Effects of temperature and irradiance on photosynthesis and growth of a green-tide-forming species (<Emphasis Type="Italic">Ulva linza</Emphasis>) in the Yellow Sea

Samples of the massive drifting green alga, Ulva linza, were collected from the coastal waters of the Yellow Sea, southwest of Korea, in early July 2009, and cultured under laboratory conditions. The effects of various temperature (10–30°C) and irradiance levels (0–1,000 μmol photons m⁻² s⁻¹) on photosynthesis, growth, and tissue nutrient content of U. linza were investigated in laboratory for both individuals of the late-stage vegetation (LSV) and the early-stage vegetation (ESV). After 1 h acclimation to various irradiance and temperature conditions, maximum gross photosynthetic rate of ESV was significantly higher than those of LSV. In the long-term (7-d) acclimation experiments to various irradiance and temperature levels, gross photosynthetic rates of ESV individuals were also significantly higher than those of LSV individuals. High photosynthetic rate of ESV individuals induced increase in mass of about 60% over the growth saturation irradiance (136 μmol photons m⁻² s⁻¹) and about 20% under low temperature conditions (10–15°C) during 7-d. The gross photosynthesis of LSV individuals was low when examined under temperature and irradiance conditions that were optimum for ESV growth. Consequently, free-floating U. linza exhibits cellular senescence beginning in early July in the Yellow Sea, and green tides formed by this species cannot be maintained beyond this time in the open sea. However, we expect that U. linza can proliferate quickly after settlement on new coastal habitats of the Yellow Sea because of the high tissue nitrogen utilization for photosynthesis in ESV, which is formed by germination of reproductive cells.     

Expression and fermentation optimization of oxidized polyvinyl alcohol hydrolase in E. coli

Oxidized polyvinyl alcohol (PVA) hydrolase (OPH) is a key enzyme in the degradation of PVA, suggesting that OPH has a great potential for application in textile desizing processes. In this study, the OPH gene from Sphingopyxis sp. 113P3 was modified, by artificial synthesis, for overexpression in Escherichia coli. The OPH gene, lacking the sequence encoding the original signal peptide, was inserted into pET-20b (+) expression vector, which was then used to transform E. coli BL21 (DE3). OPH expression was detected in culture medium in which the transformed E. coli BL21 (DE3) was grown. Nutritional and environmental conditions were investigated for improved production of OPH protein by the recombinant strain. The highest OPH activity measured was 47.54 U/mL and was reached after 84 h under optimal fermentation conditions; this level is 2.64-fold higher that obtained under sub-optimal conditions. The productivity of recombinant OPH reached 565.95 U/L/h. The effect of glycine on the secretion of recombinant OPH was examined by adding glycine to the culture medium to a final concentration of 200 mM. This concentration of glycine reduced the fermentation time by 24 h and increased the productivity of recombinant OPH to 733.17 U/L/h. Our results suggest that the recombinant strain reported here has great potential for use in industrial applications.     

Activity of Commercial Enzymes on Settlement and Adhesion of Cypris Larvae of the Barnacle Balanus amphitrite,Spores of the Green Alga Ulva linza,and the Diatom Navicula perminuta

Fouling species produce adhesive polymers during the settlement, adhesion and colonization of new surfaces in the marine environment. The present paper tests the hypothesis that enzymes of the appropriate specificity may prevent biofouling by hydrolysing these adhesive polymers. Seventeen commercially available enzyme preparations designed originally for bulk use in a range of end-use applications were tested for their effects on the settlement and/or adhesion of three major fouling species, viz. the green alga Ulva linza, the diatom Navicula perminuta and the barnacle Balanus amphitrite. The serine-proteases were found to have the broadest antifouling potential reducing the adhesion strength of spores and sporelings of U. linza, cells of N. perminuta and inhibiting settlement of cypris larvae of B. amphitrite. Mode-of-action studies on the serine-protease, Alcalase, indicated that this enzyme reduced adhesion of U. linza in a concentration-dependent manner, that spores of the species could recover their adhesive strength if the enzyme was removed and that the adhesive of U. linza and juvenile cement of B. amphitrite became progressively less sensitive to hydrolysis as they cured.     

Effect of bacterial biofilms formed on fouling-release coatings from natural seawater and Cobetia marina,on the adhesion of two marine algae

Previous studies have shown that bacterial biofilms formed from natural seawater (NSW) enhance the settlement of spores of the green alga Ulva linza, while single-species biofilms may enhance or reduce settlement, or have no effect at all. However, the effect of biofilms on the adhesion strength of algae, and how that may be influenced by coating/surface properties, is not known. In this study, the effect of biofilms formed from natural seawater and the marine bacterium Cobetia marina, on the settlement and the adhesion strength of spores and sporelings of the macroalga U. linza and the diatom Navicula incerta, was evaluated on Intersleek® 700, Intersleek® 900, poly(dimethylsiloxane) and glass. The settlement and adhesion strength of these algae were strongly influenced by biofilms and their nature. Biofilms formed from NSW enhanced the settlement (attachment) of both algae on all the surfaces while the effect of biofilms formed from C. marina varied with the coating type. The adhesion strength of spores and sporelings of U. linza and diatoms was reduced on all the surfaces biofilmed with C. marina, while adhesion strength on biofilms formed from NSW was dependent on the alga (and on its stage of development in the case of U. linza), and coating type. The results illustrate the complexity of the relationships between fouling algae and bacterial biofilms and suggest the need for caution to avoid over-generalisation.     

Characterization of gene expression of <Emphasis Type="Italic">QM</Emphasis> from <Emphasis Type="Italic">Caragana jubata</Emphasis>, a plant species that grows under extreme cold

Caragana [Caragana jubata (Pall.) Poir] is a temperate plant that thrives well under extremes of cold in high altitude of Himalaya and hence the plant is expected to be a source of genes that might play an important role in tolerance to low temperature (LT). In order to identify LT inducible gene(s), differential display of mRNA (DD) was performed using the apical buds growing under snow as well as growing in the near vicinity without snow, and a LT inducible QM gene (CjQM) homologue was identified. Realizing the importance of QM gene (which encodes human Wilms’ tumor suppressor QM protein) in aggregation of 40 and 60S ribosomal subunit and that not much has been reported on this gene in plant systems in relation to its relationship with LT, full length cDNA of CjQM was cloned through rapid amplification of cDNA ends. The gene (977 bp), encoded by small gene family, had an open reading frame of 651 bp and was found to be intronless. The gene exhibited up-regulation within 20 min of exposure to LT and abscisic acid (ABA), but no significant change in gene expression was observed in response to drought stress (DS), salicylic acid (SA) and methyl jasmonate (MJ) application. Up-regulation of CjQM was obtained in the tissues growing in situ under snow. Non-responsiveness of CjQM towards DS, SA and MJ, but up-regulation in response to LT and ABA suggested a specific regulation of the gene in Caragana under varied cues.     

Growth ofEnteromorpha linza in sewage effluent and sewage effluent-seawater mixtures

Enteromorpha linza (L.) J. Ag. was grown in full strength sewage effluent, various combinations of sewage effluent and seawater, and in natural seawater. It was found that full strength sewage effluent with a salinity of 14 supported best growth of the alga. After a 12 day cultivation period, growth ofE. linza in full strength sewage effluent and 75% sewage effluent- seawater mixture showed 3.5-fold and 2-fold increase in fresh weight over that grown in natural seawater; respectively. Uptake of PO _inf4 ^sup3– -P, NH₃-N and NO _inf3 ^sup– -N by cells ofE. linza was extremely efficient in all tested media. Data obtained from the experiments indicated that inorganic nitrogen rather than phosphorus was the limiting nutrient factor for growth ofE. linza in full strength sewage effluent and in other sewage effluent- seawater mixtures. NH₃-N at concentrations above 4.5 ppm was found to inhibit uptake of NO _inf3 ^sup– -N in the same culture medium by the algal cells. The fact that sewage grownE. linza contained comparatively much higher protein content (30.2% dry weight) than that grown in natural seawater (12.5% dry weight) leads to the conclusion that sewage grownE. linza could serve as an economically feasible feed for livestock in Hong Kong where the sewage is characterized by having a salinity of approximately 14. It is proposed that this multicellular green alga is a suitable algal species to serve the dual function of wastewater purification through the production of algal protein from sewage effluent having high salinities.     

The performance of aminoalkyl/fluorocarbon/hydrocarbon-modified xerogel coatings against the marine alga Ectocarpus crouaniorum: relative roles of surface energy and charge

The effect of a series of xerogel coatings modified with aminoalkyl/fluorocarbon/hydrocarbon groups on the adhesion of a new test species, the filamentous brown alga Ectocarpus crouaniorum, has been explored, and compared with the green alga Ulva linza. The results showed that E. crouaniorum adhered weakly to the less polar, low wettability coatings in the series, but stronger adhesion was shown on polar, higher surface energy coatings containing aminoalkyl groups. The results from a separate series of coatings tuned to have similar surface energies and polarities after immersion in artificial seawater (ASW), but widely different surface charges, demonstrated that surface charge was more important than surface energy and polarity in determining the adhesion strength of both E. crouaniorum and U. linza on xerogel coatings. No correlation was found between adhesion and contact angle hysteresis. X-ray photoelectron spectroscopy analysis of samples after immersion in ASW confirmed the presence of charged ammonium groups on the surface of the aminoalkylated coatings.     

Peroxidase from the green alga Enteromorpha linza

An enzyme displaying peroxidase activity has been extracted and purified 27-fold from the green alga Enteromorpha linza. The partially purified pre     

Enhanced production of 2,3-butanediol by engineered <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Production of 2,3-butanediol by Bacillus subtilis takes place in late-log or stationary phase, depending on the expression of bdhA gene encoding acetoin reductase, which converts acetoin to 2,3-butanediol. The present work focuses on the development of a strain of B. subtilis for enhanced production of 2,3-butanediol in early log phase of growth cycle. For this, the bdhA gene was expressed under the control of P _alsSD promoter of AlsSD operon for acetoin fermentation which served the substrate for 2,3-butanediol production. Addition of acetic acid in the medium induced the production of 2,3-butanediol by 2-fold. Two-step aerobic–anaerobic fermentation further enhanced 2,3-butanediol production by 4-fold in comparison to the control parental strain. Thus, addition of acetic acid and low dissolved oxygen in the medium are involved in activation of bdhA gene expression from P _alsSD promoter in early log phase. Under the conditions tested in this work, the maximum production of 2,3-butanediol, 2.1 g/l from 10 g/l glucose, was obtained at 24 h. Furthermore, under the optimized microaerophilic condition, the production of 2,3-butanediol improved up to 6.1 g/l and overall productivity increased by 6.7-fold to 0.4 g/l h in the engineered strain compared to that in the parental control.     

Low salinity causes oxidative stress and modulates specific antioxidant gene expression in the toxic dinoflagellate Alexandrium pacificum

Salinity is an important factor in the physiological regulation of algae; however, its influence on the genomic responses in toxic dinoflagellates is insufficiently understood. In the present study, we evaluated the effect of salinity stress on the physiology, photosynthesis, and molecular responses of the toxic dinoflagellate Alexandrium pacificum (group IV). When exposed cells to different salinities of 20–40 psu, we detected the lowest cell density (3.25?×?10³ cells mL^?1) and highest cell size (30.6 µm) at 20 psu. Photosynthesis efficiency considerably decreased at 20 and 40 psu compared to the control (33 psu). Quantitative real-time polymerase chain reaction revealed that psbA, psbD, and atpC expression levels were significantly downregulated under conditions of salinity stress for 72 h. In contrast, the expression levels of antioxidant genes MnSOD and GPx were greatly upregulated at 20 psu (13.2- and 15.2-fold changes at 6 h; 8.8- and 8.3-fold changes at 24 h, respectively). The expression levels of other antioxidant genes, CuZnSOD, GST, and APx, increased steadily over time under salinity stress. Such conditions increased the relative levels of reactive oxygen species by 2.2-fold in 6 h and 2.4-fold in 24 h at 20 psu. These results suggest that low salinity may cause cellular oxidative stress, leading to a decrease in photosynthesis and affecting specific antioxidant systems in toxic dinoflagellates.

     

Identification and expression analysis of <Emphasis Type="Italic">CjLTI</Emphasis>, a novel low temperature responsive gene from <Emphasis Type="Italic">Caragana jubata</Emphasis>

Using rapid amplification of cDNA ends, a full length cDNA (CjLTI) was cloned from apical buds of Caragana jubata, a plant species that grows under extreme cold. The cDNA obtained was 573 bp long consisting of an open reading frame of 351 bp encoding 116 amino acids. Homology analysis did not exhibit significant similarity with any sequence at NCBI database, therefore it was deduced as a novel gene. Secondary structure analysis suggested that the deduced CjLTI contained 25.86% α-helices, 4.31% β-turns, 6.90% extended strands, and 62.93% random coils. The hydropathy profile suggested CjLTI to be a hydrophobic protein having characteristic features of signal peptides at N-terminus. The gene exhibited down-regulation at 5 min of exposure to low temperature (LT, 4 ± 3°C) followed by a strong up-regulation after 15 min and onwards. Methyl jasmonate (MJ) lead to up-regulation of CjLTI starting at 5 min onwards. The gene exhibited up- and down-regulation of expression pattern in response to abscisic acid (ABA) and salicylic acid (SA). Mild drought stress slightly up-regulated gene expression and at severe drought (up to 115% reduction in leaf water potential) slight down-regulation of gene expression was observed. These results suggested CjLTI to be a LT responsive gene wherein MJ, ABA and SA pathways might be involved in regulating the gene expression.     

Effect of nitrogen limitation on oleic acid biosynthesis in <Emphasis Type="Italic">Botryococcus braunii</Emphasis>

The influence of nitrogen (N) deficiency on the cell growth and intracellular lipid production of the alga Botryococcus braunii UTEX 572 was investigated. Biomass concentration and lipid content of B. braunii cultivated in modified Chu-13 medium containing 0.04, 0.37, and 3.66 mM nitrate were 0.23–0.38 g L⁻¹ and 36–63% of dry cell weight, respectively. The specific growth rate of B. braunii reached a constant of 0.185 day⁻¹ during cultivation with an initial nitrate feed of 3.66 mM. The maximum lipid content of B. braunii was 63% with 0.04 mM nitrate. However, the maximum lipid productivity of 0.019 g L⁻¹ day⁻¹ was achieved with 0.37 mM nitrate. The level of oleic acid, an important component of biodiesel, was higher at 86% of the total fatty acids under N-limited conditions (0.04 mM nitrate) compared to 69% under N-sufficient conditions (3.66 mM nitrate). Furthermore, expression of the stearoyl-ACP desaturase gene (sad) encoding a stearoyl-ACP desaturase involved in the synthesis of oleic acid was 2.6-fold higher under N-limited conditions than under N-sufficient conditions.     

Phylogeography of the genus <Emphasis Type="Italic">Ulva</Emphasis> (Ulvophyceae,Chlorophyta), with special reference to the Japanese freshwater and brackish taxa

The nuclear-encoded ITS and associated 5.8S rDNA regions were sequenced for 72 specimens of Ulva collected from 44 rivers across Japan, including U. prolifera Müller from the Shimanto River, Kochi Prefecture, as well as 26 samples originally identified as U. linza L. from 20 coastal marine areas. Sequence data revealed that the samples fall into six distinct clades: the U. flexuosa Wulfen clade (2 samples), the Ulva linza-procera-prolifera (LPP) complex clade (75 samples), Ulva sp. 1 clade (3 samples), Ulva sp. 2 clade (7 samples), Ulva sp. 3 clade (4 samples) and Ulva sp. 4 clade (7 samples). The LPP complex contained a mixture of 26 samples collected from seashores and 49 samples obtained from rivers, including U. prolifera from the Shimanto River, and GenBank data for U. linza and U. procera Ahlner. The samples of the LPP complex differed by only 0–7 substitutions (0–1.149%). Subsequent phylogeographic analyses of the LPP complex based on the 5S rDNA spacer region revealed the presence of two further groupings: a group including 22 strictly marine littoral U. linza samples and a U. prolifera group composed of a mixture of 4 marine samples and all 49 river samples. The monophyly of all river samples indicates that adaptation to low salinity might have occurred only once in the evolutionary history of the LPP complex.     

Enhanced expression of heterologous inulinase in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> by disruption of <Emphasis Type="Italic">hap1</Emphasis> gene

Inulinase gene (Kcinu) derived from Kluyveromyces cicerisporus was expressed extracellularly in Kluyveromyces lactis using an episomal vector directed by Kcinu promoter. The influence of hap1 gene disruption on the expression of inulinase was studied. Inulinase activity in the supernatant of the recombinant Klhap1Δ strain was 391 U ml⁻¹ after cultured 120 h, which was 2.2-fold that of the wild type host. The relative inulinase mRNA level of the Klhap1Δ strain was 11.3-fold that of the wild type strain, and the expression plasmid was more stable in the mutant host. Based on these results, the disruption of hap1 facilitated the high and stable expression of inulinase controlled by Kcinu promoter in K. lactis.     

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.