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Microalgae often undergo different CO2 experiment in their habitat. To adapt to low CO2, carbon concentrating mechanism (CCM) could be launched in majority of microalgae and CCM are regulated at RNA level are well known. However, epigenetic modifications and their potential regulation of the transcription of masked genes at the genome level in response to CO2 fluctuation remain unclear. Here epigenetic regulation in response to CO2 fluctuation and epigenome-association with phenotypic plasticity of CCM are firstly uncovered in marine microalga Nannochloropsis oceanica IMET1. The result showed that lysine butyrylation (Kbu) and histone H3K9m2 modifications were present in N. oceanica IMET1. Moreover, Kbu modification positively regulated gene expression. In response to CO2 fluctuation, there were 5,438 and 1,106 genes regulated by Kbu and H3K9m2 in Nannochloropsis, respectively. Gained or lost histone methylations were closely associated with activating or repressing gene expressions. Differential modifications were mainly enriched in carbon fixation, photorespiration, photosynthesis, and lipid metabolism etc. Massive genome-wide epigenetic reprogramming was observed after N. oceanica cells shifted from high CO2 to low CO2. Particularly, we firstly noted that the transcription of the key low CO2 responsive carbonic anhydrase (CA5), a key component involved in CCM stress signaling, was potentially regulated by bivalent Kbu-H3K9m2 modifications in microalgae. This study provides novel insights into the relationship between gene transcription and epigenetic modification in Nannochloropsis, which will lay foundation on genetic improvement of CCM at epigenetic level. [ABSTRACT FROM AUTHOR]

Microalgae have emerged as a promising feedstock for the sustainable production of lipid-based products, including biofuels and omega-3 fatty acids, by utilizing CO2 as a carbon source. Glycerol-3-phosphate dehydrogenase (GPDH) is a vital enzyme in microalgal lipid synthesis, converting dihydroxyacetone phosphate into glycerol-3-phosphate, the initial substrate for the Kennedy pathway responsible for triacylglycerol production. In this study, we identified and overexpressed a chloroplastic NAD(P)+-dependent GPDH (NsGPDH1) in Nannochloropsis salina. Quantitative reverse transcription polymerase chain reaction and western blotting revealed increased NsGPDH1 expression in the transformants as compared to wild-type N.salina. This overexpression of NsGPDH1 resulted in increased glycerol production and a higher proportion of mono-unsaturated fatty acids, which are indicative of GPDH overexpression. Notably, lipid productivity in NsGPDH1-overexpressing N.salina strains increased by 40% without significant growth defects. These results highlight the effectiveness of NsGPDH1 overexpression as a strategy for enhancing lipid biosynthesis in N. salina, contributing to the advancement of microalgae-based lipid production. [ABSTRACT FROM AUTHOR]

Species in genus Nannochloropsis, especially N. oceanica and N. gaditana, have been evolving as the model microalgae for both application and theory studies. The position effect of genome integration, the carrying capability limitation of integrative vectors and the instability of non-integrative vectors have hindered Nannochloropsis genetic modification with concatenate genes and extremely long DNA fragments. The molecular tools including genetic transformation, homologous recombination, gene edition, gene stacking and episome vectors for transient gene expression and diverse reporters and selection markers have been rapidly developing in Nannochloropsis species. The construction of animal and plant artificial chromosomes with "top down" strategy has set fine examples for the construction of Nannochloropsis artificial chromosomes (NannoACs). It seems that the methods and materials to set the foundation for constructing NannoACs are at hands. In this review, we outlined the current status of transgenes in Nannochloropsis species, summarized the limitations of both integrative and non-integrative vectors, and proposed a tentative approach to construct NannoACs by doubling and stabilizing the genome first, and then truncating the natural chromosomes. NannoACs once constructed will facilitate transferring the desired traits and concatenate genes into Nannochloropsis genetic backgrounds, thus contributing towards its genetic improvement and synthetic biological studies. [ABSTRACT FROM AUTHOR]

Using microalgae as a feedstock for biofuel is a method to reduce carbon dioxide emissions. Nannochloropsis is among the most promising microalgal taxa for biofuel production. To improve the lipid productivity of the marine microalga Nannochloropsis sp. SCSIO-45217 a novel method involving phosphate (NaH2PO4·2H2O) adjustment was established. First, the effects of phosphorous concentration on the lipid accumulation and biochemical composition of Nannochloropsis sp. SCSIO-45217 was studied in a batch culture. Subsequently, to enhance lipid productivity, a fed-batch cultivation mode was established with an initial phosphate concentration of 8 mg L−1 and continuous supplementation with 1, 2 and 3 mg L−1 phosphate, respectively, beginning on the 4th day. In batch culture the lipid content increased gradually from 27.45 to 46.15% DW with decreasing phosphate concentrations. Along with the decrease in phosphate concentrations, the contents of neutral lipids, eicosapentaenoic acid (EPA) and polyunsaturated fatty acids (PUFAs) increased from 50.87 to 70.07%, 2.06 to 7.30% and 2.91 to 11.73% of the total lipid content, respectively. Lipid productivity was enhanced up to 127.20 mg L−1 day−1 in fed-batch culture, which was approximately threefold-fold higher than that of the low phosphate group (8 mg L−1). The productivity was 43.2% higher than that of the high phosphate group (40 mg L−1). These results indicated that Nannochloropsis sp. SCSIO-45217 can be a promising source for PUFAs and the optimized supply of phosphate in fed-batch cultivation is a suitable cultivation strategy for microalgal lipid production. [ABSTRACT FROM AUTHOR]

The photosynthetic apparatus is a major reactive oxygen species (ROS) proliferator, especially in high-light environments. The role of ROS in photoinhibition and photoacclimation mechanisms has been extensively explored, primarily in model plant species. However, little work has been performed on the topic in non-Archaeplastida organisms, such as the model heterokont species Nannochloropsis oceanica. To investigate the photoacclimation and damaging impact of singlet oxygen and superoxide anions on the photosynthetic apparatus of N. oceanica, we subjected cells to two doses of methyl viologen and rose bengal. Significant findings: Rose bengal (a singlet-oxygen photosensitizer) induced changes to the photosynthetic apparatus and PSII photochemistry mirroring high-light-acclimated cells, suggesting that singlet-oxygen signaling plays a role in the high-light acclimation of PSII. We further suggest that this singlet-oxygen pathway is mediated outside the plastid, given that rose bengal caused no detectable damage to the photosynthetic apparatus. Methyl viologen (a superoxide-anion sensitizer) induced an enhanced non-photochemical quenching response, similar to what occurs in high-light-acclimated cells. We propose that superoxide anions produced inside the plastid help regulate the high-light acclimation of photoprotective pathways. [ABSTRACT FROM AUTHOR]

Microalgae have been highlighted as one of the promising feedstocks for renewable fuel production owing to their high growth rate, ability to grow on wastewater, and to convert CO2 into lipids, the main source of biodiesel. However, lipid extraction from microalgal cells is cumbersome due to its thick cell wall which precludes commercial biodiesel production from microalgae, and to improve the lipid extraction efficiency, optimization of the operational parameters is crucial. One of the well-known challenges is to develop an economical technique to obtain a high lipid extraction efficiency. Hence, this study aimed to investigate the optimized conditions of lipid extraction from marine species of Nannochloropsis sp. PTCC 6016 isolated from the Persian Gulf using Soxhlet, Bligh & Dyer, and ultrasonication methods. Initially, Nannochloropsis sp. was cultivated in a 1000L open pond for the purpose of lipid evaluation and process optimization, to be transferred to a 2000 L and subsequently, a 30000L open pond. Effects of various solvent ratios, mixing time and mixing frequency, pre-treatment, and cell age have been evaluated, and the optimal conditions have been determined. Lipid extraction prediction has been investigated by artificial neural network (ANN) and support vector regression (SVR) methods. Experimental results show that the maximum extraction yield of 0.56 gr extracted lipid/gr dry biomass was obtained using Soxhlet method after 90 days of cultivation using chloroform–methanol (1:1) as extraction solvents. However, considering 10 times higher solvent used in Soxhlet method compared to Bligh and Dyer and ultrasonication methods, the ultrasonication method was the most desired option for lipid extraction efficiency of Nannochloropsis sp. in large-scale operations. In this regard, cost-effective biofuel production should be considered as the focus of future studies to achieve sustainability, energy security and addressing the climate change due to high greenhouse gas emissions. [ABSTRACT FROM AUTHOR]

The oleaginous genera Nannochloropsis and Microchloropsis are recognized for their lipid accumulation capacity. Microalgal lipid accumulation is triggered by nitrogen starvation, negatively affecting photosynthesis and growth. Moreover, light and temperature play pivotal roles in microalgal physiology, lipid accumulation and composition. This study focuses on comparing the responses of eight microalgal strains from Nannochloropsis (N. oceanica Necton, N. oceanica IMET1, Nannochloropsis. sp. CCAP211/78, N. oculata, and N. limnetica) and Microchloropsis (M. gaditana CCFM01, M.gaditana CCMP526, and M.salina) to light, temperature, and nitrogen availability. Biomass, lipid content and productivities were monitored under different light intensities (150 (LL) and 600 μmol photons m−2 s−1 (HL)) and temperatures (15, 25, 30℃) under nitrogen (N-) starvation and replete conditions. Under N-starvation and HL, N. sp. exhibited the highest lipid content (59%) and productivity (0.069 g L-1 day-1), while N. oculata had the lowest lipid content (37.5%) and productivity (0.037 g L-1 day-1) among the eight strains. Notably, M. gaditana CCFM01 achieved the highest EPA content (4.7%), contrasting with N.oceanica IMET1 lowest EPA content (2.9%) under 150 μmol photons m−2 s−1 and N-repletion. The response to temperature fluctuations under LL was strain-dependent. Microchloropsis salina and M. gaditana CCFM01 demonstrated the highest and lowest lipid productivities (0.069 g L-1 day-1 and 0.022 g L-1 day-1, respectively) at 15℃ under N-starvation. Moreover, significant EPA accumulation across various strains was observed in N. oculata (5.7%) under N-repletion at 15°C, surpassing M. gaditana CCFM01 by 40%. Ultimately, the physiological responses to cultivation conditions vary markedly among microalgal strains, even within the same genus or species. This knowledge is essential for selecting suitable strains for the efficient microalgal lipid production industry.Optimi zing cultivation conditions for the maximal lipid production in Nannochloropsis andMicrochloropsisGraphical Abstract: The oleaginous genera Nannochloropsis and Microchloropsis are recognized for their lipid accumulation capacity. Microalgal lipid accumulation is triggered by nitrogen starvation, negatively affecting photosynthesis and growth. Moreover, light and temperature play pivotal roles in microalgal physiology, lipid accumulation and composition. This study focuses on comparing the responses of eight microalgal strains from Nannochloropsis (N. oceanica Necton, N. oceanica IMET1, Nannochloropsis. sp. CCAP211/78, N. oculata, and N. limnetica) and Microchloropsis (M. gaditana CCFM01, M.gaditana CCMP526, and M.salina) to light, temperature, and nitrogen availability. Biomass, lipid content and productivities were monitored under different light intensities (150 (LL) and 600 μmol photons m−2 s−1 (HL)) and temperatures (15, 25, 30℃) under nitrogen (N-) starvation and replete conditions. Under N-starvation and HL, N. sp. exhibited the highest lipid content (59%) and productivity (0.069 g L-1 day-1), while N. oculata had the lowest lipid content (37.5%) and productivity (0.037 g L-1 day-1) among the eight strains. Notably, M. gaditana CCFM01 achieved the highest EPA content (4.7%), contrasting with N.oceanica IMET1 lowest EPA content (2.9%) under 150 μmol photons m−2 s−1 and N-repletion. The response to temperature fluctuations under LL was strain-dependent. Microchloropsis salina and M. gaditana CCFM01 demonstrated the highest and lowest lipid productivities (0.069 g L-1 day-1 and 0.022 g L-1 day-1, respectively) at 15℃ under N-starvation. Moreover, significant EPA accumulation across various strains was observed in N. oculata (5.7%) under N-repletion at 15°C, surpassing M. gaditana CCFM01 by 40%. Ultimately, the physiological responses to cultivation conditions vary markedly among microalgal strains, even within the same genus or species. This knowledge is essential for selecting suitable strains for the efficient microalgal lipid production industry.Optimi zing cultivation conditions for the maximal lipid production in Nannochloropsis andMicrochloropsis [ABSTRACT FROM AUTHOR]

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 edible oil, rapid methods are needed to determine the fatty acid composition of microalgae. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) was applied to determine the lipid and fatty acid contents of Nannochloropsis oceanica, a species rich in eicosapentaenoic acid. Samples with different intracellular components were obtained by setting different culture conditions (light intensity and nitrogen concentration). In the spectra of FTIR, some characteristic bands for lipids (3025–2800 cm−1, 1770–1710 cm−1, 1480–1350 cm−1) were identified. Partial least square regression (PLSR) models were developed to predict total biomass, lipid contents, and main fatty acid contents (palmitic acid C16:0, palmitoleic acid C16:1, eicosatetraenoic acid C20:4, eicosapentaenoic acid C20:5). Generally, the prediction ability of the model established with full spectra was slightly better than that of the model established with specific bands. In the optimized model, excellent coefficients of determination (R2 ≥ 0.90), both for calibration and prediction, were found for all variables, except for fatty acid C20:4, which was slightly worse (R2 = 0.8797). The pretreatment methods of samples were further studied. It was found that the acid-heating method (0.1 M HCl, 100 °C, 0.5 h) effectively reduced the interference of the carbohydrate in samples, thus improving the measurement of the characteristic fatty acids C20:4 and C20:5 of N. oceanica. This study showed the feasibility of FTIR as a rapid screening method for evaluating biomass and lipid content, especially some special fatty acid profile of microalgae. [ABSTRACT FROM AUTHOR]

Omega-3 fatty acids containing microalgae are considered a promising feedstock for food and nutraceutical applications. To meet the commercial and nutritional demands of essential omega-3 fatty acids, different strategies are being implemented, including supplementation of plant growth regulators (PGRs). In the present study the effects of selected, PGRs, i.e., gibberellic acid (GA), and salicylic acid (SA), and malic acid (MA; TCA cycle intermediate), were studied on growth, lipid, and omega-3 fatty acid synthesis of Nannochloropsis oceanica CASA CC201. Significant increase in growth (10 ppm GA), a 1.9-fold increase in lipid production (40 ppm MA), and a 4-fold increase in EPA production by treatment with 10 ppm MA were found. To understand the mechanism behind significant changes in growth and metabolite production, we had analyzed the generation of oxidative stress, targeted metabolite, and amino acid profile of N. oceanica under PGR treatment. To elucidate the physiological role of key metabolic pathways and contribution to lipid synthesis, the targeted metabolites were selected from glycolysis, citric acid cycle, and pentose phosphate pathway. Metabolomics analysis revealed that there is a significant increase in ATP, NADPH, NADP, and NADH at cultures treated with high concentrations of selected PGRs and significant elevation of these metabolites are associated with increased lipid production. [ABSTRACT FROM AUTHOR]

The ecological relationships between heterotrophic bacteria and marine phytoplankton are complex and multifaceted, and in some instances include the bacteria-mediated aggregation of phytoplankton cells. It is not known to what extent bacteria stimulate aggregation of marine phytoplankton, the variability in aggregation capacity across different bacterial taxa or the potential role of algogenic exopolymers in this process. Here we screened twenty bacterial isolates, spanning nine orders, for their capacity to stimulate aggregation of two marine phytoplankters, Thalassiosira weissflogii and Nannochloropsis oceanica. In addition to phytoplankton aggregation efficiency, the production of exopolymers was measured using Alcian Blue. Bacterial isolates from the Rhodobacterales, Flavobacteriales and Sphingomonadales orders stimulated the highest levels of cell aggregation in phytoplankton cultures. When co-cultured with bacteria, exopolymer concentration accounted for 34.1% of the aggregation observed in T. weissflogii and 27.7% of the aggregation observed in N. oceanica. Bacteria-mediated aggregation of phytoplankton has potentially important implications for mediating vertical carbon flux in the ocean and in extracting phytoplankton cells from suspension for biotechnological applications. [ABSTRACT FROM AUTHOR]

Solar cultivation of microalgae in photobioreactors is a valuable bioprocess for the sustainable production of commercially useful metabolites. However, the conventional culture temperature control method in solar closed photobioreactors of evaporative cooling is neither economical nor sustainable. In this study, a novel spectrally selective, insulated glazed flat plate (IGP) photobioreactor employing an infrared reflecting system embedded in the illumination surface was used for cultivation of Nannochloropsis sp. The impact of the temperature control technology on protein, lipid, carbohydrate content and fatty acid profile of Nannochloropsis sp. was investigated and compared to closed photobioreactors using passive evaporative cooling (PEC) and an infrared reflecting film (IRF) on the surface as well as an open raceway pond (ORP). Among all cultivation systems tested, the biochemical composition of biomass (mg g−1 organic biomass) showed a general trend of lipid > protein > carbohydrate, with no large variation of each across treatments. However, the areal and volumetric productivities of these constituents were significantly higher in the photobioreactors than in the ORP; results consistent with biomass productivity data. Of the major saturated and monounsaturated fatty acids present, only the proportion of C16:0, which is 24% higher in the photobioreactors than in the ORP, changed significantly among cultivation systems. The highest content of high-value dietary fatty acids, eicosapentaenoic acid (EPA, C20:5n-3; 15.5%) and ϒ-linolenic acid (C18:3n-6; 8%) were found in the ORP but were similar to that produced in the IGP (15.9 and 3.4%, respectively). Among all photobioreactors, the IGP had the least diel temperature changes and an EPA content that was 21% higher than PEC. Photobioreactors constructed with spectrally selective materials effectively allow management of internal reactor temperature with no significant negative impact on biochemical and fatty acid profiles of microalgae. [ABSTRACT FROM AUTHOR]

The areal biomass productivities (g−1 m−2 day−1) of Nannochloropsis oceanica between different sizes of way ponds were compared. Sequential batch cultivation using 2-m2, 20-m2, and 200-m2 raceway ponds with an industrial scale 4000-m2 raceway as the main culture was conducted in summer and autumn during 2017 at Whyalla, Australia. Areal productivities of sequential batch cultivation during the same culture period were 8.4 g ± 0.9 g−1 m−1 day−1 in the 2-m2 ponds, 9.3 g−1 m−1 day−1 in the 20-m2 ponds, and 8.0 g−1 m−1 day−1 in the 200-m2 ponds respectively. In parallel with the operation of the main 4000-m2 pond, some smaller scale ponds of 2, 20, and 200 m2 were operated at the same site under the same conditions. Areal productivity data of dry biomass of Nannochloropsis oceanica in each pond are very similar between industrial 4000-m2 pond and other smaller ponds. In this work, the authors demonstrate that using the growth rate and productivities of Nannochloropsis from smaller scale open ponds with the same depth is valid to estimate for large-scale ponds in excess of 4000 m2. [ABSTRACT FROM AUTHOR]

This study was conducted to determine the impacts of ZnO-, CuO-, and Fe2O3-nanoparticles (NPs) on the growth, protein content, and fatty acid profile of the green microalga Nannochloropsis oculata. The growth of alga was inhibited by the increasing concentration of NPs in the order of CuO-NPs > ZnO-NPs > Fe2O3-NPs. Protein content went up in response to ZnO- and Fe2O3-NPs and decreased after exposure to CuO-NPs. The analysis of fatty acid profile revealed the negative effect of CuO- and Fe2O3-NPs on the content of saturated fatty acids (SFAs). By contrast, the content of SFAs was enhanced in the algal cells treated with ZnO-NPs. The level of unsaturated fatty acids (USFAs) was increased upon reflection to CuO-NPs, but it was reduced after treatment with ZnO- and Fe2O3-NPs. The analysis of biodiesel indicators showed that the cloud point (CP) can be increased considerably in the algal cells exposed to ZnO- and CuO-NPs. Intriguingly, the cold filter plugging point (CFPP) of biodiesel was remarkably elevated in the treated cells with Fe2O3-NPs. Taken all together, despite the toxicity caused by high concentrations of metal oxide NPs, NPs could raise the amount of CP and CFPP and improve the oxidative stability in N. oculata biodiesel. [ABSTRACT FROM AUTHOR]

Oleaginous microalgae can accumulate large amounts of storage lipids that have many potential applications such as in the production of biodiesel and health products. All of the energy for the synthesis of lipids in microalgae is derived from photosynthesis. To date, however, the quantitative relationship between photosynthesis and lipid accumulation rate in microalgae is still unclear. In this study, Nannochloropsis sp. was selected to explore this relationship by investigating changes in lipid accumulation, photosynthetic efficiency, and the electron transport chain under nutrients limitation and replenishment. The results of the study demonstrated that lipids were the main form of storage of carbon and energy for this microalga. The alternative electron transport chain played an important role in photo-protection and lipid accumulation. The photosynthetic efficiency of this microalga showed a strong correlation with lipid accumulation rate (R2 = 0.959), with higher photosynthetic efficiency translating to higher lipid accumulation rates during the lipid accumulation stage of the growth cycle. The results of the present study indicated that nutrient depletion might not be the best signal to understand when microalgae begin to accumulate oil, as it appears that photosynthetic efficiency also has a large role in the rate of oil accumulation. Additionally, the study shows that excessive light might not be needed in later stages of growth for lipid accumulation, which in turn would alleviate the damage to microalgae caused by strong light making large-scale cultivation more cost-effective. [ABSTRACT FROM AUTHOR]

Ammonium and nitrate were compared as nitrogen sources for mixotrophic growth of Microchloropsis salina (formerly Nannochloropsis salina) on glycerol. The effects of nitrogen source and glycerol on the abundance, composition and profile of bacterial communities were examined by 16S rRNA sequencing. The effect of bacteria on culture performance was assessed by comparing ampicillin-containing and ampicillin-free cultures. The productivity of M. salina was higher with ammonium than with nitrate, and lower with bacteria present. Glycerol utilization by M. salina was minimal in the absence of a nitrogen source and the addition of glycerol increased the abundance of bacteria. Bacterial profiles were influenced by nitrogen source, with Pseudomonadaceae proliferating with ammonium, Alteromonadaceae with nitrate and Beijerinckiaceae prevalent with either. The presence of nitrate and glycerol led to aerobic-denitrifying bacteria capable of diverting both resources away from algal metabolism. These findings demonstrate significant advantages of providing ammonium during mixotrophic cultivation of M. salina on glycerol. [ABSTRACT FROM AUTHOR]

Despite the increased interest in exploring the potential of algal biomass production for food stock and renewable energy, very little work has been done in developing reliable screening protocols to enable the identification of species that are best suited to mass cultivation outdoors. Nannochloropsis is an algal genus identified as a potential source of lipids due to its ability to accumulate large quantities of these compounds, especially under nutrient-limiting conditions. The objective of the current work was to use two species of this genus, Nannochloropsis oceanica and N. oculata, as model organisms to develop a protocol that will allow the evaluation of their capacity to yield high biomass productivity under outdoor conditions. Growing the alga under different light intensities and measuring growth rate as well as a range of photosynthetic parameters based on light response curves and variable fluorescence highlighted significant differences between the two species. Our data show that N. oceanica cells have a better capacity to respond to higher light intensities, as reflected by growth measurements, photosynthetic electron transport rates, and oxygen evolution as well as their response to the very high photon flux densities expected in outdoor culture. On the other hand, N. oculata showed a higher tolerance to oxidative stress as reflected in its resistance to the reactive oxygen species generating compounds Rose Bengal (RB) and methyl viologen (MV). Based on the above evidence, we suggest that N. oceanica may perform better than N. oculata when grown under high light conditions typically found outdoors in summer, while N. oculata may perform better than N. oceanica under oxidative stress conditions usually found in outdoor cultures exposed to a combination of high light and low temperature commonly occurring in winter time. [ABSTRACT FROM AUTHOR]

Recently, we isolated a complex consisting of photosystem II (PSII) and light-harvesting complexes (LHCs) from Nannochloropsis granulata (Umetani et al. Photosynth Res 136:49-61, 2017). This complex contained stress-related protein, Lhcx, as a major component of LHC (Protein ID is Naga_100173g12.1), suggesting that non-photochemical quenching activities may be taking place in the PSII-LHC complex. In this study, we examined the energy transfer dynamics in the isolated LHCs and PSII-LHC complexes, and found substantial quenching capacity. In addition, the LHCs contained low-energy chlorophylls with fluorescence maxima at approximately 710 nm, which may enhance the quenching efficiency in the PSII-LHC. Delayed fluorescence analysis suggested that there was an approximately 50% reduction in energy trapping at the PSII reaction center in the PSII-LHC supercomplex under low-pH condition compared to neutral pH condition. Enhanced quenching may confer a survival advantage in the shallow-water habitat of Nannochloropsis. [ABSTRACT FROM AUTHOR]

The marine microalgal species Nannochloropsis gaditana has emerged as a model organism for research into algal-derived biofuels due to its ability to produce large amounts of lipids. In addition, this species has biotechnological potential owing to its ease of transformation and availability of a sequenced and annotated genome. The ability to control expression of transgenes in an inducible manner is an important requirement for many genetic engineering approaches and as such, inducible promoters are an important component of the molecular toolkit for a host organism. We designed an expression vector containing a ~ 1.3 kb region upstream of the endogenous nitrate reductase gene of N. gaditana and demonstrated this is capable of promoting the expression of a heterologous enhanced green fluorescent protein (eGFP) reporter. In the presence of ammonium, expression of the eGFP reporter was undetectable; however, in the presence of nitrate, eGFP expression is induced. In addition, we demonstrated how altering the nitrogen source of the growth media can be used to precisely control expression. We have shown that the nitrate reductase promoter can be used as a powerful molecular tool for heterologous protein expression in N. gaditana, further contributing to the development of this species as a candidate strain for biotechnology. [ABSTRACT FROM AUTHOR]

Nannochloropsis is a genus of fast-growing microalgae that are regularly used for biotechnology applications. Nannochloropsis species have a high triacylglycerol content and their polar lipids are rich in the omega-3 long-chain polyunsaturated fatty acid, eicosapentaenoic acid. Placed in the heterokont lineage, the Nannochloropsis genus has a complex evolutionary history. Genome sequences are available for several species, and a number of transcriptomic datasets have been produced, making this genus a facile model for comparative genomics. There is a growing interest in Nannochloropsis species as models for the study of microalga lipid metabolism and as a chassis for synthetic biology. Recently, techniques for gene stacking, and targeted gene disruption and repression in the Nannochloropsis genus have been developed. These tools enable gene-specific, mechanistic studies and have already allowed the engineering of improved Nannochloropsis strains with superior growth, or greater bioproduction. [ABSTRACT FROM AUTHOR]

Algae of the genus Nannochloropsis are attractive organisms for use in biotechnology due to their high lipid content. Genetic manipulation of marine Nannochloropsis species has already been reported; however, tools have not yet been developed to transform Nannochloropsis limnetica, the only known freshwater species of this genus. To establish N. limnetica as a model laboratory strain, we first tested the effects of 11 different antibiotics on growth of N. limnetica and the marine species N. oceanica and N. gaditana. These three microalgae responded very differently to antibiotic treatments, both in liquid cultures and on agar plates. In general, N. limnetica exhibited a much higher sensitivity to antibiotics than the marine strains, thus offering the potential for a large set of antibiotic resistance genes that may be applicable as artificial selection markers after transformation. We also developed a simple protocol using lysozyme to obtain high yields of viable N. limnetica protoplasts, as confirmed by flow cytometry and electron microscopy. [ABSTRACT FROM AUTHOR]

In this paper we show that the absorption spectrum of the microalgae Nannochloropsis oceanica exhibits changes in response to the modulation of incident light. A model was used to analyze the contribution of different active pigments to the total absorption in the photosynthetically active radiation region and suggested consistent diel oscillations in the optical activity of carotenoids. [ABSTRACT FROM AUTHOR]

Photosystem I (PSI) is a multi-subunit integral pigment-protein complex that performs light-driven electron transfer from plastocyanin to ferredoxin in the thylakoid membrane of oxygenic photoautotrophs. In order to achieve the optimal photosynthetic performance under ambient irradiance, the absorption cross section of PSI is extended by means of peripheral antenna complexes. In eukaryotes, this role is played mostly by the pigment-protein complexes of the LHC family. The structure of the PSI-antenna supercomplexes has been relatively well understood in organisms harboring the primary plastid: red algae, green algae and plants. The secondary endosymbiotic algae, despite their major ecological importance, have so far received less attention. Here we report a detailed structural analysis of the antenna-PSI association in the stramenopile alga Nannochloropsis oceanica (Eustigmatophyceae). Several types of PSI-antenna assemblies are identified allowing for identification of antenna docking sites on the PSI core. Instances of departure of the stramenopile system from the red algal model of PSI-Lhcr structure are recorded, and evolutionary implications of these observations are discussed. [ABSTRACT FROM AUTHOR]

Eustigmatophyte algae represent an interesting model system for the study of the regulation of the excitation energy flow due to their use of violaxanthin both as a major light-harvesting pigment and as the basis of xanthophyll cycle. Fluorescence induction kinetics was studied in an oleaginous marine alga Nannochloropsis oceanica. Nonphotochemical fluorescence quenching was analyzed in detail with respect to the state of the cellular xanthophyll pool. Two components of nonphotochemical fluorescence quenching (NPQ), both dependent on the presence of zeaxanthin, were clearly resolved, denoted as slow and fast NPQ based on kinetics of their formation. The slow component was shown to be in direct proportion to the amount of zeaxanthin, while the fast NPQ component was transiently induced in the presence of membrane potential on subsecond timescales. The applicability of these observations to other eustigmatophyte species is demonstrated by measurements of other representatives of this algal group, both marine and freshwater. [ABSTRACT FROM AUTHOR]

Oils, carbohydrates, and fats generated by microalgae are being refined in an effort to produce biofuels. The research presented here examines two marine microalgae, Nannochloropsis salina (green alga) and Phaeodactylum tricornutum (diatom), when grown with 0 (no addition), 0.5, 1.0, 2.0, and 5.0 g L NaHCO added to an f/2 medium during the growth phase (GP) and a nutrient induced (nitrate limitation) lipid formation phase (LP). We hypothesize that the addition of NaHCO is a sustainable and practical strategy to increase cellular density and concentrations of lipids in microalgae as well as the rate of lipid accumulation. In N. salina, final cell densities were significantly ( p < 0.05) higher in the NaHCO-treated cells than the control while in P. tricornutum the cell densities were higher with >[NaHCO] during the GP. During the LP, cell densities were generally higher in the NaHCO-treated cells compared with controls. F/ F (efficiency of photosystem II) patterns paralleled those for cell density with generally higher values with higher concentrations of NaHCO and significantly different values between controls and 5.0 g L NaHCO at the end of the GP ( p < 0.05). F/ F was variable between treatments in P. tricornutum (0.3-0.65) but less so in N. salina for (0.5-0.7) regardless of [NaHCO]. The lipid index (measured with Nile red), used as a proxy for triacylglycerides (TAGs), was 10.2 ± 6.5 and 4.4 ± 2.9 (fluorescence units/OD cells ×1000) for N. salina and P. tricornutum, respectively, at the end of the GP. At the end of the LP, the lipid index was eight and four times higher than during the GP in the corresponding 5.0 g L NaHCO treatments, revealing that N. salina was accumulating more lipid than P. tricornutum. Dry weights essentially doubled during LP compared with GP for N. salina; this was not the case for P. tricornutum. In general, the percentage of ash in dry weights was significantly higher in the LP relative to the corresponding GP treatments for P. tricornutum; this was not the case for N. salina. During the LP, there was also less soluble protein in N. salina compared to GP; differences were not significant in cells growing with 2.0 or 5.0 g L NaHCO. In P. tricornutum, faster growing cells had more soluble protein during the GP and LP; differences between treatments were significant. P. tricornutum generally accumulated significantly more crude protein than N. salina at higher [NaHCO]; there was three times more crude protein in the highest NaHCO (5.0 g L) treatment compared with the controls. C:N ratios (mol:mol) were similar across treatments during GP: 7.03 ± 0.12 and 10.16 ± 0.41 for N. salina and P. tricornutum, respectively. Further, C:N ratios increased with increasing [NaHCO] during LP. Species-specific fatty acid methyl ester (FAMEs) profiles were observed. While C16:0 was lower in P. tricornutum compared to N. salina, the diatom produced more C16:1 and C14 but not C18:3. Monounsaturated fatty acids (MUFA) significantly increased in N. salina in the LP compared to GP and in response to increasing [NaHCO] ( t tests; p < 0.05). Saturated fatty acids (SFA) responded similarly but to a lesser degree. There were more polyunsaturated fatty acids (PUFA) in N. salina than MUFAs or SFAs. In P. tricornutum, there were generally more SFAs, MUFAs and PUFAs in P. tricornutum during LP than GP in the corresponding NaHCO treatments. These findings reveal the importance of considering NaHCO as a supplemental carbon source in the culturing marine phytoplankton in large-scale production for biofuels. [ABSTRACT FROM AUTHOR]

The heightened awareness concerning environmental preservation, resource scarcity, food safety, and nutrition has engendered the need for a more sustainable and resource-efficient agricultural production system. In this context, microalgae offer the potential to recover nutrients from waste streams and subsequently use the microalgal biomass as a sustainable slow-release fertilizer. The aim of this study was to assess microalgal bacterial flocs treating aquaculture wastewater and marine microalgae as organic slow-release fertilizers for tomato cultivation. Comparable plant growth was observed using microalgal and commercial organic fertilizer treatments. Furthermore, the microalgal fertilizers improved the fruit quality through an increase in sugar and carotenoid content, although a lower tomato yield was obtained. An economic evaluation indicates the economic feasibility of the microalgae-based fertilizers. Further research is required to optimize the microalgae-based fertilizer composition. [ABSTRACT FROM AUTHOR]

In the present study, the potential of the microalga Pavlova viridis (= Diacronema viridis) as an n-3 polyunsaturated fatty acid (PUFA) source was evaluated and compared to Nannochloropsis sp. in diets for juvenile European sea bass ( Dicentrarchus labrax L.) (initial weight ~12.8 ± 1.7 g) in an 8-week feeding trial. Six different isoenergetic and isonitrogenous test diets were used: (1) fish oil diet (FO), major lipid source fish oil (100 %), (2) basal diet, 40 % fish oil and 60 % plant oil (in equal parts rapeseed, sunflower, and linseed oil), (3) Pavlova 50 % (P50), 50 % of the fish oil of the basal diet was substituted by lipid content of P. viridis meal, (4) Pavlova 100 % (P100), 100 % of the fish oil of the basal diet was substituted by lipid content of P. viridis meal, (5) Nannochloropsis 50 % (N50), 50 % of the fish oil of the basal diet was substituted by lipid content of Nannochloropsis sp. meal, and (6) Nannochloropsis 100 % (N100), 100 % of the fish oil of the basal diet was substituted by lipid content of Nannochloropsis sp. meal. The specific growth rate was highest and feed conversion ratio was lowest in the P100 group (SGR 1.77 ± 0.10 % day; FCR 1.17 ± 0.01), although not significantly different to the results for the FO and the other algae-groups. Furthermore, the sum of PUFA was also highest in the P100 group, followed by the P50, N100, N50, and B group (mainly due to the high content of linoleic and linolenic acids coming from plant oils and microalgal products) with the lowest levels in the FO group. The highest amounts of docosahexaenoic acid (DHA) of total fatty acids were found in the FO and B group, although not significantly higher than in groups P50 and P100. The significantly highest amount of eicosapentaenoic acid (EPA, % of total fatty acids) was in the P100 samples and the lowest amount was in samples of the basal group. The histological analyses of liver and intestine samples did not reveal any negative effects caused by the experimental treatments. Based on the basal diet, a 50 % fish oil replacement by Nannochloropsis sp. meal and a total replacement by P. viridis meal were possible without negative effects on the growth performance and nutrient utilization of juvenile sea bass. [ABSTRACT FROM AUTHOR]

Large improvements in biomass and lipid production are required to make massive scale algal biodiesel production an economic reality. The application of the biodiversity strategy to enhance algal biomass as biofuel feedstock is little. The algal diversity was manipulated in this study to investigate the effects of a combination of biodiversity complementarity and a new medium consisting of seawater and agricultural fertilizer on lipid productivity. The algae diverse community includes two strains of Dunaliella salina ( Dunaliella salina 19/30 and 19/18) and three species of Nannochloropsis ( Nannochloropsis oculata, Nannochloropsis salina, and Nannochloropsis gaditana). The results showed that the most diverse community (5 species) produced an average of sixfold more biomass in the new medium than did the standard f/2 culture medium. The most diverse polyculture had the highest growth rate (1.01 day), biomass (1.2 g L), and lipid productivity (0.31 g L day. The assessment of algal polycultures relative to monocultures is particularly interesting and novel for this biofuel field, and the observations that these polycultures resulted in significant lipid productivity improvements are very useful addition to the biofuel research. The possible mechanism (resource diversity) to explain the synergy in mixed cultures warrants further investigation. [ABSTRACT FROM AUTHOR]

We illustrate a detailed compositional characterization of hydrothermal liquefaction (HTL) oils derived from two biochemically distinct microalgae, Nannochloropsis gaditana and Chlorella sp. (DOE 1412), for a range of reaction temperature as observed by high-resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS). The unique capability to unequivocally derive molecular formulae directly from FT-ICR MS-measured mass-to-charge ratio (for several thousand compounds in each oil) shows that lipids are completely reacted/converted for any reaction temperature above 200 °C and reveals the formation of nonlipid reaction products with increasing temperature. Specifically, lipid-rich oil is obtained at low reaction temperature (<225 °C) for both microalgal strains. For positive ion mode, the major lipid components in Chlorella sp. and N. gaditana HTL oils are betaine lipids and acylglycerols, respectively. Acidic species in the HTL oils (observed by negative ion mode) are dominated by free fatty acids (FFA) regardless of reaction temperature. HTL oils obtained at higher temperatures (≥225 °C) are composed of a variety of basic nitrogen- and oxygen-containing compounds that originate from protein and carbohydrate degradation at elevated temperature. Similar structural features are observed for the abundant nitrogen heterocyclics between the two strains with slightly lower carbon number for Chlorella sp., overall. [ABSTRACT FROM AUTHOR]

Optimization of mass microalgae cultures is required for the efficient production of biodiesel feedstock in terms of total fatty acid (TFA) content and a conducive fatty acid profile. A mutant strain of Nannochloropsis sp. (MT-I5), as modified via random mutagenesis and flow cytometric cell sorting, was investigated in both a single- and two-stage cultivation using 250 L laboratory raceway ponds. Culture was based on photoautotrophic biomass production (stage 1) followed by a switch to photomixotrophic growth induced by adding sodium acetate (2 mM) (stage 2). The biomass yield of the mutant in two-stage cultivation was maintained at a level similar to that of the one-stage photoautotrophic culture, but TFA content was increased by 2.3-fold. The fatty acid profile of MT-I5 also had an increased level of desirable saturated fatty acids (SFA) for use as a biodiesel feedstock, i.e. from 43 to 48 % of TFA, as well as a decreased level of less desirable polyunsaturated fatty acids (PUFA), i.e. from 22 to 7 % of TFA. The two-stage cultivation process is of interest for the mass culture of microalgae for biofuel feedstocks, as biomass productivity can be maximized during the first stage of culture until N-starvation is achieved, followed by the enhanced synthesis of SFA in the second stage of culture by adding sodium acetate as a fixed-carbon source. [ABSTRACT FROM AUTHOR]

Translationally-controlled tumor protein (TCTP) is a eukaryote-conserved protein with crucial roles in cellular growth. It has also been proposed that plant TCTP has functions specific to plant, while no structure of TCTP from photosynthetic organism has been reported. Nannochloropsis is a photosynthetic microalga with high yield of lipid and high-value polyunsaturated fatty acid, which is promising for biodiesel production. Study of growth-related proteins may provide new clue for improving the yield of lipid. TCTP from Nannochloropsis oceanica shares low sequence identity with structure-known TCTPs. Here we reported the NMR resonance assignments of TCTP from N. oceanica for further structural and functional studies. [ABSTRACT FROM AUTHOR]

Specific growth rates (SGR) of freshwater algae species ( Chlorella vulgaris, Auxenochlorella protothecoides, and Chlorella sorokiniana) and the marine species Nannochloropsis oculata on various nitrogen sources (ammonium carbonate, ammonium chloride, sodium nitrate, and urea) could be determined by in vivo chlorophyll-a autofluorescence. These preferences could be determined before large pH changes occurred in the media, with no significant difference ( P > 0.05) between buffered and non-buffered media. In all algal species, acclimatization was observed with no significant difference ( P > 0.05) between SGRs of the second and third cultivations. ANOVA of SGRs in the acclimatized second and third cultivations revealed preferences for nitrogen sources among most of the algae; C. vulgaris preferred sodium nitrate over other nitrogen sources, A. protothecoides adapted to urea after no growth in the first cultivation, and the SGRs of N. oculata showed an aversion for sodium nitrate over other nitrogen sources ( P < 0.05). [ABSTRACT FROM AUTHOR]

Efficient production of algal biofuels could reduce dependence on foreign oil by providing a domestic renewable energy source. Moreover, algae-based biofuels are attractive for their large oil yield potential despite decreased land use and natural resource (e.g., water and nutrients) requirements compared to terrestrial energy crops. Important factors controlling algal lipid productivity include temperature, nutrient availability, salinity, pH, and the light-to-biomass conversion rate. Computational approaches allow for inexpensive predictions of algae growth kinetics for various bioreactor sizes and geometries without the need for multiple, expensive measurement systems. Parametric studies of algal species include serial experiments that use off-line monitoring of growth and lipid levels. Such approaches are time consuming and usually incomplete, and studies on the effect of the interaction between various parameters on algal growth are currently lacking. However, these are the necessary precursors for computational models, which currently lack the data necessary to accurately simulate and predict algae growth. In this work, we conduct a lab-scale parametric study of the marine alga Nannochloropsis salina and apply the findings to our physics-based computational algae growth model. We then compare results from the model with experiments conducted in a greenhouse tank and an outdoor, open-channel raceway pond. Results show that the computational model effectively predicts algae growth in systems across varying scale and identifies the causes for reductions in algal productivities. Applying the model facilitates optimization of pond designs and improvements in strain selection. [ABSTRACT FROM AUTHOR]

Carbon concentrating mechanisms (CCMs) are common among microalgae, but their regulation and even existence in some of the most promising biofuel production strains is poorly understood. This is partly because screening for new strains does not commonly include assessment of CCM function or regulation despite its fundamental role in primary carbon metabolism. In addition, the inducible nature of many microalgal CCMs means that environmental conditions should be considered when assessing CCM function and its potential impact on biofuels. In this study, we address the effect of environmental conditions by combining novel, high frequency, on-line CO gas exchange screen with microscope-based lipid characterization to assess CCM function in Nannochloropsis salina and its interaction with lipid production. Regulation of CCM function was explored by changing the concentration of CO provided to continuous cultures in airlift bioreactors where cell density was kept constant across conditions by controlling the rate of media supply. Our isotopic gas exchange results were consistent with N. salina having an inducible 'pump-leak' style CCM similar to that of Nannochloropsis gaditana. Though cells grew faster at high CO and had higher rates of net CO uptake, we did not observe significant differences in lipid content between conditions. Since the rate of CO supply was much higher for the high CO conditions, we calculated that growing cells bubbled with low CO is about 40 % more efficient for carbon capture than bubbling with high CO. We attribute this higher efficiency to the activity of a CCM under low CO conditions. [ABSTRACT FROM AUTHOR]

The Eustigmatophyceae is a class of yellow-green algae allied with the Chrysophyceae and other chlorophyll c possessing stramenopile (heterokont) algae. Some members of the class, especially the marine species of the genus Nannochloropsis, are under intense investigation for their potential for production of biofuels and beneficial fatty acids. The class has generally been thought to comprise a small number of genera and species, and these organisms were considered rare or infrequently encountered. In this study, we examined the phylogeny and diversity of this class by analysis of nuclear 18S rDNA sequence data. Our analysis included sequences from all the named members of the Eustigmatophyceae held in culture collections as well as a number of strains identified in culture collections as Xanthophyceae, new strains with features characteristic of the Eustigmatophyceae, and published data for uncultured DNA clones. The results of these analyses show that the Eustigmatophyceae is far more diverse than generally recognized. Two major lineages are supported in the class, the previously recognized order Eustigmatales and the new clade, Goniochloridales. Additional new lineages were also resolved within each of these major lineages; however, the results of our analyses were considered insufficient for naming these subordinate clades. Several of these lineages comprised only unnamed strains or uncultured DNA clones. Overall, our results indicate that the Eustigmatophyceae is a highly diverse class, with many new species, genera, and families awaiting taxonomic treatment. [ABSTRACT FROM AUTHOR]

With intensification of interest in microalgae as a source of biomass for biofuel production, rapid methods are needed for lipid screening of cultures. In this study, near-infrared reflectance spectroscopy (NIRS) was assessed as a method for analysing lipid (specifically, total fatty acid methyl esters (FAME) obtainable from processing) and biomass in late logarithmic and stationary phase cultures of the green alga Kirchneriella sp. and the eustigmatophyte Nannochloropsis sp. Culture samples were filtered, scanned by NIRS and chemically analysed; by combining these sets of information, models were developed to predict total biomass, FAME content and FAME as a percentage of dry weight in samples. Chemically derived (actual) and NIRS-predicted data were compared using the coefficient of determination ( R) and the ratio of the standard deviation (SD) of actual data to the SD of NIRS prediction (RPD). For Kirchneriella sp. samples, models gave excellent prediction ( R ≥ 0.96; RPD ≥ 4.8) for all parameters. For Nannochloropsis sp., the model metrics were less favourable ( R = 0.84-0.94; RPD = 2.5-4.2), though sufficient to provide estimations that could be useful for screening purposes. This technique may require further validation and comparison with other species, but this study shows the potential of the NIRS as a rapid screening method (e.g. up to 200 sample analyses per day) for estimating FAME or other microalgal constituents and encourages further investigation. [ABSTRACT FROM AUTHOR]

In the present paper, we investigated the physiological response of the marine microalga Nannochloropsis sp. to salt stress (13, 27, 54, and 81 g L NaCl). Increasing the sodium chloride concentration caused up to a 70 % decrease in the chlorophyll a concentration, cell growth, and net photosynthesis rate. The chlorophyll a fluorescence measurements indicated a strong reduction in the effective quantum yield of photosystem II (−60 %) and an increase in nonphotochemical quenching when the cells were exposed to NaCl concentrations greater than 27 g L (control). In contrast, the specific lipid content increased up to 80 % when the sodium chloride concentration was increased from 27 to 54-81 g L. These results are relevant for the outdoor cultivation of this microalga using open photobioreactors, in which microalgae are subjected to strong changes in salinity concentration caused by water evaporation. [ABSTRACT FROM AUTHOR]

Classical methods for protein extraction from microorganisms, used for large-scale treatments such as mechanical or chemical processes, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow-process yeast electroextraction proved that pulsed electric field technology allows us to preserve the integrity of released cytosolic proteins by keeping intact vacuole membranes. Furthermore, large volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electroextract total cytoplasmic proteins from microalgae ( Nannochloropsis salina and Chlorella vulgaris). Given that induction of electropermeabilization is under the control of the target cell size, as the mean diameter for N. salina is only 2.5 μm, we used repetitive 2-ms-long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24-h incubation period in a salty buffer. The amount of total protein released was evaluated by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris under milder electrical conditions, as expected from their larger size. This innovative technology designed in our group should become familiar in the field of microalgae biotechnology. [ABSTRACT FROM AUTHOR]

We investigated the effects of osmotic downshift induced by the transfer of Nannochloropsis oceanica CCALA 804 from artificial seawater medium (27 g L 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 s). 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 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. [ABSTRACT FROM AUTHOR]

The effect of algae with different DHA contents used for the enrichment of rotifers on the growth performance, survival, and swim bladder inflation of larval amberjack Seriola dumerili was investigated. Rotifers were enriched with freshwater Chlorella vulgaris containing three levels of DHA (rotifer containing DHA 0.04, 0.60, 1.32 g DHA 100 g DM) and Nannochloropsis (0.04 g DHA 100 g DM; 2.54 g EPA 100 gDM). The larvae were fed the enriched rotifers in triplicate from 3 days post-hatch for 7 days in static condition. The same algae used for rotifer enrichment were supplied to the larval tanks. Growth and survival rate of fish fed the rotifers enriched with Nannochloropsis were higher than those of fish fed the rotifers enriched with all three Chlorella treatments. Swim bladder inflation was lowest in fish fed the rotifers enriched with Nannochloropsis. The proportion of EPA was higher in fish fed the rotifers enriched with Nannochloropsis, while that of DHA increased proportionally with the DHA levels in the rotifers enriched with Chlorella. These results suggest that rotifers enriched with Nannochloropsis (EPA-rich rotifers) are effective to enhance growth and survival, but DHA instead of EPA is essential to improve the swim bladder inflation in amberjack larvae. [ABSTRACT FROM AUTHOR]

We describe the preliminary extractions from a pilot-scale supercritical carbon dioxide (SC-CO) extractor for the isolation of algal lipids suitable for small-scale conversion to liquid hydrocarbon fuels. Flowable oils were recovered from SC-CO extractions of lyophilized Nannochloropsis granulata. The extracted oils were determined to be composed primarily of triacylglycerols (TAG) by liquid chromatography-mass spectrometry analysis. Gravimetric lipid yield was increased significantly from 15.56 to 28.45 mg g ash-free dry weight (AFDW) with an increase in temperature from 50°C to 70°C, at 35 MPa over 270 min. Varying pressure had no significant effect on lipid yield. Liquid chromatography-mass spectrometry analysis of the SC-SO extracts indicated that the TAG profile remained constant regardless of extraction pressure, and analysis of fatty acid methyl esters (FAME) revealed a uniform profile throughout all extraction conditions. Our optimized gravimetric lipid yields from N. granulata (28.45 mg g AFDW) were approximately half of the yields obtained by Soxhlet extraction with hexane (57.53 mg g AFDW); however, the FAME yields were similar regardless of extraction technique (18.23 mg FAME g and 17.35 mg FAME g AFDW from SC-CO extraction and hexane extraction, respectively). Further extractions with Botryococcus braunii indicated that fatty acid extraction by SC-CO was as efficient as hexane extraction. These results highlight the suitability of SC-CO for large-scale oil extraction of microalgae for biofuel or biojet analyses due to its selectivity for TAG extraction. [ABSTRACT FROM AUTHOR]

To elucidate possible causes of skeletal malformations in larval Japanese flounder, we reared larvae fed rotifers enriched with three types of preserved Nannochloropsis (fresh, refrigerated, and frozen). The incidence of malformations at 50 days post hatch ranged from 14.5 to 38.5 % within the three experimental groups, and elongation of the lower jaw (LJ) was the most frequently observed malformation, ranging from 68 to 89 % of total malformations. We also investigated larval parentages using microsatellite markers. Parentage analysis of the fresh Nannochloropsis group showed that one sire and a pair generated significant numbers of LJ-elongated individuals. In the refrigerated Nannochloropsis group, one dam and two sires generated significant numbers of LJ-elongated individuals. In the frozen Nannochloropsis group, no broodstocks or pairs generated significant numbers of LJ-elongated individuals. Our results suggest that LJ elongation in Japanese flounder likely results from the application of different types of preserved Nannochloropsis during rotifer feeding stage. However, there is also some level of genetic influence associated with this deformity. [ABSTRACT FROM AUTHOR]

Nannochloropsis, a green microalga, is a source for commercially valuable compounds as extensively described and, in particular, is recognised as a good potential source of eicosapentaenoic acid (20:5ϖ3), an important polyunsaturated fatty acid for human consumption for prevention of several diseases. Climate change might include variation in the ultraviolet (UV) levels as one of the consequences derived from the anthropogenic activity. This paper shows the response of Nannochloropsis cultures exposed for 7 days to UV-A (320-400 nm) added to photosynthetically active radiation (PAR; 400-700 nm). Growth rates and photosynthetic activity were assessed to determine the impact of UV-A increased levels on the cell growth and basic metabolism activity. Xanthophyll pigments (zeaxanthin and violaxanthin), carotenoids (canthaxanthin and β-carotene) and polyunsaturated fatty acids (myristic, palmitic, palmitoleic, arachidonic and eicosapentaenoic acids) were measured for assessing the antioxidant response of the microalgae to added UV-A radiation to PAR. The results show that the modulated use of UV-A radiations can lead to increased growth rates, which are sustained in time by an increased light transduction activity. The expected antioxidant response to the incident UV-A radiation consisted of increases in zeaxanthin and β-carotene contents-synthesis of antioxidant carotenoids-and increases in the saturated fatty acids to polyunsaturated fatty acids ratio. The results suggest that modulated UV-A radiation can be used as a tool to stimulate value molecules accumulation in microalgae through an enhanced both light transduction process and antioxidant response, while sustaining cell growth. [ABSTRACT FROM AUTHOR]

In order to develop a practical approach for fast and non-destructive assay of total fatty acid (TFA) and pigments in the biomass of the marine microalga Nannochloropsis sp. changes in TFA, chlorophyll, and carotenoid contents were monitored in parallel with the cell suspension absorbance. The experiments were conducted with the cultures grown under normal (complete nutrient f/2 medium at 75 μmol PAR photons/(m s)) or stressful (nitrogen-lacking media at 350 μmol PAR photons/(m s)) conditions. The reliable measurement of the cell suspension absorbance using a spectrophotometer without integrating sphere was achieved by deposition of cells on glass-fiber filters in the chlorophyll content range of 3-13 mg/L. Under stressful conditions, a 30-50% decline in biomass and chlorophyll, retention of carotenoids and a build-up of TFA (15-45 % of dry weight) were recorded. Spectral regions sensitive to widely ranging changes in carotenoid-to-chlorophyll ratio and correlated changes of TFA content were revealed. Employing the tight inter-correlation of stress-induced changes in lipid metabolism and rearrangement of the pigment apparatus, the spectral indices were constructed for non-destructive assessment of carotenoid-to-chlorophyll ratio (range 0.3-0.6; root mean square error (RMSE) = 0.03; r = 0.93) as well as TFA content of Nannochloropsis sp. biomass (range 5.0-45%; RMSE = 3.23 %; r = 0.89) in the broad band 400-550 nm normalized to that in chlorophyll absorption band (centered at 678 nm). The findings are discussed in the context of real-time monitoring of the TFA accumulation by Nannochloropsis cultures under stressful conditions. [ABSTRACT FROM AUTHOR]

As large-scale outdoor production cannot be done in complete containment, cultures are (more) open for bacteria, which may affect the productivity and stability of the algae production process. We investigated the bacterial diversity in two indoor reactors and four pilot-scale outdoor reactors for the production of Nannochloropsis sp. CCAP211/78 spanning four months of operation from July to October. Illumina sequencing of 16S rRNA gene amplicons demonstrated that a wide variety of bacteria were present in all reactor types, with predominance of Bacteroidetes and Alphaproteobacteria. Bacterial communities were significantly different between all reactor types (except between the horizontal tubular reactor and the vertical tubular reactor) and also between runs in each reactor. Bacteria common to the majority of samples included one member of the Saprospiraceae family and one of the NS11-12_marine group (both Bacteroidetes). Hierarchical clustering analysis revealed two phases during the cultivation period separated by a major shift in bacterial community composition in the horizontal tubular reactor, the vertical tubular reactor and the raceway pond with a strong decrease of the Saprospiraceae and NS11-12_marine group that initially dominated the bacterial communities. Furthermore, we observed a less consistent pattern of bacterial taxa appearing in different reactors and runs, most of which belonging to the classes Deltaproteobacteria and Flavobacteriia. In addition, canonical correspondence analysis showed that the bacterial community composition was significantly correlated with the nitrate concentration. This study contributes to our understanding of bacterial diversity and composition in different types of outdoor reactors exposed to a range of dynamic biotic and abiotic factors.Key points• Reactor types had significantly different bacterial communities except HT and VT• The inoculum source and physiochemical factors together affect bacterial community• The bacterial family Saprospiraceae is positively correlated to microalgal growthKey points• Reactor types had significantly different bacterial communities except HT and VT• The inoculum source and physiochemical factors together affect bacterial community• The bacterial family Saprospiraceae is positively correlated to microalgal growth [ABSTRACT FROM AUTHOR]

Phytoplankton used in fish hatcheries is mass-cultured in the open air and usually contains large numbers of bacteria. In commercial fish production, the phytoplankton cultures are usually added into the larval rearing tanks; however, the numbers and types of bacteria introduced into the rearing tanks simultaneously are unknown. In this study, the bacterial community structures in Nannochloropsis sp. cultures were analyzed by using fluorescence in situ hybridization (FISH). A direct viable count (DVC)-FISH analysis was also performed as DVC is useful for the detection of actively growing cells. Total numbers of bacteria in Nannochloropsis sp. cultures ranged from 7.72 × 105−2.39 × 106 cells/mL. High proportions of the total bacteria (31.6–53.6%) in the Nannochloropsis sp. cultures showed growth potential. DVC-FISH analysis revealed that α-proteobacteria and the Cytophaga– Flavobacterium cluster were abundant in the bacterial community of actively growing cells. Thus, the high growth potentials of the distinct bacterial communities in Nannochloropsis sp. culture must influence the bacterial communities in larval rearing tanks. [ABSTRACT FROM AUTHOR]

Pigment composition and its variation with culture agewere analyzed in six strains of Nannochloropsis(Eustigmatophyceae). The capacity for accumulationof the ketocarotenoids astaxanthin and canthaxanthinwas higher in N. salina and N. gaditanathan in the other strains studied here. Theinfluence of salinity (15 to 100 practical units) onpigment production was studied in N. gaditana,where a defined pattern of variation could not befound apart from a notable increase in zeaxanthin at100‰. In cultures grown in a photobioreactor and athigh cell densities of about 109 cells mL-1,pigment production reached: 350 mg L-1 forchlorophyll a, 50 mg L-1 for violaxanthin,5 mg L-1 for canthaxanthin, 3 mg L-1 forastaxanthin. The highest contents of canthaxanthin andastaxanthin obtained in experiments with N.gaditana were 19.4 and 14.6 ng pigment (106cells)-1, respectively, which accounts for 0.7%dry weight. By means of xanthophyll cycle inductionthrough exposure of cells to high irradiance and at40 °C, conversion of violaxanthin intozeaxanthin may attain up to 70% of the violaxanthincontent, which corresponds to 0.6% dry weight. Theresults indicate that interest in Nannochloropsis as a source of valuable pigments isnot related to its capacity for single pigmentaccumulation, but the availability of a range ofpigments such as chlorophyll a, zeaxanthin,canthaxanthin and astaxanthin, each with highproduction levels. [ABSTRACT FROM AUTHOR]

Inorganic carbon (Ci) uptake and efflux has been investigated in the marine microalga Nannochloropsis gaditana Lubian by monitoring CO2 fluxes in cell suspensions using mass spectrometry. Addition of H13CO3- to cell suspensions in the dark caused a transient increase in the CO2 concentration in the medium far in excess of the equilibrium CO2 concentration. The magnitude of this release was dependent on the length of time the cells had been kept in the dark. Once equilibrium between the Ci species had been achieved, a CO2 efflux was observed after saturating light intensity was applied to the cells. External carbonic anhydrase (CA) was not detected nor does this species demonstrate a capacity to take up CO2 by active transport. Photosynthetic O2 evolution and the release CO2 in the dark depend on HCO3- uptake since both were inhibited by the anion exchange inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). The bicarbonate uptake mechanism requires light but can also continue for short periods in the dark. Ethoxyzolamide, a CA inhibitor, markedly inhibited CO2 efflux in the dark, indicating that CO2 efflux was dependent upon the intracellular dehydration of HCO3-. These results indicate that Nannochloropsis possesses a bicarbonate uptake system which causes the accumulation of high intracellular Ci levels and an internal CA which maintains the equilibrium between CO2 and HCO3- and thus causes a subsequent release of CO2 to the external medium. [ABSTRACT FROM AUTHOR]

The vitamin content in four Australian microalgae, a Nannochloropsis-like sp., Pavlova pinguis, Stichococcus sp. and Tetraselmis sp., were examined. These were grown under a 12:12 h light:dark regimen (100 μmol photon m−2s−1) and harvested during late-logarithmic phase. Typically, the content showed a two- to three fold range between the species. When expressed on a dry weight basis, the content of ascorbate ranged from 1.3 to 3.0 mg g−1, β-carotene from 0.37 to 1.05 mg g−1, α-tocopherol from 0.07 to 0.29 mg g−1, thiamine from 29 to 109 μg g−1, riboflavin from 25 to 50 μg g−1, total folates from 17 to 24 μg g−1, pyridoxine from 3.6 to 17 μg g−1, cobalamin from 1.70 to 1.95 μg g−1 and biotin from 1.1 to 1.9 μg g−1. Retinol was detected only in Tetraselmis sp. (2.2 μg g−1); any vitamins D2 or D3 were below the detection limit (≤0.45 μg g−1). Nannochloropsis sp. was also grown under a 24:0 h light:dark light cycle and harvested at stationary phase. The content of most vitamins in Nannochloropsis sp. cultures differed significantly, and the degree of variation was similar to that observed between the four species grown under 12:12 h light:dark regimen (100 μmol photon m−2s−1) and harvested during late-logarithmic phase. Thiamine content was also examined in six non-Australian strains commonly used in aquaculture, Chaetoceros muelleri, Thalassiosira pseudonana, Nannochloris atomus, Nannochloropsis oculata, Isochrysis sp. (T.ISO) and Pavlova lutheri. Values (average 61 μg g−1; range 40 to 82) were similar to those in the Australian strains (average 61 μg g−1; range 29 to 109) and increased during stationary phase (average 94 μg g−1; 38 to 131). Comparison of the data with the known nutritional requirements for marine fish species and prawns suggests that the microalgae should provide excess or adequate levels of the vitamins for aquaculture food chains. The data may be used to guide the content of vitamins included in micro-diets developed as replacements for live diets. [ABSTRACT FROM AUTHOR]

Inorganic-carbon transport was investigated in the eukaryotic marine microalgae Stichococcus minor, Nannochloropsis oculata and a Monallantus sp. Photosynthetic O evolution at constant inorganic-carbon concentration but varying pH showed that S. minor had a greater capacity for CO rather than HCOutilization but for N. oculata and Monallantus HCOwas the preferred source of inorganic carbon. All three microalgae had a low affinity for CO as shown by the measurement of inorganic-carbon-dependent photosynthetic O evolution at pH 5.0. At pH 8.3, where HCOis the predominant form of inorganic carbon, the concentration of inorganic carbon required for half-maximal rate of photosynthetic O evolution [ K (CO)] was 53 μM for Monallantus sp. and 125 μM for N. oculata, values compatible with HCOtransport. Neither extra- nor intracellular carbonic anhydrase was detected in these three microalgal species. It is concluded that these microalgae lack a specific transport system for CO but that HCOtransport occurs in N. oculata and Monallantus, and in the absence of intracellular carbonic anhydrase the conversion of HCOto CO may be facilitated by the internal pH of the cell. [ABSTRACT FROM AUTHOR]