Growth, lipid production and metabolic adjustments in the euryhaline eustigmatophyte Nannochloropsis oceanica CCALA 804 in response to osmotic downshift

We investigated the effects of osmotic downshift induced by the transfer of Nannochloropsis oceanica CCALA 804 from artificial seawater medium (27 g L^?1 NaCl) to the same medium without NaCl or freshwater modified BG-11 medium (mBG-11) as a function of photosynthetically active radiation (170, 350, or 700 μmol photon m^–2 s^–1). Alterations in growth, total fatty acid (FA) content and FA composition of individual lipid classes, and in relative contents of metabolites relevant to osmotic adjustments were studied. Cells displayed remarkable tolerance to the osmotic downshift apart from some swelling, with no substantial lag or decline in cell division rate. Biomass accumulation and chlorophyll a content were enhanced upon downshifting, especially under the highest irradiance. The highest chlorophyll a and eicosapentaenoic acid (EPA) biomass and culture contents were determined in the cultures grown in mBG-11. Two days after transfer to 0 g L^-1 NaCl, the proportion in total acyl lipids of the major chloroplast galactolipid monogalactosyldiacylglycerol, a major depot of EPA, increased twofold, along with a modest change in the proportion of digalactosyldiacylglycerol (DGDG). EPA percentage decreased in DGDG and increased in the extraplastidial lipid phosphatidylethanolamine. Metabolite profiling by GC–MS analysis revealed a sharp decrease in metabolites potentially involved in osmoregulation, such as mannitol and proline, while proline-cycle intermediates and some free sugars increased. The stress-induced polyamine spermidine decreased ca. one order of magnitude, while its catabolic product—the non-protein amino acid γ-amino butyric acid—increased twofold, as did the stress-related sugars trehalose and talose. Biochemical mechanisms governing osmotic plasticity and implications for optimization of EPA production by N. oceanica CCALA 804 under variable cultivation conditions are discussed.     

Cell surface and cellular debris-associated heat-stable lipolytic enzyme activities of the marine alga Nannochloropsis oceanica

Microbial surface display of lipases can be effectively employed for the development of whole-cell biocatalysts for industrial bioconversions. In the present work, we report for the first time the presence of thermostable lipolytic enzyme activities against p-nitrophenyl laurate, both on the cell surface and the cellular debris fraction of the marine microalga Nannochloropsis oceanica (strain CCMP1779). Whole cell-associated lipolytic activity (WCLA) shows a 2.5-fold stimulation after heat treatment at 100?°C for 60?min, while the activity of the respective cell debris is retained for 15?min. In contrast, heat treatment renders the soluble fraction of the disrupted cells inactive. The progress curve of cellular debris-associated lipase activity is biphasic and levels off very fast. Treatment with the surfactants SDS, Triton X-100 and CHAPS, which are known to inhibit lipase activity in various degrees, results in a loss of both cell bound and cell debris lipolytic activities (CDLA). The highest whole cell lipase catalytic efficiency was observed against p-nitrophenyl butyrate and the optimum pH for hydrolysis was determined at pH 7.0. Both unheated and heated undisrupted whole cell biocatalysts are also catalytically active against olive oil. High-salt concentrations (1M NaCl) lead to about 50% whole cell enzyme inhibition whereas the activity of heated cells increases. These findings offer novel insight into the biocatalytic properties and the biotechnological applicability of microalgal lipases from N. oceanica.     

Zeocin treatment significantly elevated transformation efficiency of Nannochloropsis oceanica

Journal of Applied Phycology - Genetic transformation is critical to microalgal genetic engineering which generally prefers a high transformation efficiency. Here, we describe a simple and...     

Cryopreservation of the unicellular marine alga, Nannochloropsis oculata

In microalgal culture collections, as in many biological resource centres, cryoconservation is the most attractive method for the long-term, secure storage of living material. Nannochloropsis oculata, a marine unicellular alga, is of interest in the field of biotechnology due to its high lipid content. Of various cryoprotectants tested for their toxicity and for their ability to prevent cryoinjury, glycerol (final concentration 1.1 M) was the most efficient. When glycerol-treated cultures were submitted to a strictly regulated cooling rate (-3 degrees C min(-1)), they attained the control culture density within 13 d after thawing.     

Architecture of the Golgi apparatus of a scale-forming alga: biogenesis and transport of scales

Mechanisms of transport of secretory products across the Golgi apparatus (GA) as well as of scale formation in prymnesiophytes have remained controversial. We have used a quantitative morphological approach to study formation and transport of scales across the GA in haploid cells of Pleurochrysis sp. The GA of these cells differs from the GA of higher plants in at least six morphological characteristics. Our results show that scales form in the trans-Golgi network (TGN) and transit the TGN in heretofore unrecognized prosecretory vesicles. Prosecretory vesicles differentiate into secretory vesicles prior to exocytosis of scales to the cell surface. Because prosecretory vesicles are only fragments of TGN cisternae, the classical model of cisternal progression is not a valid mechanism of transport in this alga. TGN transport vesicles are also involved in scale formation; however, the role of tubular connections between cisternae of a single stack-TGN unit is not clear. The relationship of two morphological types of cisternal dilations to a membrane-associated, bottlebrush-shaped macromolecule of novel morphology suggests a new hypothesis for the biogenesis of scales.     

Lipidomic analysis can distinguish between two morphologically similar strains of Nannochloropsis oceanica

The two morphologically similar microalgae NMBluh014 and NMBluh‐X belong to two different strains of Nannochloropsis oceanica. They possess obviously different feeding effects on bivalves, but are indistinguishable by 18S rRNA and morphological features. In this work, lipidomic analysis followed by principal component analysis and orthogonal projections to latent structures discriminant analysis provided a clear distinction between these strains. Metabolites that definitively contribute to the classification were selected as potential biomarkers. The most important difference in polar lipids were sulfoquinovosyldiacylglycerol (containing 18:1/16:0 and 18:3/16:0) and monogalactosyldiacylglycerol (containing 18:3/16:3 and 20:5/14:0), which were detected only in NMBluh‐X. Additionally, an exhaustive qualitative and quantitative profiling of the neutral lipid triacylglycerol (TAG) in the two strains was carried out. The predominant species of TAG containing 16:1/16:1/16:1 acyl groups was detected only in NMBluh‐X with a content of ~93.67 ± 11.85 nmol · mg^?1 dry algae at the onset of stationary phase. Meanwhile, TAG containing 16:0/16:0/16:0 was the main TAG in NMBluh014 with a content of 40.25 ± 3.92 nmol · mg^?1. These results provided the most straightforward evidence for differentiating the two species. The metabolomic profiling indicated that NMBluh‐X underwent significant chemical and physiological changes during the growth process, whereas NMBluh014 did not show such noticeable time‐dependent metabolite change. This study is the first using Ultra Performance Liquid Chromatography coupled with Electrospray ionization‐Quadrupole‐Time of Flight Mass Spectrometry (UPLC‐Q‐TOF‐MS) for lipidomic profiling with multivariate statistical analysis to explore lipidomic differences of plesiomorphous microalgae. Our results demonstrate that lipidomic profiling is a valid chemotaxonomic tool in the study of microalgal systematics.     

Cadmium-induced changes in antioxidant enzymes from the marine alga Nannochloropsis oculata

Cadmium-induced oxidative stress symptoms such as lipid peroxidation and H₂O₂ production were examined in the marine alga Nannochloropsis oculata. Changes in antioxidant enzyme levels and isozyme patterns were also examined. Increasing concentrations of Cd produced growth inhibition. Among the responses to added Cd, the H₂O₂ content and malonyldialdehyde accumulation increased significantly, indicating a state of oxidative stress. In the case of ascorbate peroxidase activity the increase was about 2.5 times and a marked induction of the isozyme APX2 contributed to this increase. Guaiacol peroxidase activity increased about 4-fold, this being due mainly to the isozyme GPX3. Catalase activity increased slightly, whereas superoxide dismutase and glutathione reductase activity decreased markedly. Alterations of antioxidant enzyme levels and isozyme pattern changes in Cd-treated alga suggest that they might be involved in the heavy metal tolerance in this alga.     

Determination of intracellular lipid and main fatty acids of Nannochloropsis oceanica by ATR-FTIR spectroscopy

Journal of Applied Phycology - Fatty acid profile was one of the important characteristic parameters of edible oil. With the intensification of interest in microalgae as a potential source of...     

Adaptation to shade of the light-harvesting apparatus in Silene dioica

Abstract. The physiological characteristics and photo-system composition of the photosynthetic apparatus of Silene dioica , a woodland plant, grown in sun and natural shade are examined. As expected, shade leaves exhibited lower chlorophyll a/b ratios, light saturated rates of CO₂ assimilation (A_sat), dark respiration (R_d,) and light compensation points ( Г ), with both sun and shade leaves having similar absorptances and quantum yields of CO₂ assimilation (φ). Shade leaves were able to utilize far-red light for electron transport and carbon assimilation and reach the compensation point. Sun leaves in far-red light had a rate of carbon assimilation equivalent to their dark respiration rate. Chlorophyll fluorescence kinetics from leaves at 77 K together with analyses of thylakoid contents of photosystems (PS) I and II and the light-harvesting cholorphyll a/b protein complex associated with PSII (LHCII) demonstrated that the antenna size of PSII was similar in thylakoids of sun and shade leaves, but shade leaves contained ca. 20% more PSII and ca. 12% less PSI complexes. The increased PSII/PSI ratio in shade leaves accounted for their ability to achieve the compensation point in far-red light. An important feature of photosynethic shade adaptation in S. dioica is an increase in the PSII/PSI ratio and not an increase in the antenna size of PSII. The adaptive response of sun leaves when placed in a shade environment was rapid and had a half-time of ca. 18h.     

Cell wall and organelle modifications during nitrogen starvation in Nannochloropsis oceanica F&M-M24

Nannochloropsis oceanica F&M-M24 is able to increase its lipid content during nitrogen starvation to more than 50% of the total biomass. We investigated the ultrastructural changes and the variation in the content of main cell biomolecules that accompany the final phase of lipid accumulation. Nitrogen starvation induced a first phase of thylakoid disruption followed by chloroplast macroautophagy and formation of lipid droplets. During this phase, the total amount of proteins decreased by one-third, while carbohydrates decreased by 12–13%, suggesting that lipid droplets were formed by remodelling of chloroplast membranes and synthesis of fatty acids from carbohydrates and amino acids. The change in mitochondrial ultrastructure suggests also that these organelles were involved in the process. The cell wall increased its thickness and changed its structure during starvation, indicating that a disruption process could be partially affected by the increase in wall thickness for biomolecules recovery from starved cells. The wall thickness in strain F&M-M24 was much lower than that observed in other strains of N. oceanica, showing a possible advantage of this strain for the purpose of biomolecules extraction. The modifications following starvation were interpreted as a response to reduction of availability of a key nutrient (nitrogen). The result is a prolonged survival in quiescence until an improvement of the environmental conditions (nutrient availability) allows the rebuilding of the photosynthetic apparatus and the full recovery of cell functions.

     

Excitation energy transfer in the far-red absorbing violaxanthin/vaucheriaxanthin chlorophyll a complex from the eustigmatophyte alga FP5

Photosynthesis Research - This work highlights spectroscopic investigations on a new representative of photosynthetic antenna complexes in the LHC family, a putative violaxanthin/vaucheriaxanthin...     

Ionic liquid-based microwave-assisted extraction of lipid and eicosapentaenoic acid from Nannochloropsis oceanica biomass: experimental optimization approach

Journal of Applied Phycology - Lipids and eicosapentaenoic acid (EPA) compounds from Nannochloropsis oceanica biomass were extracted using a combination of ionic liquids (ILs) and...