Profiles of glycerolipids in Pyropia haitanensis and their changes responding to agaro-oligosaccharides

A sensitive method based on electrospray ionization tandem mass spectrometry was used to profile glycerolipids in Pyropia haitanensis and their changes responding to agaro-oligosaccharides. Ten monogalactosyldiacylglycerols (MGDGs), twelve digalactosyldiacylglycerols (DGDGs), five sulfoquinovosyldiacylglycerols (SQDGs), five phosphatidylglycerols (PGs), fifteen phosphatidylcholins (PCs), three phosphatidic acids (PAs), and three phosphatidylethanolamines (PEs) were identified in P. haitanensis. We found the SQDG was the most abundant species, followed by MGDG, DGDG, PG, PC, PE, and PA of the total glycerolipids. The predominant lipid species contained C20 fatty acids at sn-1/sn-2 positions, which was different from higher plants. Changes in membrane lipid species occurred when P. haitanensis were treated with agaro-oligosaccharides. At first, agaro-oligosaccharides induced an increase in total glycerolipids including the galactolipids such as MGDG (20:5/20:5) and phospholipids such as PC (18:3/20:5), suggesting that agaro-oligosaccharides caused changes of lipids in chloroplasts and plasma membrane. With increased treatment time, a large decline in major plasma membrane lipids (PCs and PEs) was observed, but not galactolipids (MGDGs and DGDGs), suggesting that the lipid changes occurred mainly at the plasma membrane after prolonged treatment.     

The lipidome of the photosynthetic bacterium Rhodobacter sphaeroides R26 is affected by cobalt and chromate ions stress

A detailed characterization of membrane lipids of the photosynthetic bacterium Rhodobacter (R.) sphaeroides was accomplished by thin-layer chromatography coupled with matrix-assisted laser desorption ionization mass spectrometry. Such an approach allowed the identification of the main membrane lipids belonging to different classes, namely cardiolipins (CLs), phosphatidylethanolamines, phosphatidylglycerols (PGs), phosphatidylcholines, and sulfoquinovosyldiacylglycerols (SQDGs). Thus, the lipidomic profile of R. sphaeroides R26 grown in abiotic stressed conditions by exposure to bivalent cobalt cation and chromate oxyanion, was investigated. Compared to bacteria grown under control conditions, significant lipid alterations take place under both stress conditions; cobalt exposure stress results in the relative content increase of CLs and SQDGs, most likely compensating the decrease in PGs content, whereas chromate stress conditions result in the relative content decrease of both PGs and SQDGs, leaving CLs unaltered. For the first time, the response of R. sphaeroides to heavy metals as Co²⁺ and CrO₄ ^2? is reported and changes in membrane lipid profiles were rationalised.     

Regulation of lipid metabolism in the snow alga Chlamydomonas nivalis in response to NaCl stress: An integrated analysis by cytomic and lipidomic approaches

The cultures of the snow alga Chlamydomonas nivalis in the exponential phage were stressed by NaCl (up to 1.5%) for 0～48 h, followed by Nile Red staining-based cytomic analysis (flow cytometry and confocal laser scanning microscopy). The fluorescent intensities of total lipids, and neutral and polar lipids increased to the maximum within 7 h in the NaCl stressed cells with the highest increase in total lipids by 2-fold (0.75%-NaCl for 7 h), the highest increase in neutral lipids by 68-fold (1%-NaCl for 7 h) and the highest increase in polar lipids by 10-fold (1.25%-NaCl for 5 h), respectively. Seven types and 22 kinds of polar lipid molecules were selected and identified as biomarkers by UPLC/Q-TOF-MS-based lipidomic analysis, which demonstrated differences in total lipids between the stress group (0.75%-NaCl for 7 h) and the control. The biological roles of the biomarkers in the alga under NaCl stress were discussed. The integrated approach based on “omics” technologies developed in the present work is validated as a powerful tool to successfully reveal the regulation of lipid metabolism in microalgae in response to stress stimulation.     

Polyunsaturated molecular species of galactolipids: Markers of zooxanthellae in a symbiotic association of the soft coral Capnella sp. (Anthozoa: Alcyonacea)

A method that uses marker fatty acids (FAs) is widely applied in investigations of trophic and symbiotic relationships. In a search for new lipid markers, we determined the total lipid FA composition, as well as the composition of molecular species of mono- and digalactosyl diacylglycerols (MGDGs and DGDGs), which are specific galactolipids of thylakoid membranes, in zooxanthellae (endosymbiotic dinoflagellates) of the tropical soft coral Capnella sp. Some FAs of zooxanthellae were suggested for use as marker polyunsaturated FAs (PUFAs). Thirteen molecular species of MGDGs and ten molecular species of DGDGs were detected using the method of high-resolution tandem mass spectrometry. All marker PUFAs of zooxanthellae were found in acyl groups of galactolipids. The major molecular species of DGDGs (18:4/18:4, 18:4/20:5, and 16:2/22:6) and the unique molecular species of MGDGs (16:4/18:5) were described. The identification of several polyunsaturated molecular species of galactolipids that contain marker FAs allowed us to propose that this lipid group be used as molecular lipid markers of zooxanthellae for the study of symbiont–host interactions in soft corals.     

Nutrient resupplementation arrests bio-oil accumulation in Phaeodactylum tricornutum

Phaeodactylum tricornutum is a marine diatom in the class Bacillariophyceae and is important ecologically and industrially with regards to ocean primary production and lipid accumulation for biofuel production, respectively. Triacylglyceride (TAG) accumulation has been reported in P. tricornutum under different nutrient stresses, and our results show that lipid accumulation can occur with nitrate or phosphate depletion. However, greater lipid accumulation was observed when both nutrients were depleted as observed using a Nile Red assay and fatty acid methyl ester (FAME) profiles. Nitrate depletion had a greater effect on lipid accumulation than phosphate depletion. Lipid accumulation in P. tricornutum was arrested upon resupplementation with the depleted nutrient. Cells depleted of nitrogen showed a distinct shift from a lipid accumulation mode to cellular growth post-resupplementation with nitrate, as observed through increased cell numbers and consumption of accumulated lipid. Phosphate depletion caused lipid accumulation that was arrested upon phosphate resupplementation. The cessation of lipid accumulation was followed by lipid consumption without an increase in cell numbers. Cells depleted in both nitrate and phosphate displayed cell growth upon the addition of both nitrate and phosphate and had the largest observed lipid consumption upon resupplementation. These results indicate that phosphate resupplementation can shut down lipid accumulation but does not cause cells to shift into cellular growth, unlike nitrate resupplementation. These data suggest that nutrient resupplementation will arrest lipid accumulation and that switching between cellular growth and lipid accumulation can be regulated upon the availability of nitrogen and phosphorus.     

Culture media optimization for growth and phycoerythrin production from Porphyridium purpureum

Porphyridium spp. is a red micro alga and is gaining importance as a source of valuable products viz., phycobiliproteins (PB), sulfated exopolysaccharides, and polyunsaturated fatty acids with potential applications in the food and pharmaceutical industries. In the present study, the effects of the major media constituents of Porphyridium species were studied using response surface methodology (RSM) on biomass yield, total PB and the production of phycoerythrin (PE). A second order polynomial can be used to predict the PB and PE production in terms of the independent variables. The independent variables such as the concentrations of sodium chloride, magnesium sulfate, sodium nitrate, and dipotassium hydrogen phosphate influenced the total PB and PE production. The optimum conditions showed that total PB was 4.8% at the concentration of sodium chloride 26.1 g/L, magnesium sulfate 5.23 g/L, sodium nitrate 1.56 g/L, and dipotassium hydrogen phosphate 0.034 g/L. In case of optimum PE production (3.3%), the corresponding values are 29.62, 6.11, 1.59, and 0.076 g/L, respectively. PE production depends greatly on the concentrations of chloride, nitrate, and sulfate as well as phosphate of which the former possess the maximum effect.     

The polar lipid composition of Mesorhizobium ciceri

The extractable lipid composition of Mesorhizobium ciceri strain HAMBI 1750 grown in a phosphate sufficient medium (79CA) is reported. Cardiolipin (CL—27% of total lipids), phosphatidylglycerol (PG—18%), phosphatidylethanolamine (PE—1%), phosphatidylcholine (PC—30%) and two methylated derivatives of PE, i.e. phosphatidyl-N, N-dimethylethanolamine (DMPE—1%) and phosphatidyl-N-monomethylethanolamine (MMPE—1%), were found to make up the phospholipids of the analysed bacteria. Nonphosphorus, ornithine-containing lipid (OL—10%) was also detected. Polar groups of phospholipids were predominantly acylated with cis-11,12-methyleneoctadecanoyl (lactobacillic) residues, whereas the ornithine lipid contained mainly 3-hexadecanoyloxy-11,12-methyleneoctadecanoic acid bound to the α-amino group.     

Effect of phosphate deprivation on enzymes of the cyclic adenosine monophosphate metabolism in rat proximal convoluted and proximal straight tubules.

Phosphate deprivation causes a resistance to the phosphaturic effect of parathyroid hormone (PTH). The present study determined whether acute phosphate deprivation alters basal or stimulated activities of key enzymes of the cyclic adenosine monophosphate (cAMP) metabolism in microdissected proximal convoluted and proximal straight tubules, since blunted cAMP levels in these proximal subsegments might account for refractoriness to the effect of PTH on phosphate reabsorption in the proximal convoluted and proximal straight tubule segments. In the proximal convoluted tubules of rats fed a normal-phosphate diet (NPD), PTH increased the adenylate cyclase activity by tenfold. In the proximal convoluted tubule of rats fed a low-phosphate diet (LPD), PTH also increased the adenylate cyclase activity by tenfold. In addition, forskolin increased the adenylate cyclase activity to levels similar to PTH in the proximal convoluted tubule of rats fed NPD or LPD. In the proximal straight tubule of rats fed NPD, PTH resulted in an approximately fivefold increase in adenylate cyclase activity. In the proximal straight tubule of rats fed LPD, PTH resulted in a fourfold increase in adenylate cyclase activity. The forskolin-stimulated adenylate cyclase activity was markedly decreased (46%) in the proximal straight tubule of phosphate-deprived rats. The cAMP-phosphodiesterase activity in the proximal convoluted tubule was significantly increased by 26% in phosphate-deprived rats. The cAMP-phosphodiesterase activities in the proximal straight tubules from rats fed NPD or LPD were similar. We conclude that distinct differences in key enzymes of cAMP metabolism exist in the proximal convoluted and proximal straight tubule subsegments. Further, phosphate deprivation affects the cAMP-phosphodiesterase and adenylate cyclase activities differently in these nephron subsegments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diversity and distribution of a key sulpholipid biosynthetic gene in marine microbial assemblages

Sulphoquinovosyldiacylglycerols (SQDG) are polar sulphur‐containing membrane lipids, whose presence has been related to a microbial strategy to adapt to phosphate deprivation. In this study, we have targeted the sqdB gene coding the uridine 5′‐diphosphate‐sulphoquinovose (UDP‐SQ) synthase involved in the SQDG biosynthetic pathway to assess potential microbial sources of SQDGs in the marine environment. The phylogeny of the sqdB‐coding protein reveals two distinct clusters: one including green algae, higher plants and cyanobacteria, and another one comprising mainly non‐photosynthetic bacteria, as well as other cyanobacteria and algal groups. Evolutionary analysis suggests that the appearance of UDP‐SQ synthase occurred twice in cyanobacterial evolution, and one of those branches led to the diversification of the protein in members of the phylum Proteobacteria. A search of homologues of sqdB‐proteins in marine metagenomes strongly suggested the presence of heterotrophic bacteria potential SQDG producers. Application of newly developed sqdB gene primers in the marine environment revealed a high diversity of sequences affiliated to cyanobacteria and Proteobacteria in microbial mats, while in North Sea surface water, most of the detected sqdB genes were attributed to the cyanobacterium Synechococcus sp. Lipid analysis revealed that specific SQDGs were characteristic of microbial mat depth, suggesting that SQDG lipids are associated with specific producers.     

Systematic investigation of biomass and lipid productivity by microalgae in photobioreactors for biodiesel application

We describe a methodology to investigate the potential of given microalgae species for biodiesel production by characterizing their productivity in terms of both biomass and lipids. A multi-step approach was used: determination of biological needs for macronutrients (nitrate, phosphate and sulphate), determination of maximum biomass productivity (the “light-limited” regime), scaling-up of biomass production in photobioreactors, including a theoretical framework to predict corresponding productivities, and investigation of how nitrate starvation protocol affects cell biochemical composition and triggers triacylglycerol (TAG) accumulation. The methodology was applied to two freshwater strains, Chlorella vulgaris and Neochloris oleoabundans, and one seawater diatom strain, Cylindrotheca closterium. The highest total lipid content was achieved with N. oleoabundans (25-37% of DW), while the highest TAG content was found in C. vulgaris (11-14% of DW). These two species showed similar TAG productivities.     

Macronutrients requirements of the dinoflagellate Protoceratium reticulatum

The basal L1 medium was found to be unsatisfactory for culturing the red tide dinoflagellate Protoceratium reticulatum at a high growth rate and biomass yield. The L1 medium enhanced with phosphate to a total concentration of 217 μM supported the highest attainable growth rate and biomass yield. Once the phosphate concentration exceeded 6× L1, phosphate inhibited the dinoflagellate growth and negatively affected cell viability. At the optimal phosphate concentration of 217 μM, an increase in nitrate concentration over the range of 882–8824 μM, did not affect cell growth and yield. Nitrate did not inhibit growth at any of the concentrations used. Clearly, the basal nitrate level in L1 is sufficient for effectively culturing P. reticulatum. At the ranges of phosphate and nitrate concentrations tested, cell volume was not sensitive to the concentration of nutrients but the concentration of phosphate affected both the specific cell number and cell volume growth rates. Elevated levels of nutrients supported their intracellular accumulation. Cell-specific production of yessotoxin was not influenced by concentration of phosphate in the culture medium, but elevated (>1764 μM) nitrate concentration did enhance the yessotoxin level. Phosphate concentration that maximized biomass yield also maximized volumetric production of yessotoxin in the culture broth.     

Lipid and fatty acids composition of photoautotrophically and heterotrophically grownChlamydomonas reinhardtii

Monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and sulfoquinovosyl diacylglycerol (SQDG) are the most abundant lipid classes present in both the autotrophically and heterotrophically grownChlamydomonas reinhardtii. However, phosphatidylcholine (PC) and diacylglycerol (N,N,N-trimethyl)-homoserine (DGTS) were absent in both alga types. The polyne index B was higher in heterotrophic than photoautotrophic algae, but the unsaturation index was higher in photoautotrophic algae PI, PE and DGDG. The proportion of linolenic acid decreased under heterotrophy with compensatory increases in hexadecadienoic (16 : 3), oleic (18 : 1) and linoleic (18 : 3) acids.     

Adsorption of nitrogen and phosphorus by intact cells and cell wall polysaccharides of Microcystis

Microcystis blooms can move vertically and horizontally in natural water bodies, which often causes a rapid change of nutritional environment around Microcystis cells. To evaluate the capability of Microcystis capturing nitrogen (N) and phosphorus (P) when environmental nutrient levels change, we studied N and P adsorption of two different forms of Microcystis aeruginosa strains, a colonial strain XW01 and a unicellular strain PCC 7806, and a green alga Chlorella pyrenoidosa to different concentrations of nitrate, ammonium and phosphate in 30 min. The results showed that XW01 had much stronger adsorption capacity than PCC7806 and Chlorella. As main components of the cell wall, the polysaccharides of XW01 displayed different adsorption capacities in different N and P concentrations, their adsorption capabilities rose higher with the N or P concentration increase. Comparing with pH 7.0, XW01 could adsorb much more ammonia and phosphate in alkaline condition (pH 9.0), although the nitrate adsorption decreased a little.     

Mechanism of proton-linked nitrate uptake in Cyanidium caldarium,an acidophilic non-vacuolated alga

The unicellular non-vacuolated alga Cyanidium caldarium, grown under conditions of nitrogen limitation, possesses two permease systems for nitrate uptake, one of which, the so-called ‘high-affinity nitrate uptake system’, enables the alga to take up nitrate through a mechanism involving cotransport of protons. Measurements of nitrate and proton stoichiometry, and determination of the kinetic parameters of uptake in cells resuspended in medium adjusted at different pH values, are consistent with a mechanism of uptake in which two protons for each nitrate ion are transported across the plasmalemma. Furthermore, kinetic data suggest that the carrier first binds nitrate and, subsequently, protons. Permutations of this binding sequence do not agree with the experimental results.     

Spatial and seasonal variations in the biofunctional lipid substances (fucoxanthin and fucosterol) of the laboratory-grown edible Japanese seaweed (Sargassum horneri Turner) cultured in the open sea

This work studied the effect of spatial and seasonal differences on the accumulation of functional lipid components in Sargassum horneri (Turner), an edible Japanese seaweed popularly called Akamoku. S. horneri obtained from Samenoura bay area of Japan was laboratory cultured to evaluate the effect of temperature on the accumulation of total lipids (TL), fucoxanthin (Fx) and fucosterol (Fs) by the alga. The laboratory cultured 3 month old S. horneri were cultured in the open sea in two different geographical locations off Usujiri and Matsushima to evaluate the monthly variations, over a year, in their TL, Fx and Fs contents. S. horneri grown off the Usujiri area accumulated the maximum TL close to 193 mg g⁻¹ dry weight during the coldest part of the year. Fx and Fs contributed 5.6% and 16.2% of the TL in S. horneri harvested off Usujiri in February. Further, in spite of being the same species and parent stock, S. horneri grown off the Matsushima area accumulated less TL, Fx and Fs as compared to their Usujiri counterparts. Our study clearly indicates the role of temperature and light apart from nutritional profile and depth of waters where the seaweed was grown on the accumulation of functional lipid components in S. horneri.     

Thermal acclimation in clownfish: An integrated biomarker response and multi-tissue experimental approach

The effects of increased temperature were tested in Amphiprion ocellaris, using a cellular diagnostics approach (in several tissues) combined with an organismal approach (body condition). Clownfish were exposed to a one month experiment following two temperature treatments: control (26 °C) and elevated temperature (30 °C). Fish were sampled at 0, 7, 14, 21 and 28 days for (1) assessment of stress biomarkers (catalase, lipid peroxidation, glutathione-S-transferase, superoxide dismutase, acetylcholinesterase, heat shock protein 70 kDa and ubiquitin – in brain, gills, liver, intestine and muscle), (2) estimation of integrated biomarker response index based on the biomarkers tested and (3) assessment of Fulton’s K index. Results show all biomarkers except acetylcholinesterase responded consistently and significantly to elevated temperature across tissue types suggesting they are suitable indicators of thermal stress in A. ocellaris. Biomarker levels were tissue-specific, and in addition, the most reactive tissues to temperature were muscle, gills and liver which suggest that highly oxygenated tissues seem to be the most responsive under thermal stress. The most responsive sampling times to increased temperature were T7 and T28: thermal stress was observed after 7 days of exposure (biomarker levels increased), then a pattern of decrease in biomarker levels towards the end of the experiment was observed, which may suggest fish were able to acclimate to exposure conditions. This indicates that A. ocellaris probably lives far from its upper thermal limit and is capable of adjusting the protein quality control system and enzymes’ activities to protect cell functions under elevated temperatures. The temperature treatment did not significantly influence body condition of the animals but biomarkers were negatively correlated to wet body weight. This suggests that thermal acclimation incurs at some energetic cost. In conclusion, these results suggest that this coral reef fish species presents a significant acclimation potential under ocean warming scenarios of +4 °C.     

Specificity for Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate of Nitrate Reductase from the Salt-tolerant Alga Dunaliella parva

Nitrate reductase of the salt-tolerant alga Dunaliella parva could utilize NADPH as well as NADH as an electron donor. The two pyridine nucleotide-dependent activities could not be separated by either ion exchange chromatography on DEAE-cellulose or gel filtration on Sepharose 4B. The NADPH-dependent activity was not inhibited by phosphatase inhibitors. NADPH was not hydrolyzed to NADH and inorganic phosphate in the course of nitrate reduction. Reduction of nitrate in vitro could be coupled to a NADPH-regenerating system of glycerol and NADP-dependent glycerol dehydrogenase. It is concluded that the nitrate reductase of D. parva will function with NADPH as well as NADH. This is a unique characteristic not common to most algae.     

Nitrate and phosphate uptake kinetics of the harmful diatom Eucampia zodiacus Ehrenberg,a causative organism in the bleaching of aquacultured Porphyra thalli

The diatom Eucampia zodiacus Ehrenberg is a harmful diatom which indirectly causes bleaching of aquacultured Nori (Porphyra thalli) through competitive utilization of nutrients during bloom events. In the present study, we experimentally investigated the nitrate (N) and phosphate (P) uptake kinetics of E. zodiacus, Harima-Nada strain. Maximum uptake rates (ρ_max), which were obtained by short-term experiments, were 0.777 and 0.916 pmol cell^?1 h^?1 for nitrate and 0.244 and 0.550 pmol cell^?1 h^?1 for phosphate at 9 and 20 °C, respectively. The half-saturation constants for uptake (K_s) were 2.59 and 2.92 μM N and 1.83 and 4.85 μM P at 9 and 20 °C, respectively. Although the maximum specific uptake rate (V_max; V_max = ρ_max/Q₀, Q₀; minimum cell quota) and V_max/K_s for nitrate at 9 °C are about 1/2 of those obtained at the optimum temperature (20 °C), they are still higher than those obtained for many other phytoplankton at their optimum temperature conditions for uptake. These results suggest that E. zodiacus utilizes nitrogen efficiently at low water temperature, and it is one of the important factors causing the serious damage to Porphyra thalli by bleaching due of this species. For phosphate, the K_s values of E. zodiacus were higher than those reported for other species; the V_max and V_max/K_s values were much lower than those of other diatoms such as Skeletonema costatum (Greville) Cleve. These results suggest that E. zodiacus is disadvantaged compared to other diatom species during competitive utilization of phosphate.     

Adaptation to phosphate deprivation in osteoblast-like cells

The rat osteosarcoma cell line UMR-106–01 has an osteoblast-like phenotype. When grown in monolyer culture these cells transport inroganic phosphate and L-alanine via Na⁺-dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60–70 per cent increase in Na⁺-dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na⁺-dependent alanine uptake and Na⁺-independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased V_max and was blocked completely by pretreatment with cycloheximide (70 μM). In these cells recovery of intracellular pH after acidification with NH₄Cl is due primarily to the Na⁺/H⁺ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate-deprived cells compared to controls indicating that Na⁺/H⁺ exchange was inhibited during phosphate deprivation.