Online micro solid phase extraction coupled with ultra-performance liquid chromatography-tandem mass spectrometry for trace analysis of endogenous plant hormones in Ulva linza.

OBJECTIVE: Ulva linza (L.) is a species of green algae widely distributed in China. We aimed to establish a sensitive online analytical method for quantification of endogenous phytohormones in fresh minute seaweed samples. METHOD: The method for quantification of endogenous plant hormones in fresh minute samples was developed based on a homemade online micro solid phase extraction (m-SPE) system coupled with an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) platform. The online m-SPE instrument injected the eluent of m-SPE directly onto the LC separation column, improving the utilization of samples and saving time. The m-SPE column, of which the effective size was 9.6 × 2 mm i.d., was filled with 19 mg of C18 (10 µm). RESULTS: Under optimized conditions, the limits of detection were 0.002-0.060 ng ml-1 for five plant hormones. The actual sample recoveries of phytohormones were 76.4-103.4% and the coefficients of variance were below 14.1%. The temporal distribution of these endogenous plant hormones of U. linza during different growth periods is described. CONCLUSION: The proposed online m-SPE method was successfully applied to quantification of endogenous acidic and alkaline plant hormones in U. linza. It provides important information for the further study of the physiological and ecological effects of plant hormones in lower algal species.

Low Fouling Polysulfobetaines with Variable Hydrophobic Content.

Amphiphilic polymer coatings combining hydrophilic elements, in particular zwitterionic groups, and hydrophobic elements comprise a promising strategy to decrease biofouling. However, the influence of the content of the hydrophobic component in zwitterionic coatings on the interfacial molecular reorganization dynamics and the anti-fouling performance is not well understood. Therefore, coatings of amphiphilic copolymers of sulfobetaine methacrylate 3-[N-2'-(methacryloyloxy)ethyl-N,N-dimethyl]-ammonio propane-1-sulfonate (SPE) are prepared which contain increasing amounts of hydrophobic n-butyl methacrylate (BMA). Their fouling resistance is compared to that of their homopolymers PSPE and PBMA. The photo-crosslinked coatings form hydrogel films with a hydrophilic surface. Fouling by the proteins fibrinogen and lysozyme as well as by the diatom Navicula perminuta and the green algae Ulva linza is assessed in laboratory assays. While biofouling is strongly reduced by all zwitterionic coatings, the best fouling resistance is obtained for the amphiphilic copolymers. Also in preliminary field tests, the anti-fouling performance of the amphiphilic copolymer films is superior to that of both homopolymers. When the coatings are exposed to a marine environment, the reduced susceptibility to silt incorporation, in particular compared to the most hydrophilic polyzwitterion PSPE, likely contributes to the improved fouling resistance.

Topographic cues guide the attachment of diatom cells and algal zoospores.

Surface topography plays a key role in the colonization of substrata by the colonizing stages of marine fouling organisms. For the innovation of marine antifouling coatings, it is essential to understand how topographic cues affect the settlement of these organisms. In this study, tapered, spiked microstructures and discrete honeycombs of varying feature dimensions were designed and fabricated in order to examine the influence of topography on the attachment of zoospores of the green macroalga Ulva linza and cells of the diatom (microalga) Navicula incerta. Contrasting results were obtained with these two species of algae. Indeed, the preferred location of cells of N. incerta was dominated by attachment point theory, which suggested a positive correlation between the density of cells adhering and the amount of available attachment points, while the settlement of spores of U. linza was mainly regulated by both Wenzel roughness and local binding geometry.

The performance of hybrid titania/silica-derived xerogels as active antifouling/fouling-release surfaces against the marine alga Ulva linza: in situ generation of hypohalous acids.

Mixed titania/silica xerogels were prepared using titanium tetraisopropoxide (TTIP) and tetraethoxy orthosilicate (TEOS). Xerogel properties were modified by incorporating n-octyltriethoxysilane (C8). The xerogels catalyze the oxidation of bromide and chloride with hydrogen peroxide (H2O2) to produce hypohalous acids at pH 7 and pH 8. The antifouling/ fouling-release performance of a TTIP/C8/TEOS xerogel in the presence and absence of H2O2 was evaluated for the settlement of zoospores of the marine alga Ulva linza and for the removal of sporelings (young plants). In the absence of H2O2, differences in the settlement of zoospores and removal of sporelings were not significant relative to a titanium-free C8/TEOS xerogel. Addition of H2O2 gave a significant reduction in zoospore settlement and sporeling removal relative to the C8/TEOS xerogel and relative to peroxide-free conditions. The impact of TTIP on xerogel characteristics was evaluated by comprehensive contact angle analysis, scanning electron microscopy, and X-ray photoelectron spectroscopy.

Charged hydrophilic polymer brushes and their relevance for understanding marine biofouling.

The resistance of charged polymers to biofouling was investigated by subjecting cationic (PDMAEMA), anionic (PSPMA), neutral (PHEMA-co-PEG10MA), and zwitterionic (PSBMA) brushes to assays testing protein adsorption; attachment of the marine bacterium Cobetia marina; settlement and adhesion strength of zoospores of the green alga Ulva linza; settlement of barnacle (Balanus amphitrite and B. improvisus) cypris larvae; and field immersion tests. Several results go beyond the expected dependence on direct electrostatic attraction; PSPMA showed good resistance towards attachment of C. marina, low settlement and adhesion of U. linza zoospores, and significantly lower biofouling than on PHEMA-co-PEG10MA or PSBMA after a field test for one week. PDMAEMA showed potential as a contact-active anti-algal coating due to its capacity to damage attached spores. However, after field testing for eight weeks, there were no significant differences in biofouling coverage among the surfaces. While charged polymers are unsuitable as antifouling coatings in the natural environment, they provide valuable insights into fouling processes, and are relevant for studies due to charging of nominally neutral surfaces.

Effects of surface-active block copolymers with oxyethylene and fluoroalkyl side chains on the antifouling performance of silicone-based films.

Block copolymers made from a poly(dimethyl siloxane) (Si) and a poly(meth)acrylate carrying oxyethylene (EG) or fluoroalkyl (AF) side chains were synthesized and incorporated as surface-active components into a silicone matrix to produce cross-linked films with different surface hydrophilicity/phobicity. Near-edge X-ray absorption fine structure (NEXAFS) studies showed that film surfaces containing Si-EG were largely populated by the siloxane, with the oxyethylene chains present only to a minor extent. In contrast, the fluorinated block was selectively segregated to the polymer-air interface in films containing Si-AF as probed by NEXAFS and X-ray photoelectron spectroscopy (XPS) analyses. Such differences in surface composition were reflected in the biological performance of the coatings. While the films with Si-EG showed a higher removal of both Ulva linza sporelings and Balanus amphitrite juveniles than the silicone control, those with Si-AF exhibited excellent antifouling properties, preventing the settlement of cyprids of B. amphitrite.

Holographic microscopy provides new insights into the settlement of zoospores of the green alga Ulva linza on cationic oligopeptide surfaces.

Interaction of zoospores of Ulva linza with cationic, arginine-rich oligopeptide self-assembled monolayers (SAMs) is characterized by rapid settlement. Some spores settle (ie permanently attach) in a 'normal' manner involving the secretion of a permanent adhesive, retraction of the flagella and cell wall formation, whilst others undergo 'pseudosettlement' whereby motile spores are trapped (attached) on the SAM surface without undergoing the normal metamorphosis into a settled spore. Holographic microscopy was used to record videos of swimming zoospores in the vicinity of surfaces with different cationic oligopeptide concentrations to provide time-resolved insights into processes associated with attachment of spores. The data reveal that spore attachment rate increases with increasing cationic peptide content. Accordingly, the decrease in swimming activity in the volume of seawater above the surface accelerated with increasing surface charge. Three-dimensional trajectories of individual swimming spores showed a 'hit and stick' motion pattern, exclusively observed for the arginine-rich peptide SAMs, whereby spores were immediately trapped upon contact with the surface.

Antifouling properties of oligo(lactose)-based self-assembled monolayers.

The antifouling (AF) properties of oligo(lactose)-based self-assembled monolayers (SAMs), using four different proteins, zoospores of the green alga Ulva linza and cells of the diatom Navicula incerta, were investigated. The SAM-forming alkylthiols, which contained 1, 2 or 3 lactose units, showed significant variation in AF properties, with no differences in wettability. Non-specific adsorption of albumin and pepsin was low on all surfaces. Adsorption of lysozyme and fibrinogen decreased with increasing number of lactose units in the SAM, in agreement with the generally observed phenomenon that thicker hydrated layers provide higher barriers to protein adsorption. Settlement of spores of U. linza followed an opposite trend, being greater on the bulkier, more hydrated SAMs. These SAMs are more ordered for the larger saccharide units, and it is therefore hypothesized that the degree of order, and differences in crystallinity or stiffness between the surfaces, is an important parameter regulating spore settlement on these surfaces.

Conditioning of self-assembled monolayers at two static immersion test sites along the east coast of Florida and its effect on early fouling development.

Among the first events after immersion of surfaces in the ocean is surface 'conditioning'. Here, the accumulation and composition of the conditioning films formed after immersion in the ocean are analyzed. In order to account for different surface chemistries, five self-assembled monolayers that differ in resistance to microfouling and wettability were used. Water samples from two static immersion test sites along the east coast of Florida were collected at two different times of the year and used for experiments. Spectral ellipsometry revealed that conditioning films were formed within the first 24 h and contact angle goniometry showed that these films changed the wettability and rendered hydrophobic surfaces more hydrophilic and vice versa. Infrared reflection adsorption spectroscopy showed that the composition of the conditioning film depended on both the wettability and immersion site. Laboratory and field assays showed that the presence of a conditioning film did not markedly influence settlement of microorganisms.

Flash extraction of ulvan polysaccharides from marine green macroalga Ulva linza and evaluation of its antioxidant and gut microbiota modulation activities.

In this study, flash extraction was used to rapidly extract water-soluble polysaccharides from Ulva linza. The optimal extraction process for the flash extraction was determined by Box-Behnken design with extraction temperature 80 °C, extraction time 117 s, liquid-solid ratio 46:1 (mL/g) and a corresponding yield of 18.5 %. The crude Ulva linza polysaccharides (CULP) were subsequently isolated by chromatography technology to obtain purified Ulva linza polysaccharide (ULP) and characterized by monosaccharide composition and molecular weight determination analysis. Furthermore, the antioxidant bioactivity of ULP was studied and the results revealed that it had a good scavenging effect on DPPH, ABTS and OH, with IC50 values of 149.2 µg/mL, 252.5 µg/mL and 1073 µg/mL, respectively. After in vitro fermentation by human fecal microbiota, the pH value of fermentation culture significantly decreased to 5.06, suggesting that ULP could be hydrolyzed and utilized by gut microbiota. The abundance of beneficial bacteria including Bacteroides, Parabacteroides and Faecalibacterium was improved. Meanwhile, the relative abundance of Prevotella, Blautia and Ruminococcus was decreased, and the low ratio of these organisms might reveal positive effects on maintaining the balance of gut microbial biodiversity. These results suggested that the composition of the human gut microbiota could be modulated by ULP, and ULP might possess the potential to maintain gut homeostasis and improve human intestinal health.

The characterisation of Wickerhamomyces anomalus M15, a highly tolerant yeast for bioethanol production using seaweed derived medium.

Advanced generation biofuels have potential for replacing fossil fuels as society moves forward into a net-zero carbon future. Marine biomass is a promising source of fermentable sugars for fermentative bioethanol production; however the medium derived from seaweed hydrolysis contains various inhibitors, such as salts that affected ethanol fermentation efficiency. In this study the stress tolerance of a marine yeast, Wickerhamomyces anomalus M15 was characterised. Specific growth rate analysis results showed that Wickerhamomyces anomalus M15 could tolerate up to 600 g/L glucose, 150 g/L xylose and 250 g/L ethanol, respectively. Using simulated concentrated seaweed hydrolysates, W. anomalus M15's bioethanol production potential using macroalgae derived feedstocks was assessed, in which 5.8, 45.0, and 19.9 g/L ethanol was produced from brown (Laminaria digitata), green (Ulva linza) and red seaweed (Porphyra umbilicalis) based media. The fermentation of actual Ulva spp. hydrolysate harvested from United Kingdom shores resulted in a relatively low ethanol concentration (15.5 g/L) due to challenges that arose from concentrating the seaweed hydrolysate. However, fed-batch fermentation using simulated concentrated green seaweed hydrolysate achieved a concentration of 73 g/L ethanol in fermentations using both seawater and reverse osmosis water. Further fermentations conducted with an adaptive strain W. anomalus M15-500A showed improved bioethanol production of 92.7 g/L ethanol from 200 g/L glucose and reduced lag time from 93 h to 24 h in fermentation with an initial glucose concentration of 500 g/L. The results indicated that strains W. anomalus M15 and W. anomalus M15-500A have great potential for industrial bioethanol production using marine biomass derived feedstocks. It also suggested that if a concentrated high sugar content seaweed hydrolysate could be obtained, the bioethanol concentration could achieve 90 g/L or above, exceeding the minimum industrial production threshold.

Ocean acidification exacerbates copper toxicity in both juvenile and adult stages of the green tide alga Ulva linza.

The toxicity of heavy metals to coastal organisms can be modulated by changes in pH due to progressive ocean acidification (OA). We investigated the combined impacts of copper and OA on different stages of the green macroalga Ulva linza, which is widely distributed in coastal waters, by growing the alga under the addition of Cu (control, 0.125 (medium, MCu), and 0.25 (high) µM, HCu) and elevated pCO2 of 1,000 µatm, predicted in the context of global change. The relative growth rates decreased significantly in both juvenile and adult thalli at HCu under OA conditions. The net photosynthetic and respiration rates, as well as the relative electron transfer rates for the adult thalli, also decreased under the combined impacts of HCu and OA, although no significant changes in the contents of photosynthetic pigments were detected. Our results suggest that Cu and OA act synergistically to reduce the growth and photosynthetic performance of U. linza, potentially prolonging its life cycle.

Allelopathic effects of Ulva linza on marine phytoplankton and identification of the allelochemicals.

Green tides have been increasing in frequency and severity in coastal areas in recent years, and thus, the adverse effects of green tides on the environment have attracted much research attention. Allelopathy is one of the most significant effects of green tide algae on the surrounding organisms. In this study, a series of experiments were developed to fully investigate the allelopathic effects of the green tide alga Ulva linza on two common coastal phytoplankton species and to isolate and identify the chemical compositions of the allelochemicals. Our results indicated that the fresh tissue, dry powder, and aqueous extracts all inhibited the population growth of the two experimental phytoplankton species, indicating the occurrence of allelopathy; further analysis of the allelochemicals identified multiple kinds of fatty acids as allelopathic compounds, in which the most abundant ones were hexadecanoic acid; 9Z, 12Z, 15Z-octadecatetrienoic acid; and 9E-octadecenoic acid. Our findings enrich the database for research on allelopathy between marine green tide algae and phytoplankton. Both algae and phytoplankton could increase their own competitive abilities during bloom formation, thereby changing the phytoplankton community structure.

Effect of Dipole Orientation in Mixed, Charge-Equilibrated Self-assembled Monolayers on Protein Adsorption and Marine Biofouling.

While zwitterionic interfaces are known for their excellent low-fouling properties, the underlying molecular principles are still under debate. In particular, the role of the zwitterion orientation at the interface has been discussed recently. For elucidation of the effect of this parameter, self-assembled monolayers (SAMs) on gold were prepared from stoichiometric mixtures of oppositely charged alkyl thiols bearing either a quaternary ammonium or a carboxylate moiety. The alkyl chain length of the cationic component (11-mercaptoundecyl)-N,N,N-trimethylammonium, which controls the distance of the positively charged end group from the substrate's surface, was kept constant. In contrast, the anionic component and, correspondingly, the distance of the negatively charged carboxylate groups from the surface was varied by changing the alkyl chain length in the thiol molecules from 7 (8-mercaptooctanoic acid) to 11 (12-mercaptododecanoic acid) to 15 (16-mercaptohexadecanoic acid). In this way, the charge neutrality of the coating was maintained, but the charged groups exposed at the interface to water were varied, and thus, the orientation of the dipoles in the SAMs was altered. In model biofouling studies, protein adsorption, diatom accumulation, and the settlement of zoospores were all affected by the altered charge distribution. This demonstrates the importance of the dipole orientation in mixed-charged SAMs for their inertness to nonspecific protein adsorption and the accumulation of marine organisms. Overall, biofouling was lowest when both the anionic and the cationic groups were placed at the same distance from the substrate's surface.

Future CO2-induced seawater acidification mediates the physiological performance of a green alga Ulva linza in different photoperiods.

Photoperiods have an important impact on macroalgae living in the intertidal zone. Ocean acidification also influences the physiology of macroalgae. However, little is known about the interaction between ocean acidification and photoperiod on macroalgae. In this study, a green alga Ulva linza was cultured under three different photoperiods (L: D = 8:16, 12:12, 16:8) and two different CO2 levels (LC, 400 ppm; HC, 1,000 ppm) to investigate their responses. The results showed that relative growth rate of U. linza increased with extended light periods under LC but decreased at HC when exposed to the longest light period of 16 h compared to 12 h. Higher CO2 levels enhanced the relative growth rate at a L: D of 8:16, had no effect at 12:12 but reduced RGR at 16:8. At LC, the L: D of 16:8 significantly stimulated maximum quantum yield (Yield). Higher CO2 levels enhanced Yield at L: D of 12:12 and 8:16, had negative effect at 16:8. Non-photochemical quenching (NPQ) increased with increasing light period. High CO2 levels did not affect respiration rate during shorter light periods but enhanced it at a light period of 16 h. Longer light periods had negative effects on Chl a and Chl b content, and high CO2 level also inhibited the synthesis of these pigments. Our data demonstrate the interactive effects of CO2 and photoperiod on the physiological characteristics of the green tide macroalga Ulva linza and indicate that future ocean acidification may hinder the stimulatory effect of long light periods on growth of Ulva species.

Model Amphiphilic Block Copolymers with Tailored Molecular Weight and Composition in PDMS-Based Films to Limit Soft Biofouling.

A set of controlled surface composition films was produced utilizing amphiphilic block copolymers dispersed in a cross-linked poly(dimethylsiloxane) network. These block copolymers contained oligo(ethylene glycol) (PEGMA) and fluoroalkyl (AF6) side chains in selected ratios and molecular weights to control surface chemistry including antifouling and fouling-release performance. Such properties were assessed by carrying out assays using two algae, the green macroalga Ulva linza (favors attachment to polar surfaces) and the unicellular diatom Navicula incerta (favors attachment to nonpolar surfaces). All films performed well against U. linza and exhibited high removal of attached sporelings (young plants) under an applied shear stress, with the lower molecular weight block copolymers being the best performing in the set. The composition ratios from 50:50 to 60:40 of the AF6/PEGMA side groups were shown to be more effective, with several films exhibiting spontaneous removal of the sporelings. The cells of N. incerta were also removed from several coating compositions. All films were characterized by surface techniques including captive bubble contact angle, atomic force microscopy, and near edge X-ray absorption fine structure spectroscopy to correlate surface chemistry and morphology with biological performance.

Complete mitochondrial genome of Ulva linza, one of the causal species of green macroalgal blooms in Yellow Sea, China.

Ulva linza, a green macroalgae, is one of the causal species of the world's largest macroalgal blooms in the Yellow Sea, China. In this study, we sequenced and annotated the complete mitochondrial genome of U.linza (GenBank accession no. KU189740). The genome consists of circular chromosomes of 70 858 bp and encodes a total of 28 protein-coding genes including eight rps genes, three rpl genes, five atp genes, three cox genes, eight nad genes and cob gene. Phylogenetic analysis showed U. linza clustered into Ulvophyceae clade and had close genetic relationship with algae Ulva prolifera.

Occurrence of the PsbS and LhcSR products in the green alga Ulva linza and their correlation with excitation pressure.

To avoid photoinhibition, plants have developed diverse photoprotection mechanisms. One of the short-term high light protection mechanisms in plants is non-photochemical quenching (NPQ), which dissipates the absorbed light energy as thermal energy. In the green alga, Ulva linza, the kinetics of NPQ starts with an initial, quick rise followed by a decline, and then a second and higher rise at longer time periods. During the whole phase, NPQ is triggered and controlled by ΔpH, then strengthened and modulated by zeaxanthin. Light-harvesting complex (LHC) family members are known to play crucial roles in this mechanism. The PSBS protein, a member of the LHC family that was thought to be present exclusively in higher plants, has been identified for the first time in U. linza. The expression of both PSBS and LHCSR was up-regulated during high light conditions, and LHCSR increased more than PSBS. Both LHCSR and PSBS-dependent NPQ may be important strategies for adapting to the environment, and they have undoubtedly played a role in their evolution.