Continuous production of oligoglucuronans by immobilized glucuronan lyase

A glucuronan lyase was incubated with sepharose matrices pre-activated with N-hydroxysuccinimide (NHS), cyanogen bromide (CNBr) or epoxy. The CNBr- and NHS-activated gels showed satisfactory immobilization yields whereas no enzyme could be immobilized using the epoxy coupling group. Glucuronan lyase immobilized on CNBr-gel ensured rapid conversion of several glucuronans into oligomers with an enzymatic activity identical to that of the free enzyme. As classically observed when using free enzymes, the acetylation degree of glucuronan limited enzyme activity. Nevertheless, this immobilized system made it easier to obtain accurately oligomers with different polymerization degrees, notably in modifying the glucuronans residence times in column. Thus oligoglucuronan pools with polymerization degrees between 2 and 25 could be obtained with a productivity ranging from 120 mg h^?1 to 1.2 g h^?1 using 0.9 ml of chromatographic gel with immobilized glucuronan lyase. This methodology opens the way to continuous and large oligoglucuronan productions.     

Purification and characterization of a novel glucuronan lyase from Trichoderma sp. GL2

The filamentous fungus Trichoderma sp. GL2 produces an extracellular glucuronan lyase (GL) when grown on glucuronan as the sole carbon source. In this paper, we report the purification to electrophoretical homogeneity of this polysaccharide lyase by size exclusion chromatography and anion exchange chromatography. The purified GL, classified as an endopolyglucuronate lyase, is a monomer with an apparent molecular weight of 27 kDa and an isoelectric point of 6.95. Despite an inhibition of the activity when polysaccharide substrates were substituted by acetates, the enzyme was active toward glucuronans (acetylated or not) and ulvan, leading to various (4,5)-unsaturated products as oligoglucuronans (acetylated or deacetylated), highly acetylated low-molecular-weight (LMW) glucuronans, and LMW ulvans.     

Production of glucuronan oligosaccharides using a new glucuronan lyase activity from a Trichoderma sp. strain

Sinorhizobium meliloti M5N1CS synthesizes a homopolymer of glucuronic acids beta-(1,4) linked and variably C2 and/or C3O-acetylated. To obtain beta-Delta-(4,5)-unsaturated oligoglucuronans, various acetylated forms of this bacterial polymer were cleaved by a Trichoderma sp. GL2 glucuronan lyase. Oligomers with polymerization degrees up to 8 were then produced, purified by liquid chromatography (size exclusion and anions exchange) and characterized using 1H NMR and ESI-Q/TOF-MS. Finally, the production (in gram quantity) of pure unsaturated oligoglucuronans non-acetylated (di- and trisaccharide) was investigated thanks to the complete depolymerization of deacetylated glucuronan.     

Marine macroalgae as a source for osmoprotection for Escherichia coli

At elevated osmolarity of the mineral medium M63, marine macroalgae constitute important osmoprotectants and nutrients sources for Escherichia coli. Growth of bacterial population (16 strains) was improved by supplementing M63 salts medium with either aqueous or ethanolic algal extracts obtained from Ascophyllum nodosum, Fucus serratus, Enteromorpha ramulosa, Ulva lactuca, and Palmaria palmata. In their presence, growth was still observed even at 1.02 m NaCl. Furthermore, the E. coli ZB400 growth in presence of whole macroalgae thalli in M63/0.85 m NaCI reached its maximum within 24 h (5 × 10⁷ – 5 × 10⁸ colony-forming units [CFU] per milliliter). In the presence of A. nodosum, bacterial growth was inhibited. In the same experimental conditions, ethanolic extracts improved E. coli growth significantly, because the yield reached 10¹¹ CFU per milliliter. Ulva lactuca and P. palmata allowed the better growth. The Dragendorff-positive compounds extracted from bacterial cells growing on each ethanolic extract exhibited an osmoprotective effect as proved by a disk-diffusion assay. On the other hand, the -onium compounds (quaternary ammonium [betaines] and tertiary sulphonium) and total free amino acid contents of U. lactuca ethanolic extracts were higher than in others. Fucaceae extracts demonstrated especially high protein content. Algal extracts constitute not only an appreciable osmoprotection source for E. coli but also nutrient sources. Correspondence to: J. Minet     

Studies on fungal polysaccharide XVII. A new glucuronan “protuberic acid” produced by a fungus Kobayashi Nipponica

A water-soluble glucuronan “protuberic acid”, [α]_d²² −83.6° and purified from Kobayashia Nipponica, and its physicochemical properties were investigated.The purified protuberic acid was homogeneous as shown by zone electrophoresis, gel filtration over Sepharose 4B, and ultracentrifugation. The sedimentation coefficient was 1.8 S and its intrinsic viscosity was 1.1 dl/g. By gel filtration the molecular weight was estimated to be about 170 000. The results of periodate oxidation, methylation analysis, and partial acid hydrolysis indicated that this acidic polysaccharide has a linear structure of mainly 1,4-linkages and containing an acid-labile linkage. Reduced protuberic acid, [α]_d²² −44°, is also described.     

Uptake kinetics and assimilation of phosphorus byCatenella nipaeandUlva lactucacan be used to indicate ambient phosphate availability

Uptake, assimilation and compartmentation of phosphate were studied in the opportunist green macroalgaUlva lactucaand the estuarine red algal epiphyteCatenella nipae. The Michaelis–Menten model was used to describe uptake rates of inorganic phosphate (P_i) at different concentrations. Maximum uptake rates (V _max) of P-starved material exceededV _maxof P-enriched material; this difference was greater forC. nipae. Uptake and allocation of phosphorus (P) to internal pools was measured using trichloroacetic acid (TCA) extracts and³²P. Both species demonstrated similar assimilation paths: when P-enriched, most³²P accumulated as free phosphate. When unenriched,³²P was rapidly assimilated into the TCA-insoluble pool.C. nipaeconsistently assimilated more³²P into this pool thanU. lactuca, indicatingC. nipaehas a greater P-storage capacity. In both species,³²P release data showed two internal compartments with very different biological half-lives. The rapidly exchanging compartment had a short half-life of 2 to 12 min, while the slowly exchanging compartment had a much longer half-life of 12 days in P-starvedC. nipaeor 4 days in P-starvedU. lactuca. In both species, the slowly exchanging compartment accounted for more than 90% of total tissue.U. lactucaandC. nipaeresponded differently to high external P_i.U. lactucarapidly took up P_i, transferring this P_iinto tissue phosphate and TCA-soluble P in a few hours (90% of total P).C. nipaetook up P_iat lower rates and stored much of this P in less mobile TCA-insoluble forms. Long-term storage of refractory forms of P makesC. nipaea useful bioindicator of the prevailing conditions of P_iavailability over at least the previous 7 days, whereas the P-status ofU.lactucamay reflect conditions over no more than the previous few hours or days.C. nipaeis a more useful bioindicator for P status of estuarine and marine waters thanU. lactuca.     

Chemical and physical-chemical characteristics of dietary fibres from Ulva lactuca (L.) Thuret and Enteromorpha compressa (L.) Grev.

Dietary fibres from Ulva lactuca (L.) Thuret (sea lettuce) and Enteromorpha compressa (L.) Grev. (A.O. nori) were measured according to a ‘standard’ method and a ‘physiological’ protocol simulating the gastric and intestinal environments. U. lactuca contained 15.8–8.0% soluble and 24.2–32.6% insoluble fibres according to the ‘standard’ and ‘physiological’ methods, respectively. For E. compressa, these values were 14.9–15.9 and 21.6–28.7%, respectively. For both algae, the composition suggests that the soluble fibres were xylorhamnoglycuronans sulphates and insoluble fibres were essentially composed of glucans. No marked chemical compositional variation was observed between soluble fractions extracted under the simulated gastric and intestinal conditions. Fibres in both algae are hydrophilic but the water holding capacities were higher after extraction of soluble fibres (5.5–9.5 g g⁻¹ for the dry algae; 14.0–16.0 g g⁻¹ for the standard insoluble fibres). Water soluble fibres demonstrated low intrinsic viscosities at 37 °C in buffers, particularly those from E. compressa (36.0–36.5 ml g⁻¹), and was affected by pH for those of U. lactuca (147.5 ml g⁻¹ at pH 3.0 and 175.0 ml g⁻¹ at pH 7.3).     

Improvement of lipid profile and antioxidant of hypercholesterolemic albino rats by polysaccharides extracted from the green alga Ulva lactuca Linnaeus

Sulfated polysaccharides from Ulva lactuca were extracted in hot water and precipitated by ethanol then orally gavaged to rats fed on a hypercholesterolemic diet for 21 days to evaluate the antihypercholesterolemic and antioxidant actions. Atorvastatine Ca (Lipitor) was used as a reference drug. The intragastric administration of U. lactuca extract to hypercholesterolemic rats caused significant decrease of serum total lipids, triglycerides, total cholesterol, LDL-cholesterol and vLDL-cholesterol levels. Whereas, HDL-cholesterol concentration was markedly increased by 180%. Aqueous extract showed a significant ameliorative action on elevated atherogenic index, creatine kinase and lactate dehydrogenase activities of hypercholesterolemic group. Furthermore, serum activities of transaminases and alkaline phosphatase were also improved. High fat diet intake caused a highly significantly elevated serum urea, creatinine concentration. These effects were reversed by oral administration of U. lactuca extract. Sulfates polysaccharides extract of U. lactuca ameliorate hepatic enzymatic (catalase, glutathione peroxidase and superoxide dismutase), non-enzymatic (reduced glutathione & total thiol) antioxidant defenses and thiobarbituric acid reactive substances. In conclusion, the tested U. lactuca polysaccharides extract has potent hypocholesterolemic and antioxidant effects in experimentally-induced hypercholesterolemic animal model.     

Gracilaria conferta and its epiphytes: 3. Allelopathic inhibition of the red seaweed byUlva cf.lactuca

In a previous study (Svirski et al., 1993), it was found that growth inhibition ofGracilaria spp., when cultured in the presence ofUlva cf.lactuca, was not due to shading or nutrient depletion, but seemed to be caused by competition for inorganic carbon or some type of allelopathy. In the present study, we attempted to differentiate between these two possible influences by (1) growing the two algae in biculture under various conditions, but keeping inorganic carbon levels constant and measuring net photosynthesis, respiration and growth rates, and by (2) measuring growth rates ofGracilaria spp. in the presence of extracts derived from media previously used to growUlva cf.lactuca.Both net photosynthesis and growth rates ofGracilaria spp. in biculture were inhibited, despite CO₂ (and also HCO₃ ^–) levels being kept constantly high in the culture media. It is likely that these responses were due to markedly enhanced rates of dark respiration inGracilaria spp. when grown together withUlva cf.lactuca. Growth ofGracilaria spp. was also inhibited by extracts derived from seawater in whichUlva cf.lactuca had previously been grown. The strong inhibition by ethyl acetate and chloroform extracts indicate an allelopathic effect onGracilaria spp.     

Production of oligoglucuronans by enzymatic depolymerization of nascent glucuronan

An original bioreactor process for production of oligoglucuronans was developed using the Sinorhizobium meliloti M5N1CS strain that produces glucuronan. This anionic homopolysaccharide was composed of beta-D-(1,4)-glucopyranosyluronic residues variably O-acetylated at C-3 and/or C-2 positions according to culture conditions. It was depolymerized during its biosynthesis by addition of a fungal glucuronan lyase activity in broths. After purification by tangential ultrafiltration and low-pressure liquid chromatography, (1)H NMR and ESI-Q/TOF-MS characterized the poly- and oligoglucuronic acid fractions. This enzymatic bioreactor strategy authorized the production in gram quantity of an unsaturated and no acetylated oligoglucuronan with a degree of polymerization of 3.     

Lead,mercury, and certain nutrient elements in ulva lactuca (linnaeus) from Ras Beirut,Lebanon

Collections of Ulva lactuca (Linnaeus) at nine locations along the coast of Ras Beirut, Lebanon were analysed for lead, mercury, phosphate, calcium, magnesium, iron, copper, and zinc. Low lead concentrations, the same in all samples, could possibly indicate that U. lactuca has some means by which lead uptake and toxicity can be controlled. Phosphate levels may be a contributing factor to this process.Concentrations of all other elements seemed relatively uniform with few exceptions. This suggests that U. lactuca is subject to similar environmental conditions and element exposure at each of the collecting sites and might be capable of maintaining biochemical stability under high levels of stress.Further studies of toxic and nutrient elements as to their individual and combined effects in U. lactuca are needed.c/o Sturdy Memorial Hospital, Attleboro, Massachusetts U.S.A.     

Gracilaria and its epiphytes: 4. The response of two Gracilaria species to Ulva lactuca in a bacteria-limited environment

The responses of Gracilaria lemaneiformis, an easily epiphytized host,and the relatively resistant G. cornea mutant, to the green alga Ulva lactuca were studied using biculture experiments with and withoutantibiotics. Both Gracilaria species grown with and without U.lactuca showed different levels of growth rate, release of hydrogenperoxide and of halogenated hydrocarbons. These quantitative differencesled to a successful response against Ulva lactuca in the case of G.cornea mutant and to a failure in response in the case of G.lemaneiformis. The response of each Gracilaria species to U.lactuca was qualitatively similar to its response to bacteria. This suggeststhe involvement of oligosaccharide elicitors produced in the presence ofepiphytes and bacteria. A clear Gracilaria inhibition was demonstratedwith extracts of the culture medium. It appears that hydrogen peroxide,halogenated hydrocarbons and oligosaccharides may be components of theinhibitory activity of the extracts. The responses of Gracilaria speciesto the presence of U. lactuca suggest the characterization of adefence response.     

Evidence for inhibition of bacterial luminescence by allelochemicals from <Emphasis Type="Italic">Fibrocapsa japonica</Emphasis> (Raphidophyceae), and the role of light and microalgal growth rate

The marine microalga Fibrocapsa japonica Toriumi and Takano (Raphidophyceae) produces haemolysins, neurotoxins and reactive oxygen species (ROS). To quantify potential effects of such bioactive compounds on surrounding organisms the marine bacterium Vibrio fischeri was exposed to F. japonica culture samples. Inhibition of V. fischeri ‘s natural luminescence, indicative of impaired metabolism, was related to the number of F. japonica cells added. The effect was fast, within 15 min. It was caused by one, possibly several, excreted substances that were less active after heating. Freezing of culture supernatant partly inactivated these substances, but ROS-scavenging enzymes had no effect. Light enhanced the V. fischeri luminescence inhibition in two ways. The direct effect of light on the action of F. japonica luminescence inhibiter(s) could be described by a saturation curve with maximum effect above 20 μmol photons m⁻² s⁻¹. Light also had an indirect effect: biomass production, dependent on light availability, was closely related to the amount of inhibiting compound(s) produced by the alga. Algal growth rate, rather than its cell density, determined the bacterial luminescence inhibition per F. japonica cell, resulting in a 5-fold stronger inhibition at maximum growth rates compared to cells that barely grew during the stationary growth phase. The bioassay with F. japonica and V. fischeri has allowed quantification of the negative effects on bacteria in the microalgal microenvironment. The results presented here suggest that at favourable growth conditions F. japonica releases bioactive compounds that improve its competitive abilities.     

Differential growth response of <Emphasis Type="Italic">Ulva lactuca</Emphasis> to ammonium and nitrate assimilation

Controlled cultivation of marine macroalgal biomass such as Ulva species, notably Ulva lactuca, is currently studied for production of biofuels or functional food ingredients. In a eutrophic environment, this macrophyte is exposed to varying types of nutrient supply, including different and fluctuating levels of nitrogen sources. Our understanding of the influences of this varying condition on the uptake and growth responses of U. lactuca is limited. In this present work, we examined the growth response of U. lactuca exposed to different sources of nitrogen (NH₄⁺; NO₃⁻; and the combination NH₄NO₃) by using photo-scanning technology for monitoring the growth kinetics of U. lactuca. The images revealed differential increases of the surface area of U. lactuca disks with time in response to different N-nutrient enrichments. The results showed a favorable growth response to ammonium as the nitrogen source. The NH₄Cl and NaNO₃ rich media (50 μM of N) accelerated U. lactuca growth to a maximum specific growth rate of 16.4 ± 0.18% day⁻¹ and 9.4 ± 0.72% day⁻¹, respectively. The highest biomass production rate obtained was 22.5 ± 0.24 mg DW m⁻²·day⁻¹. The presence of ammonium apparently discriminated the nitrate uptake by U. lactuca when exposed to NH₄NO₃. Apart from showing the significant differential growth response of U. lactuca to different nitrogen sources, the work exhibits the applicability of a photo-scanning approach for acquiring precise quantitative growth data for U. lactuca as exemplified by assessment of the growth response to two different N-sources.     

The effect of water flow on photosynthetic processes of the alga Ulva lactuca L.

Photosynthetic rates of aquatic macrophytes are affected by the diffusion of carbon through the boundary layer which decreases with increasing flow velocities. This is shown by a significant increase in photosynthetic rates of Ulva lactuca fronds exposed to friction velocities (u _*) between 0.0 and 0.3 cm s^–1. No further increase in photosynthetic rates is observed at u _* values between 0.3 and 1.0 cm s^–1. Stagnating flow conditions at saturating light levels caused a 29% reduction in photosynthetic rates. Experiments in a closed system indicate that Ulva may utilize internal carbon sources during periods of stagnation. On a subtidal rock directly exposed to wave action, Ulva lactuca is not exposed to such conditions while in a wave flushed intertidal pool stagnation occurs during very short time intervals based on in situ measurements of u _* levels on fronds of this species using a microprocessor-controlled, autonomous recorder and hot-film sensors.     

Effects of wild and farm-grown macroalgae on the growth of juvenile South African abalone Haliotis midae Linnaeus

The effect of various macroalgal diets on the growth of grow-out (>20 mm shell length) South African abalone Haliotis midae was investigated on a commercial abalone farm. The experiment consisted of four treatments: fresh kelp blades (Ecklonia maxima (Osbeck) Papenfuss) (c. 10% protein); farmed, protein-enriched Ulva lactuca Linnaeus (c. 26% protein) grown in aquaculture effluent; wild U. lactuca (c. 20% protein); and a combination diet of kelp blades + farmed U. lactuca. Abalone grew best on the combination diet (0.423 ± 0.02% weight d^?1 SGR [specific growth rate]; 59.593 ± 0.02 ?m d^?1 DISL [daily increment in shell length]; 1.093 final CF [condition factor]) followed by the kelp only diet (0.367 ± 0.02% weight d^?1 SGR; 53.148 ± 0.02 ?m d^?1 DISL; 1.047 final CF), then the farmed, protein-enriched U. lactuca only diet (0.290 ± 0.02% weight d^?1 SGR; 42.988 ± 0.03 um d”¹ DISL; 1.013 final CF) that in turn outperformed the wild U. lactuca only diet (-0.079 ± 0.01% weight d^?1 SGR; 3.745 ± 0.02 ?m d”^?1 DISL; 0.812 final CF). The results suggest that protein alone could not have accounted for the differences produced by the varieties of U. lactuca and that the gross energy content is probably important.     

Algal biomass: A sustainable,economical and renewable approach for microbial production of pectinolytic enzymes using submerged and solid state fermentation techniques

In the past decade, algal waste has been used as useful natural resource for production of enormous range of products that have wide economical and commercial importance. Pectinases are group of enzymes that have wide commercial applications. Hence, current study was designed to utilize algal biomass for the production of pectinases using submerged (SmF) and solid state fermentation (SSF) techniques. Different algal sources including brown (Dictyopteris polypodioides, Sargassum wightii and Dictyopteris divaricata) and green algae (Ulva lactuca and Codium tomentosum) were used and U. lactuca was found to be the most suitable substrate. Several bacterial and fungal strains were screened and among them Bacillus licheniformis KIBGE-IB4 was selected based on maximum pectinase production. SmF and SSF were studied utilizing U. lactuca as a substrate and results revealed that enzyme production was favoured by SmF (2457?±?3.31?U?mg^?1) as compared to SSF (1432?±?1.46?U?mg^?1). Parametric optimization of pectinase production indicated that B. licheniformis KIBGE-IB4 requires 10.0?g L^–1 U. lactuca as a biomass in the medium with a pH 7.0 when incubated at 37?°C for 24 hours. Likewise, production of pectinase using algal resource was also compared with that of the conventional agricultural biomass and it was observed that when U. lactuca was used, the selected bacterial isolate produced a higher yield of enzyme than sugarcane bagasse and rice husk. Hence, it is anticipated that algal biomass can be efficiently utilized as an environmental friendly bioresource for the production of industrially important hydrolytic enzymes.     

Energy from algae using microbial fuel cells

Bioelectricity production from a phytoplankton, Chlorella vulgaris, and a macrophyte, Ulva lactuca was examined in single chamber microbial fuel cells (MFCs). MFCs were fed with the two algae (as powders), obtaining differences in energy recovery, degradation efficiency, and power densities. C. vulgaris produced more energy generation per substrate mass (2.5 kWh/kg), but U. lactuca was degraded more completely over a batch cycle (73 ± 1% COD). Maximum power densities obtained using either single cycle or multiple cycle methods were 0.98 W/m² (277 W/m³) using C. vulgaris, and 0.76 W/m² (215 W/m³) using U. lactuca. Polarization curves obtained using a common method of linear sweep voltammetry (LSV) overestimated maximum power densities at a scan rate of 1 mV/s. At 0.1 mV/s, however, the LSV polarization data was in better agreement with single‐ and multiple‐cycle polarization curves. The fingerprints of microbial communities developed in reactors had only 11% similarity to inocula and clustered according to the type of bioprocess used. These results demonstrate that algae can in principle, be used as a renewable source of electricity production in MFCs. Biotechnol. Bioeng. 2009;103: 1068–1076. © 2009 Wiley Periodicals, Inc.     

THE PERFORMANCE OF SPRAY‐IRRIGATED ULVA LACTUCA (ULVOPHYCEAE,CHLOROPHYTA) AS A CROP AND AS A BIOFILTER OF FISHPOND EFFLUENTS1

The seaweed Ulva lactuca L. was spray cultured by mariculture effluents in a mattress‐like layer, held in air on slanted boards by plastic netting. Air‐agitated seaweed suspension tanks were the reference. Growth rate, yield, and ammonia‐N removal rate were 11.8% · d^?1, 171 g fresh weight (fwt) · m^?2 · d^?1, and 5 g N · m^?2 · d^?1, respectively, by the spray‐cultured U. lactuca, and 16.9% · d^?1, 283 g fwt · m^?2 · d^?1, and 7 g N · m^?2 · d^?1, respectively, by the tank U. lactuca. Biomass protein content was similar in both treatments. Dissolved oxygen in the fishpond effluent water was raised by >3 mg · L^?1 and pH by up to half a unit, upon passage through both culture systems. The data suggest that spray‐irrigation culture of U. lactuca in this simple green‐mattress‐like system supplies the seaweed all it needs to grow and biofilter at rates close to those in standard air‐agitated tank culture.     

Effect of UV radiation on photoinhibition of marine macrophytes in culture systems

The present study examined the effect of UV andphotosynthetically active radiation (PAR) onphotoinhibition and recovery in the Phaeophyte Macrocystis pyrifera, the Rhodophyte Chondruscrispus and the Chlorophyte Ulva lactuca underoutdoor culture conditions. There was an increase inphotoinhibition as a consequence of high exposure toUV-B radiation in M. pyrifera, however, highlevels of PAR accounted for most of thephotoinhibition in C. crispus and U.lactuca. Photodamage by UV-A, UV-B and PAR wascompletely repaired within 5 h and effective quantumyield reached pretreatment values in the three speciesstudied. Species were less susceptible tophotoinhibition after being incubated for 5 d underhigh exposures of natural irradiance suggesting aphotoadaptive process. The recovery of the effectivequantum yield was impaired by long exposure to highlevels of UV-B in C. crispus and UV-A, UV-B andPAR in M. pyrifera. This suggests a differentkind of damage by UV-A and PAR radiation, one to thephotosynthetic apparatus and another which affects therepair mechanism of some species. There was anincrease in UV-absorption ( 330 nm) in M. pyrifera and C. crispus within four days ofthe initiation of the experiment suggesting that thesespecies photoprotect their photosynthetic system whenexposed to elevated UV and PAR levels.