Enhancement of BODIPY505/515 lipid fluorescence method for applications in biofuel-directed microalgae production

This paper describes a microalgal cell lipid fluorescence enhancement method using BODIPY(505/515), which can be used to screen for lipids in wild-type microalgae and to monitor lipid content within microalgae production processes to determine optimal harvesting time. The study was based on four microalgae species (Dunaliella teteriolecta, Tetraselmis suecica, Nannochloropsis oculata, and Nannochloris atomus) selected because of their inherent high lipid content. An extended analysis was carried out with N. oculata due to the depressed fluorescence observed when compared with the other experimental strains. BODIPY(505/515) lipid fluorescence was determined for two solvent pre-treatment methods (DMSO and glycerol) and four staining condition parameters (analysis time, staining temperature, dye concentration, and algal cell concentration). It was found that lipid fluorescence of thick cell-walled microalgae, such as N. oculata, is significantly enhanced by both the pre-treatment methods and staining condition parameters, thereby significantly enhancing lipid fluorescence by ca. 800 times the base autofluorescence. The lipid fluorescence enhancement method provides a quick and simple index for in vivo Flow Cytometry quantification of total lipid contents for purposes of species screening or whole culture monitoring in biofuel-directed microalgae production.     

Rapid estimation of lipid content in an Antarctic ice alga (Chlamydomonas sp.) using the lipophilic fluorescent dye BODIPY505/515

Chlamydomonas sp. ICE-L, isolated from Antarctic coastal marine environments, was selected as a high lipid producer, which may be useful for biodiesel production. The lipophilic fluorescent dye BODIPY505/515 was used to determine the algal lipid content. Lipid bodies stained with BODIPY505/515 have a characteristic green fluorescence, and their volumes were determined using the sphere volume formula. In this study, lipid accumulation by Chlamydomonas ICE-L was analyzed under different cultivation conditions (nitrogen deficiency and UV-B radiation). The results demonstrated that nitrogen deficiency and UV-B radiation could significantly promote the accumulation of lipid content per cell. The highest yields of total lipid content (reaching 84?μL?L^?1) were obtained in full Provasoli medium after 12?days of cultivation, but not in the nitrogen-deficient medium. The inoculum used in this experiment was obtained from the late-exponential growth phase. The main reason was that the cell numbers in nitrogen-deficient medium had not increased and total lipid contents were offset by the lower growth rate. Considering the high lipid content in Chlamydomonas sp. ICE-L, this alga might be a promising alternative species for production of microalgal oil for the production of renewable biodiesel in the future.     

Effects of agitation on the microalgae Phaeodactylum tricornutum and Porphyridium cruentum

The effect of mechanical agitation on the microalgae Phaeodactylum tricornutum and Porphyridium cruentum was investigated in aerated continuous cultures with and without the added shear protectant Pluronic F68. Damage to cells was quantified through a decrease in the steady state concentration of the biomass in the photobioreactor. For a given aeration rate, the steady state biomass concentration rose with increasing rate of mechanical agitation until an upper limit on agitation speed was reached. This maximum tolerable agitation speed depended on the microalgal species. Further increase in agitation speed caused a decline in the steady state concentration of the biomass. An impeller tip speed of >1.56 m s^–1 damaged P. tricornutum in aerated culture. In contrast, the damage threshold tip speed for P. cruentum was between 2.45 and 2.89 m s^–1. Mechanical agitation was not the direct cause of cell damage. Damage occurred because of the rupture of small gas bubbles at the surface of the culture, but mechanical agitation was instrumental in generating the bubbles that ultimately damaged the cells. Pluronic F68 protected the cells against damage and increased the steady state concentration of the biomass relative to operation without the additive. The protective effect of Pluronic was concentration-dependent over the concentration range of 0.01–0.10% w/v.     

Physiological and molecular biological characterization of intracellular carbonic anhydrase from the marine diatom Phaeodactylum tricornutum

A single intracellular carbonic anhydrase (CA) was detected in air-grown and, at reduced levels, in high CO(2)-grown cells of the marine diatom Phaeodactylum tricornutum (UTEX 642). No external CA activity was detected irrespective of growth CO(2) conditions. Ethoxyzolamide (0.4 mM), a CA-specific inhibitor, severely inhibited high-affinity photosynthesis at low concentrations of dissolved inorganic carbon, whereas 2 mM acetazolamide had little effect on the affinity for dissolved inorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism in P. tricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, and two-step column chromatography on diethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. The purified CA was shown, by SDS-PAGE, to comprise an electrophoretically single polypeptide of 28 kD under both reduced and nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplification of cDNA ends method and indicated that the cDNA encodes 282 amino acids. Comparison of this putative precursor sequence with the N-terminal amino acid sequence of the purified CA indicated that it included a possible signal sequence of up to 46 amino acids at the N terminus. The mature CA was found to consist of 236 amino acids and the sequence was homologous to beta-type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acid residues that may constitute a CO(2)-binding site appeared to be unique among the beta-CAs so far reported.     

Effects of Mn2+ on neutral lipid content,C4 pathway,and related gene expression in Phaeodactylum tricornutum

Journal of Applied Phycology - The neutral lipids from the diatom Phaeodactylum tricornutum are a valuable substance with a vital role in biofuel. Increasing the neutral lipid content of P....     

Phototaxis, gravitaxis and vertical migrations in the marine dinoflagellate Prorocentrum micans

Abstract The phototactic orientation of the marine dinoflagellate Prorocentrum micans was studied at three different ages and at several light intensities. High irradiances caused the cells to show negative phototaxis and low irradiances caused positive phototaxis. The precision of negative phototaxis reached a maximum in the early afternoon, while the precision of positive phototaxis was found to peak in the morning and at night. The cells also showed a pronounced negative gravitactic orientation, which had a maximum in precision in the early afternoon. The degree of gravitaxis was found to be constant over time when the cells were confined to a closed cuvette for up to 9 h. As a consequence of the orientation strategies, populations of Prorocentrum micans showed daily vertical migrations in a 3-m Plexiglas column. They accumulated in the top layers in the afternoon and were almost randomly distributed during the rest of the day.     

尼罗红荧光法快速检测培养过程中湛江等鞭金藻的中性脂

研究一种快速准确测定微藻中中性脂的方法。湛江等鞭金藻是一种中性脂含量高且具有开发潜力的能源微藻。以湛江等鞭金藻为实验对象,首先优化尼罗红染色的条件。当二甲基亚砜体积分数为2.0%、尼罗红质量浓度为1.00μg/m L、细胞密度为1.0×106个/m L、激发波长为480 nm、检测波长为580 nm时,优化的染色时间为10min。其次测定了背景荧光对检测的影响。结果表明,在不同细胞状态下,背景荧光强度大约是微藻内荧光强度的20%左右,可以忽略。最后比较了尼罗红荧光法和重量法。结果表明,荧光强度与中性脂含量的相关系数R2=0.946 8,虽然两者相关性并不十分高,但作为一种快速测定微藻中中性脂的方法,尼罗红荧光法依然是研究微藻培养过程中中性脂含量变化的有效方法。     

Fluorometric determination of the neutral lipid content of microalgal cells using Nile Red

The fluorophore Nile Red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) has been used to determine neutral lipid in microalgal cells. Cellular fluorescence of stained cells and gravimetrically or chromatographically determined lipid were linearly correlated when Nile Red was excited at 488 – 525 nm and the fluorescent emision measured at 570 – 600 nm. Nile Red is a vital stain which allowed flow cytometric sorting of live microalgal populations based on their lipid content.     

Microwave-assisted Nile red method for in vivo quantification of neutral lipids in microalgae

In vivo determination of neutral lipids with Nile red fluorescence has been used as a rapid screening method for certain types of microalgae, but has been unsuccessful in others, particularly those with thick, rigid cell walls that prevent penetration of the fluorescence dye into the cell. To solve the problem, a microwave-assisted Nile red staining method for microalgal lipid determination was developed. In a two-step staining protocol, 50 and 60 s were selected as the optimal microwave times for the pretreatment and staining process, respectively. Moreover, several calibration methods for quantitative analysis of neutral lipids in microalgae were investigated and compared with conventional gravimetric methods. Factors that affected the in vivo quantification of cellular neutral lipids were also investigated. Application of the new method for detection and quantification of neutral lipids in a number of green microalgae was demonstrated.     

Application of the standard addition method for the absolute quantification of neutral lipids in microalgae using Nile red

Microalgae are considered one of the best candidates for biofuel production due to their high content in neutral lipids, therefore, an accurate quantification of these lipids in microalgae is fundamental for the identification of the better candidates as biodiesel source.Nile red is a fluorescent dye widely employed for the quantification of neutral lipids in microalgae. Usually, the fluorescence intensity of the stained samples is correlated to the neutral lipid content determined with standard methods, in order to draw a standard curve and deduce the neutral lipids concentration of the unknown samples positioning their fluorescence intensity values on the curve.Standard methods used for the neutral lipids determination are laborious and often implying solvent extraction and/or other transformation (i.e. saponification or transesterification) of the sample. These methods are also time consuming and may give rise to an underestimation of the lipid content due to variable extraction yields.The approach described in this paper combines the standard addition method and the fluorometric staining using Nile red, avoiding the association of traditional neutral lipids quantification methods to the fluorometric determination. After optimization of instrument parameters and staining conditions, a linear correlation between the fluorescence intensity of each sample stained with the Nile red and its neutral lipids content deduced with the standard addition method was identified. The obtained curve allowed the direct determination of neutral lipids content maintaining a linearity range from 0.12 to 12 μg of neutral lipids per ml of sample, without need of pre-concentration. This curve was then used in the quantification of the neutral lipids content in culture of Skeletonema marinoi (Bacillariophyceae) at different days from the inoculum. This method was also successfully applied on Chaetoceros socialis (Bacillariophyceae) and Alexandrium minutum (Dinophyceae).     

Circadian variability in the photobiology of Phaeodactylum tricornutum: pigment content

Circadian variations of pigment content in the diatom Phaeodactylumtricornutum were analyzed in different light regimes. The studywas aimed at discerning the role of putative endogenous controlsfrom the constraint imposed by the alternation of light (L)and dark (D) periods. Our experiments showed that in a typicalLD cycle of illumination, pigment synthesis follows the somaticgrowth of the cell, both arresting during D periods. In particular,the diurnal increase of chlorophyll a content was proportionalto the increase in cell size and preceding cell division, occurringat night. By contrast, diadinoxanthin and ß–carotene displayed different phases, which is likely to be relatedto their involvement in photoprotection mechanisms. The experimentsalso showed that the synthesis of both photosynthetic and photoprotectivepigments was dependent not only on light availability and thephasing of somatic growth, but also responded to other internalregulation. Over the time scale of the experiments (hours todays), the removal of LD–DL triggers impaired cell physiology,whereas the circadian patterns in pigment synthesis persisted.Our results support the hypothesis that an internal regulationof cell biosynthetic machinery can improve phytoplankton fitness,even in high variable environments such as the oceanic mixedlayers. Therefore, we suggest that phytoplankton growth dependsnot only on the availability of external resources, but alsoon internal regulatory mechanisms whose unveiling would furtherour understanding of phytoplankton diversity and dynamics.     

Isolation and characterization of carbonic anhydrases from the marine diatom Phaeodactylum tricornutum

Carbonic anhydrase (CA) isozymes were identified and isolated from three strains of Phaeodactylum tricornutum [University of Texas Culture Collection (UTEX 640), North Eastern Pacific Culture Collection at the University of British Columbia B31 and Culture Collection of Algae and Protozoa 1052/1A]. External (CA_ext) and internal CA activity was detected by potentiometric assay of intact cells and cell homogenates of air and high CO₂-grown cells. CA_ext was detected only in UTEX 640 grown under CO₂-limited conditions and present in trace amounts in cells grown on high CO₂. CA isozymes in cells extracts were separated by cellulose acetate electrophoresis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All three strains had two CA bands in common, while UTEX 640 had a third, faster-running band which was absent from extracts of high CO₂-grown cells and thus was the external isozyme. The internal CA isoforms of the UTEX 640 strain were shown to have molecular masses of 28 and 25 kDa, and the external 24 kDa. A fourth CA_ext isozyme with a molecular weight of 23.5 kDa was later detected using a polyclonal CA antibody. The CA isozymes were low-CO₂-inducible proteins because Western blot analysis, using a polyclonal antibody, indicated that CA expression was repressed in high CO₂-grown cells. CA localization, using both immunofluorescence and immunogold techniques, with air-grown cells indicated that the CA_ext was located in the periplasmic space and on the cell membrane, whereas in high CO₂-grown cells only internal CA was detected.     

Monitoring cell‐specific neutral lipid accumulation in Phaeodactylum tricornutum (Bacillariophyceae) with Nile Red staining – a new method for FlowCAM

With the fluorescent stain Nile Red (NR), phytoplankton lipid accumulation can be monitored quickly and in situ. In the light of recent results in phytoplankton diversity research, there is also a need for cell‐ and species‐specific lipid measurement techniques. The objective of this work was to investigate whether cell‐specific phytoplankton lipid accumulation could be monitored with the image‐based particle analyzer FlowCAM? and NR staining. Applying Phaeodactylum tricornutum as a model species, we compared the FlowCAM method to two established lipid quantification methods: spectrofluorometric NR fluorescence measurement and total lipid analysis by gas chromatography. The experiment was carried out in batch cultures under nitrogen limitation to induce lipid accumulation. We showed significant correlation between the three different lipid quantification methods confirming the applicability of the novel FlowCAM method in cell‐specific and near real‐time lipid quantification. Furthermore, with the method described here, the lipid content of taxonomically distinguished cells can eventually be measured from multispecies cultures, opening several new possibilities to study species‐specific responses to stress conditions and the complementarity effect.     

Positional distribution of fatty acids in lipids of the marine diatom Phaeodactylum tricornutum

The composition of fatty acids and lipids in the marine diatom, Phaeodactylum tricornutum was determined. The Lipids consisted of monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulphoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphtidylinositol, triacylglycerol and minor unidentified ones. At the early stationary phase of growth, the total fatty acids were mainly 20:5, 16:1, 16:0 and 16:3. 20:5 was distributed in polar lipids, particularly in monogalactosyldiacylglycerol, phosphatidylcholine and phosphatidylglycerol. This fatty acid was exclusively located at the sn-1 position of the glycerol moiety in all polar lipids except for phosphatidylcholine. In phosphatidylcholine 20:5 was distributed at both the sn-1 and sn-2 positions. 16:3 was concentrated at the sn2 position of monogalactosyldiacylglycerol and trans-16:1 (n-13) was dominant at the sn-2 position of phosphatidylglycerol. C₁₈ fatty acids, the minor fatty acids in P. tricornutum, were confined to the sn-2 position of phosphatidylcholine.     

Proteomes reveal the lipid metabolic network in the complex plastid of Phaeodactylum tricornutum

Phaeodactylum tricornutum plastid is surrounded by four membranes, and its protein composition and function remain mysterious. In this study, the P. tricornutum plastid-enriched fraction was obtained and 2850 proteins were identified, including 92 plastid-encoded proteins, through label-free quantitative proteomic technology. Among them, 839 nuclear-encoded proteins were further determined to be plastidial proteins based on the BLAST alignments within Plant Proteome DataBase and subcellular localization prediction, in spite of the strong contamination by mitochondria-encoded proteins and putative plasma membrane proteins. According to our proteomic data, we reconstructed the metabolic pathways and highlighted the hybrid nature of this diatom plastid. Triacylglycerol (TAG) hydrolysis and glycolysis, as well as photosynthesis, glycan metabolism, and tocopherol and triterpene biosynthesis, occur in the plastid. In addition, the synthesis of long-chain acyl-CoAs, elongation, and desaturation of fatty acids (FAs), and synthesis of lipids including TAG are confined in the four-layered-membrane plastid based on the proteomic and GFP-fusion localization data. The whole process of generation of docosahexaenoic acid (22:6) from palmitic acid (16:0), via elongation and desaturation of FAs, occurs in the chloroplast endoplasmic reticulum membrane, the outermost membrane of the plastid. Desaturation that generates 16:4 from 16:0 occurs in the plastid stroma and outer envelope membrane. Quantitative analysis of glycerolipids between whole cells and isolated plastids shows similar composition, and the FA profile of TAG was not different. This study shows that the diatom plastid combines functions usually separated in photosynthetic eukaryotes, and differs from green alga and plant chloroplasts by undertaking the whole process of lipid biosynthesis.     

Growth kinetics and nitrogen-nutrition of the marine diatom Phaeodactylum tricornutum in continuous dialysis culture

The growth kinetics and nitrogen (N)-nutrition of the marine pennate diatom Phaeodactylum tricornutum Bohlin were determined in continuous dialysis culture at different cell densities. Inflow nutrient medium was supplied as natural unenriched estuarine seawater to a dialysis culture system with a high ratio of membrane surface area/culture volume (Am/Vc). Under the experimental conditions, the supply of inorganic macronutrients (NO ₃ ^? + NO ₄ ^? and PO ₄ ^?3 ) by diffusion (Nd) was markedly greater than that provided by the dilution (FfCN) of the culture (Nd ? FfCN), thereby establishing an inverse relationship between the cell density and the dilution rate (D). This continuous dialysis system allows for the maintenance of prolonged growth (> two weeks) at various cell densities (1.4 to 27.2 × 10⁹ cells 1^?1) within a range of dilution rates between 0.30 to 1.08 d^?1. In high cell density cultures, where the extracellular medium was characterized as nutrient deficient, a lower growth rate (μ_e) was exhibited than in cultures with lower cell densities. The growth rate (μ_e) remained equivalent to the dilution rate (D) throughout the culture cycle, indicating that equilibrated growth was achieved. High cell density cultures yielded higher productivity (P), relative to that of cultures grown at lower cell densities, in terms of cell-N and ?C produced per unit time. However, cell quotas of both N and C declined with increasing cell concentrations. Denser cultures were characterized by an enhanced N-conversion efficiency (Y_N) and a higher cellular N/C atomic ratio. The nutritional response of this diatom in dense cultures reveals an efficient use of N-nutrients, presumably as a result of cellular nutrient adaptation to oligotrophic conditions.