothermal vent archaea to tolerate otherwise toxic metal concentrations in their natural environment.     

Capsular polysaccharides secreted by building façade colonisers: characterisation and adsorption to surfaces

Abstract

Exopolymers secreted by algal and cyanobacterial strains isolated from building façades were imaged by microscopy techniques. They were extracted and characterised to investigate their possible contribution to interactions with solid surfaces. The polymers were polysaccharides, with anionic and hydrophobic properties varying between the various strains. Capsular polysaccharides extracted from a strain of Klebsormidium flaccidum adsorbed in higher amounts on hydrophobic than on hydrophilic surfaces. These results tend to confirm the hypothesis that exopolymers are important in the colonisation process of microorganisms to surfaces.     

Did shifting seawater sulfate concentrations drive the evolution of deep-sea methane-seep ecosystems?

The origin and evolution of the faunas inhabiting deep-sea hydrothermal vents and methane seeps have been debated for decades. These faunas rely on a local source of sulfide and other reduced chemicals for nutrition, which spawned the hypothesis that their evolutionary history is independent from that of photosynthesis-based food chains and instead driven by extinction events caused by deep-sea anoxia. Here I use the fossil record of seep molluscs to show that trends in body size, relative abundance and epifaunal/infaunal ratios track current estimates of seawater sulfate concentrations through the last 150 Myr. Furthermore, the two main faunal turnovers during this time interval coincide with major changes in seawater sulfate concentrations. Because sulfide at seeps originates mostly from seawater sulfate, variations in sulfate concentrations should directly affect the base of the food chain of this ecosystem and are thus the likely driver of the observed macroecologic and evolutionary patterns. The results imply that the methane-seep fauna evolved largely independently from developments and mass extinctions affecting the photosynthesis-based biosphere and add to the growing body of evidence that the chemical evolution of the oceans had a major impact on the evolution of marine life.     

Cultivation and metabolic insights of an uncultured clade,Bacteroidetes VC2.1 Bac22 (Candidatus Sulfidibacteriales ord. nov.), from deep-sea hydrothermal vents

Bacteroidetes VC2.1 Bac22 (referred to as VC2.1) is an uncultured clade that is widely distributed in marine ecosystems, including hydrothermal vents, oxygen-minimum zones and other anoxic, sulfide-rich environments. However, the lack of cultured representatives and sequenced genomes of VC2.1 limit our understanding of its physiology, metabolism and ecological functions. Here, we obtained a stable co-culture of VC2.1 with autotrophic microbes by establishing an autotrophy-based enrichment from a hydrothermal vent chimney sample. We recovered a high-quality metagenome-assembled genome (MAG) that belonged to VC2.1. Phylogenetic analyses of both 16S rRNA genes and conserved protein markers suggested that VC2.1 belongs to a novel order in the Bacteroidetes phylum, which we named Candidatus Sulfidibacteriales. The metabolic reconstruction of this MAG indicated that VC2.1 could utilize polysaccharides, protein polymers and fatty acids as well as flexibly obtain energy via NO/N₂O reduction and polysulfide reduction. Our results reveal the ecological potential of this novel Bacteroidetes for complex organic carbons mineralization and N₂O sinks in deep-sea hydrothermal vents. Furthermore, guided by the genome information, we designed a new culture medium in which starch, ammonium and polysulfide were used as the carbon source, nitrogen source and electron acceptor respectively, to isolate VC2.1 successfully.     

Sulfide Ameliorates Metal Toxicity for Deep-Sea Hydrothermal Vent Archaea

The chemical stress factors for microbial life at deep-sea hydrothermal vents include high concentrations of heavy metals and sulfide. Three hyperthermophilic vent archaea, the sulfur-reducing heterotrophs Thermococcus fumicolans and Pyrococcus strain GB-D and the chemolithoautotrophic methanogen Methanocaldococcus jannaschii, were tested for survival tolerance to heavy metals (Zn, Co, and Cu) and sulfide. The sulfide addition consistently ameliorated the high toxicity of free metal cations by the formation of dissolved metal-sulfide complexes as well as solid precipitates. Thus, chemical speciation of heavy metals with sulfide allows hydrothermal vent archaea to tolerate otherwise toxic metal concentrations in their natural environment.     

Influence of subtilisin on the adhesion of a marine bacterium which produces mainly proteins as extracellular polymers

Aims:  The nature of exopolymers involved in the adhesion of a marine biofilm-forming bacterium Pseudoalteromonas sp. D41 was investigated to evaluate and understand the antifouling potential of subtilisin.
Methods and Results:  The exopolymers of D41 produced by fermentation were analysed by FTIR and SDS-PAGE showing the presence of polysaccharides, glycoproteins and proteins. A high content of proteins was detected both in soluble and capsular fractions. The microscopic observations of fluorescamine and calcofluor stained adhered D41 indicated mainly the presence of proteins in exopolymers produced during adhesion. Subtilisin, the broad spectrum protease, tested in natural sea water and in polystyrene microplates showed that antifouling activity was higher in the prevention of bacterial adhesion than in the detachment of adhered D41 cells.
Conclusions:  Overall, these results demonstrate the involvement of proteins in Pseudoalteromonas sp . D41 adhesion and confirm the high antifouling potential of subtilisin.
Significance and Impact of the Study:  This study emphasizes the major role of proteins instead of polysaccharides, thus extending our knowledge regarding the nature of extracellular polymers involved in bacterial adhesion. Furthermore, the high antifouling potential of subtilisin evaluated in the very first stages of fouling, bacterial adhesion, could lead to less toxic compounds than organometallic compounds in antifouling paint.     

Characterizing the distribution and rates of microbial sulfate reduction at Middle Valley hydrothermal vents

Few studies have directly measured sulfate reduction at hydrothermal vents, and relatively little is known about how environmental or ecological factors influence rates of sulfate reduction in vent environments. A better understanding of microbially mediated sulfate reduction in hydrothermal vent ecosystems may be achieved by integrating ecological and geochemical data with metabolic rate measurements. Here we present rates of microbially mediated sulfate reduction from three distinct hydrothermal vents in the Middle Valley vent field along the Juan de Fuca Ridge, as well as assessments of bacterial and archaeal diversity, estimates of total biomass and the abundance of functional genes related to sulfate reduction, and in situ geochemistry. Maximum rates of sulfate reduction occurred at 90 °C in all three deposits. Pyrosequencing and functional gene abundance data revealed differences in both biomass and community composition among sites, including differences in the abundance of known sulfate-reducing bacteria. The abundance of sequences for Thermodesulfovibro-like organisms and higher sulfate reduction rates at elevated temperatures suggests that Thermodesulfovibro-like organisms may have a role in sulfate reduction in warmer environments. The rates of sulfate reduction presented here suggest that—within anaerobic niches of hydrothermal deposits—heterotrophic sulfate reduction may be quite common and might contribute substantially to secondary productivity, underscoring the potential role of this process in both sulfur and carbon cycling at vents.     

Fluorescent derivatization of polysaccharides and carbohydrate-containing biopolymers for measurement of enzyme activities in complex media

Fluorescence derivatization provides a means of tracing the dynamics of polysaccharides even in the presence of high concentrations of other organic compounds or salts. A method of labeling polysaccharides with fluoresceinamine was extended to polysaccharides of a wide range of chemical composition, and alternative means of preparation were established for polysaccharides not initially amenable to column chromatography. The polysaccharides were activated with cyanogen bromide, coupled to fluoresceinamine, and separated from unreacted fluorophore via gel filtration chromatography or dialysis. Since the resulting derivatized polysaccharides proved to be stable to further physical and chemical manipulation, methods were also developed for re-activation and labeling with a second fluorophore, as well as for tethering the labeled polysaccharides to agarose beads. As an example of the application of this approach, five distinct fluorescently-labeled polysaccharides (pullulan, laminarin, xylan, chondroitin sulfate, and alginic acid) were used to investigate the activities and structural specificities of extracellular enzymes produced in situ by marine microbial communities, providing a means of measuring specifically the activities of endo-acting extracellular enzymes and avoiding use of low molecular mass substrate proxies. These labeled polysaccharides could be used to explore the dynamics of polysaccharides in other types of complex media, as well as to investigate the activities and specificities of endo-acting enzymes in other systems.     

Phylogenetic diversity of nitrogenase (nifH) genes in deep-sea and hydrothermal vent environments of the Juan de Fuca Ridge

The subseafloor microbial habitat associated with typical unsedimented mid-ocean-ridge hydrothermal vent ecosystems may be limited by the availability of fixed nitrogen, inferred by the low ammonium and nitrate concentrations measured in diffuse hydrothermal fluid. Dissolved N2 gas, the largest reservoir of nitrogen in the ocean, is abundant in deep-sea and hydrothermal vent fluid. In order to test the hypothesis that biological nitrogen fixation plays an important role in nitrogen cycling in the subseafloor associated with unsedimented hydrothermal vents, degenerate PCR primers were designed to amplify the nitrogenase iron protein gene nifH from hydrothermal vent fluid. A total of 120 nifH sequences were obtained from four samples: a nitrogen-poor diffuse vent named marker 33 on Axial Volcano, sampled twice over a period of 1 year as its temperature decreased; a nitrogen-rich diffuse vent near Puffer on Endeavour Segment; and deep seawater with no detectable hydrothermal plume signal. Subseafloor nifH genes from marker 33 and Puffer are related to anaerobic clostridia and sulfate reducers. Other nifH genes unique to the vent samples include proteobacteria and divergent ARCHAEA: All of the nifH genes from the deep-seawater sample are most closely related to the thermophilic, anaerobic archaeon Methanococcus thermolithotrophicus (77 to 83% amino acid similarity). These results provide the first genetic evidence of potential nitrogen fixers in hydrothermal vent environments and indicate that at least two sources contribute to the diverse assemblage of nifH genes detected in hydrothermal vent fluid: nifH genes from an anaerobic, hot subseafloor and nifH genes from cold, oxygenated deep seawater.     

Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate reduction is used in industrial wastewater treatment to remove large concentrations of sulfate along with the chemical oxygen demand (COD) and heavy metals. The most common approach to the process is with anaerobic bioreactors in which sulfidogenic sludge is obtained through adaptation of predominantly methanogenic granular sludge to sulfidogenesis. This process may take a long time and does not always eliminate the competition for substrate due to the presence of methanogens in the sludge. In this work, we propose a novel approach to obtain sulfidogenic sludge in which hydrothermal vents sediments are the original source of microorganisms. The microbial community developed in the presence of sulfate and volatile fatty acids is wide enough to sustain sulfate reduction over a long period of time without exhibiting inhibition due to sulfide. This protocol describes the procedure to generate the sludge from the sediments in an upflow anaerobic sludge blanket (UASB) type of reactor. Furthermore, the protocol presents the procedure to demonstrate the capability of the sludge to remove by reductive dechlorination a model of a highly toxic organic pollutant such as trichloroethylene (TCE). The protocol is divided in three stages: (1) the formation of the sludge and the determination of its sulfate reducing activity in the UASB, (2) the experiment to remove the TCE by the sludge, and (3) the identification of microorganisms in the sludge after the TCE reduction. Although in this case the sediments were taken from a site located in Mexico, the generation of a sulfidogenic sludge by using this procedure may work if a different source of sediments is taken since marine sediments are a natural pool of microorganisms that may be enriched in sulfate reducing bacteria.     

Reductive coupling of aldehydes by h(2) s in aqueous solutions, a C--C bond forming reaction of prebiotic interest

We report here a novel reductive coupling reaction of conjugated, non- or poorly enolizable aldehydes induced by H(2) S and operative in aqueous solutions under prebiotically relevant conditions. This reaction leads from retinal to β-carotene, and from benzylic aldehydes to the corresponding diarylethylenes. This novel reaction also opens a new potentially prebiotic pathway leading from glyoxylic acid to various compounds that are involved in the reductive tricarboxylic acid cycle. This C--C bond forming reaction of prebiotic interest might have been operative, notably, in the sulfide-rich environments of hydrothermal vents, which have been postulated as possible sites for the first steps of organic chemical evolution.     

Chemical characterization of exopolysaccharides from the marine fouling diatom amphora coffeaeformis

Amphora coffeaeformis cells were grown in batch cultures under continuous illumination at 18°C for 10 d. Algal cells were removed by centrifugation, lyophilized and used for the extraction of exopolysaccharides using either 0.05 M EDTA, 1 M NaOH or 1.5 M NaCl. The 1.5 M NaCl treatment removed most exopolysaccharides. Glucose (81%) was the most abundant monosaccharide in the exopolysaccharides. The chemical composition data suggest that the exopolymers were acidic sulphated polysaccharides containing high concentrations of pyruvate (22%) and uronic acids (18%). These polysaccharides may play an important role in metal complexation and protection from desiccation in A. coffeaeformis.     

Chemolithotrophic Sulfur-Oxidizing Bacteria from the Galapagos Rift Hydrothermal Vents

Three distinct physiological types of sulfur-oxidizing bacteria were enriched and isolated from samples collected at several deep-sea hydrothermal vents (2,550 m) of the Galapagos Rift ocean floor spreading center. Twelve strains of the obligately chemolithotrophic genus Thiomicrospira were obtained from venting water and from microbial mats covering surfaces in the immediate vicinity of the vents. From these and other sources two types of obligately heterotrophic sulfur oxidizers were repeatedly isolated that presumably oxidized thiosulfate either to sulfate (acid producing; 9 strains) or to polythionates (base producing; 74 strains). The former were thiobacilli-like, exhibiting a thiosulfate-stimulated increase in growth and CO₂ incorporation, whereas the latter were similar to previously encountered pseudomonad-like heterotrophs. The presence of chemolithotrophic sulfur-oxidizing bacteria in the sulfide-containing hydrothermal water supports the hypothesis that chemosynthesis provides a substantial primary food source for the rich populations of invertebrates found in the immediate vicinity of the vents.     

Prebiotic Synthesis of Glycine from Ethanolamine in Simulated Archean Alkaline Hydrothermal Vents

Submarine hydrothermal vents are generally considered as the likely habitats for the origin and evolution of early life on Earth. In recent years, a novel hydrothermal system in Archean subseafloor has been proposed. In this model, highly alkaline and high temperature hydrothermal fluids were generated in basalt-hosted hydrothermal vents, where H₂ and CO₂ could be abundantly provided. These extreme conditions could have played an irreplaceable role in the early evolution of life. Nevertheless, sufficient information has not yet been obtained for the abiotic synthesis of amino acids, which are indispensable components of life, at high temperature and alkaline condition. This study aims to propose a new method for the synthesis of glycine in simulated Archean submarine alkaline vent systems. We investigated the formation of glycine from ethanolamine under conditions of high temperature (80–160 °C) and highly alkaline solutions (pH = 9.70). Experiments were performed in an anaerobic environment under mild pressure (0.1–8.0 MPa) at the same time. The results suggested that the formation of glycine from ethanolamine occurred rapidly and efficiently in the presence of metal powders, and was favored by high temperatures and high pressures. The experiment provides a new pathway for prebiotic glycine formation and points out the phenomenal influence of high-temperature alkaline hydrothermal vents in origin of life in the early ocean.     

STRUCTURE AND DISTRIBUTION OF GLUCOMANNAN AND SULFATED GLUCAN IN THE CELL WALLS OF THE RED ALGA KAPPAPHYCUS ALVAREZII (GIGARTINALES, RHODOPHYTA)

Two new polysaccharides were isolated from the cell walls of the carrageenan producing red seaweed Kappaphycus alvarezii (Doty) Doty. They were characterized by chemical analyses, enzymatic degradations, and nuclear magnetic resonance spectroscopy. One was a 4.0 M NaOH soluble β-(1,4)- d -glucomannan that mostly precipitated upon neutralization and dialysis. It was composed of about 82 residues, and 70% of its glucose and mannose were released by a commercial cellulase enzyme complex. The disaccharide β- d -Man (1→4) d -Glc was recovered from the hydrolysate during the first hours of degradation and confirmed the chemical structure of the polysaccharide. The other polysaccharide was extracted with 1.5 M NaOH and was identified as a sulfated glucan of degree of polymerization of about 180 1,4-linked β-glucose containing 10% 1,3-linkages. The sulfate was located on C-6 of 64% of the 4-linked glucose residues. A third alkali-soluble polysaccharide rich in galactose was also detected. The distribution of the glucomannan and galactose containing polysaccharides was inversely related to the algal cell size. Potential functions of these alkali-soluble polymers are discussed in the context of cell wall polysaccharide assembly.