Metabolic and morphological changes of an oil accumulating trebouxiophycean alga in nitrogen-deficient conditions

Oil-rich algae have promising potential for a next-generation biofuel feedstock. Pseudochoricystis ellipsoidea MBIC 11204, a novel unicellular green algal strain, accumulates a large amount of oil (lipids) in nitrogen-deficient (–N) conditions. Although the oil bodies are easily visualized by lipophilic staining in the cells, little is known about how oil bodies are metabolically synthesized. Clarifying the metabolic profiles in –N conditions is important to understand the physiological mechanisms of lipid accumulations and will be useful to optimize culture conditions efficiently produce industrial oil. Metabolome and lipidome profiles were obtained, respectively, using capillary electrophoresis- and liquid chromatography-mass spectrometry from P. ellipsoidea in both nitrogen-rich (+N; rapid growth) and –N conditions. Relative quantities of more than 300 metabolites were systematically compared between these two conditions. Amino acids in nitrogen assimilation and N-transporting metabolisms were decreased to 1/20 the amount, or less, in –N conditions. In lipid metabolism, the quantities of neutral lipids increased greatly in –N conditions; however, quantities of nearly all the other lipids either decreased or only changed slightly. The morphological changes in +N and –N conditions were also provided by microscopy, and we discuss their relationship to the metabolic changes. This is the first approach to understand the novel algal strain’s metabolism using a combination of wide-scale metabolome analysis and morphological analysis.     

Growth promotion of Chlorella ellipsoidea by co-inoculation with Brevundimonas sp. isolated from the microalga

Eight bacterial strains identified as P1, P2, Y1, Y2, W1, W2, G, and R were isolated from a long-term laboratory culture of the green alga Chlorella ellipsoidea. Although it is unknown how these bacterial strains have been maintained with the C. ellipsoidea culture, all appeared to promote the growth of C. ellipsoidea. Co-inoculation of each bacterial strain with C. ellipsoidea resulted in 0.5–3 times greater algal growth than that of C. ellipsoidea alone. The most effective bacterium (i.e., strain P1) was selected and further characterized. Biochemical analysis and transmission electron microscopy revealed that strain P1 is closely related to the genus Brevundimonas. Sequence analysis of the 16S rRNA of strain P1 showed 99.9 and 99.4% nucleotide sequence identity to that of B. nasdae and B. vesicularis, respectively. In addition to the growth promotion of C. ellipsoidea by strain P1, the growth of strain P1 was also significantly enhanced by co-culturing with C. ellipsoidea, indicating a symbiotic relationship between the bacterium and alga. Scanning electron microscopy showed the direct adhesion of strain P1 cells to the surface of C. ellipsoidea cells, as well as the development of abundant crinkles on the surface of co-cultured C. ellipsoidea cells. Handling editor: J. Padisak     

The role of diatom glucose-6-phosphate dehydrogenase on lipogenic NADPH supply in green microalgae through plastidial oxidative pentose phosphate pathway

Commercial production of biofuel from oleaginous microalgae is often impeded by their slow growth rate than other fast-growing algal species. A promising strategy is to genetically engineer the fast-growing algae to accumulate lipids by expressing key lipogenic genes from oleaginous microalgae. However, lacking of strong expression cassette to transform most of the algal species and potential metabolic target to engineer lipid metabolism has hindered its biotechnological applications. In this study, we engineered the oxidative pentose phosphate pathway (PPP) of green microalga Chlorella pyrenoidosa for lipid enhancement by expressing a glucose-6-phosphate dehydrogenase (G6PD) from oleaginous diatom Phaeodactylum tricornutum. Molecular characterization of transformed lines revealed that heterologous PtG6PD was transcribed and expressed successfully. Interestingly, subcellular localization analyses revealed that PtG6PD was targeted to chloroplasts of C. pyrenoidosa. PtG6PD expression remarkably elevated NADPH content and consequently enhanced the lipid content without affecting growth rate. Collectively, this report represents a promising candidate to engineer lipid biosynthesis in heterologous hosts with notable commercial significance, and it highlights the potential role of plastidial PPP in supplying lipogenic NADPH in microalgae.

     

Improvement of lipid production in the marine strains Alexandrium minutum and Heterosigma akashiwo by utilizing abiotic parameters

Two different strains of microalgae, one raphidophyte and one dinoflagellate, were tested under different abiotic conditions with the goal of enhancing lipid production. Whereas aeration was crucial for biomass production, nitrogen deficiency and temperature were found to be the main abiotic parameters inducing the high-level cellular accumulation of neutral lipids. Net neutral lipid production and especially triacylglycerol (TAG) per cell were higher in microalgae (>200% in Alexandrium minutum, and 30% in Heterosigma akashiwo) under treatment conditions (25°C; 330 μM NaNO₃) than under control conditions (20°C; 880 μM NaNO₃). For both algal species, oil production (free fatty acids plus TAG fraction) was also higher under treatment conditions (57 mg L⁻¹ in A. minutum and 323 mg L⁻¹ in H. akashiwo). Despite the increased production and accumulation of lipids in microalgae, the different conditions did not significantly change the fatty acids profiles of the species analyzed. These profiles consisted of saturated fatty acids (SAFA) and polyunsaturated fatty acids (PUFA) in significant proportions. However, during the stationary phase, the concentrations per cell of some PUFAs, especially arachidonic acid (C20:4n6), were higher in treated than in control algae. These results suggest that the adjustment of abiotic parameters is a suitable and one of the cheapest alternatives to obtain sufficient quantities of microalgal biomass, with high oil content and minimal changes in the fatty acid profile of the strains under consideration.     

Transformation of Lipid Bodies Related to Hydrocarbon Accumulation in a Green Alga,Botryococcus braunii (Race B)

The colonial microalga Botryococcus braunii accumulates large quantities of hydrocarbons mainly in the extracellular space; most other oleaginous microalgae store lipids in the cytoplasm. Botryococcus braunii is classified into three principal races (A, B, and L) based on the types of hydrocarbons. Race B has attracted the most attention as an alternative to petroleum by its higher hydrocarbon contents than the other races and its hydrocarbon components, botryococcenes and methylsqualenes, both can be readily converted into biofuels. We studied race B using fluorescence and electron microscopy, and clarify the stage when extracellular hydrocarbon accumulation occurs during the cell cycle, in a correlation with the behavior and structural changes of the lipid bodies and discussed development of the algal colony. New accumulation of lipids on the cell surface occurred after cell division in the basolateral region of daughter cells. While lipid bodies were observed throughout the cell cycle, their size and inclusions were dynamically changing. When cells began dividing, the lipid bodies increased in size and inclusions until the extracellular accumulation of lipids started. Most of the lipids disappeared from the cytoplasm concomitant with the extracellular accumulation, and then reformed. We therefore hypothesize that lipid bodies produced during the growth of B. braunii are related to lipid secretion. New lipids secreted at the cell surface formed layers of oil droplets, to a maximum depth of six layers, and fused to form flattened, continuous sheets. The sheets that combined a pair of daughter cells remained during successive cellular divisions and the colony increased in size with increasing number of cells.     

Yarrowia lipolytica as a model for bio-oil production

The yeast Yarrowia lipolytica has developed very efficient mechanisms for breaking down and using hydrophobic substrates. It is considered an oleaginous yeast, based on its ability to accumulate large amounts of lipids. Completion of the sequencing of the Y. lipolytica genome and the existence of suitable tools for genetic manipulation have made it possible to use the metabolic function of this species for biotechnological applications. In this review, we describe the coordinated pathways of lipid metabolism, storage and mobilization in this yeast, focusing in particular on the roles and regulation of the various enzymes and organelles involved in these processes. The physiological responses of Y. lipolytica to hydrophobic substrates include surface-mediated and direct interfacial transport processes, the production of biosurfactants, hydrophobization of the cytoplasmic membrane and the formation of protrusions. We also discuss culture conditions, including the mode of culture control and the culture medium, as these conditions can be modified to enhance the accumulation of lipids with a specific composition and to identify links between various biological processes occurring in the cells of this yeast. Examples are presented demonstrating the potential use of Y. lipolytica in fatty-acid bioconversion, substrate valorization and single-cell oil production. Finally, this review also discusses recent progress in our understanding of the metabolic fate of hydrophobic compounds within the cell: their terminal oxidation, further degradation or accumulation in the form of intracellular lipid bodies.     

PRODUCTION AND CELLULAR LOCALIZATION OF NEUTRAL LONG‐CHAIN LIPIDS IN THE HAPTOPHYTE ALGAE ISOCHRYSIS GALBANA AND EMILIANIA HUXLEYI1

Isochrysis galbana Parke, Emiliania huxleyi (Lohm.) Hay and Mohler, and some related prymnesiophyte algae produce as neutral lipids a set of polyunsaturated long‐chain (C_37–39) alkenones, alkenoates, and alkenes (PULCA). These biomarkers are widely used for paleothermometry, but the biosynthesis and cellular location of these unique lipids remain largely unknown. By staining with the fluorescent lipophilic dye Nile Red, we found that I. galbana and E. huxleyi, like many other algae, package their neutral lipid into cytoplasmic vesicles or lipid bodies. We found that these lipid bodies increase in abundance under nutrient limitation and disappear under prolonged darkness and show that this pattern correlates well with the concentration of PULCA as measured by TLC. In addition, we show that lipid vesicles purified by sucrose density gradient centrifugation consist predominantly of PULCA. We also found significant pools of neutral lipid associated with chloroplasts, and PULCA component profiles in lipid vesicles and chloroplasts are similar. Examination of cell ultrastructure shows conspicuous cytoplasmic and chloroplast lipid bodies, and we suggest that PULCA may be synthesized in chloroplasts and then exported to cytoplasmic lipid bodies for storage and eventual metabolism. Our results connect and extend prior observations of lipid bodies and membrane‐unbound PULCA in I. galbana and E. huxleyi, as well as the behavior of PULCA during nutrient and light stress.     

Fatty acid composition and lipid peroxidation of soft-shelled turtle, Pelodiscus sinensis, fed different dietary lipid sources

Juvenile soft-shelled turtles (Pelodiscus sinensis) were fed 7 diets containing 8% of lard, soybean oil, olive oil, menhaden fish oil, or mixtures of 1 to 1 ratio of fish oil and lard, soybean oil, olive oil for 10 weeks. Growth and muscle proximate compositions of the turtles were not affected by different dietary treatments (p>0.05). Fatty acid profiles in muscle polar lipids, muscle non-polar lipids, and liver polar lipids reflected the fatty acid composition of dietary lipid source. Turtles fed diets containing fish oil generally contained significantly higher (p<0.05) proportion of highly unsaturated fatty acids (HUFA) in both polar and non-polar lipids of muscle and polar fraction of liver lipids than those fed other oils. Non-polar fraction of liver lipids from all groups of turtles contained less than 1% of HUFA. All turtles contained relatively high proportions of oleic acid in their lipids regardless of the dietary lipid source. Further, lipid peroxidation in both muscle tissue and liver microsomes of turtles fed fish oil as the sole lipid source was greater (p<0.05) than those fed fish oil-free diets. Turtles fed olive oil as the sole lipid source had the lowest lipid peroxidation rate among all dietary groups. The results indicate that dietary n-3 HUFA may not be crucial for optimal growth of soft-shelled turtles although they may be used for metabolic purpose. Further, high level of dietary HUFA not only increases the HUFA content in turtle tissues, but also enhances the susceptibility of these tissues to lipid peroxidation.     

Evaluating the usability of 19 effluents for heterotrophic cultivation of microalgal consortia as biodiesel feedstock

A key reason inhibiting commercialization of algal oil as biodiesel feedstock, is cultivation cost. For this reason, the usability of 19 readily available industrial effluents (autoclaved and non-autoclaved) to support heterotrophic growth and lipid accumulation was evaluated using six mixed algal cultures. Autoclaved whey effluent was the best with 14.32 g biomass L^?1, 13.23% lipids, resulting in a lipid production of 1.91 g lipids L^?1. Biomass production and lipid accumulation were in many cases inverse, e.g. mixed algal culture termed TUT4 accumulating 84.25% lipids on autoclaved acid mine drainage, with very little biomass produced. Biomass production was dependent on the effluent type, whereas the lipid accumulation was influenced mostly by the specific mixed algal cultures. The fatty acid composition of the algal oil (fish cannery and whey effluents) showed high saturation, leading to acceptable cetane numbers, kinematic viscosity, good oxidative stability, but poor cold flow properties.     

Proteomic analysis provides new insights into the adaptive response of a dinoflagellate Prorocentrum donghaiense to changing ambient nitrogen

Nitrogen (N) is the major nutrient limiting phytoplankton growth and productivity over large ocean areas. Dinoflagellates are important primary producers and major causative agents of harmful algal blooms in the ocean. However, very little is known about their adaptive response to changing ambient N. Here, we compared the protein profiles of a marine dinoflagellate Prorocentrum donghaiense grown in inorganic N‐replete, N‐deplete and N‐resupplied conditions using 2‐D fluorescence differential gel electrophoresis. The results showed that cell density, chlorophyll a and particulate organic N contents presented low levels in N‐deplete cells, while particulate organic carbon content and glutamine synthetase (GS) activity maintained high levels. Comparison of the protein profiles of N‐replete, N‐deplete and N‐resupplied cells indicated that proteins involved in photosynthesis, carbon fixation, protein and lipid synthesis were down‐regulated, while proteins participating in N reallocation and transport activity were up‐regulated in N‐deplete cells. High expressions of GS and 60 kDa chaperonin as well as high GS activity in N‐deplete cells indicated their central role in N stress adaptation. Overall, in contrast with other photosynthetic eukaryotic algae, P. donghaiense possessed a specific ability to regulate intracellular carbon and N metabolism in response to extreme ambient N deficiency.     

Physiological and biochemical studies on Egyptian fresh water algae

Summary Comparative study of the different methods of extraction and estimation of the algal nitrogenous constituents mainly proteins of an Egyptian strain ofChlorella ellipsoidea had been carried out. This study also involves the assay of the different fractions of the soluble nitrogenous compounds.     

Bacterial influence on alkenones in live microalgae

The microalga Emiliania huxleyi produces alkenone lipids that are important proxies for estimating past sea surface temperatures. Field calibrations of this proxy are robust but highly variable results are obtained in culture. Here, we present results suggesting that algal‐bacterial interactions may be responsible for some of this variability. Co‐cultures of E. huxleyi and the bacterium Phaeobacter inhibens resulted in a 2.5‐fold decrease in algal alkenone‐containing lipid bodies. In addition levels of unsaturated alkenones increase in co‐cultures. These changes result in an increase in the reconstructed growth temperature of up to 2°C relative to axenic algal cultures.     

Betaine lipids in chlorarachniophytes

Evolving from the endosymbiosis of a green algal cell by a filose amoeba or amoeboflagellate, the chimearic chlorarachniophytes combine unique features retained from both of their ancestral units. They have preserved from the endosymbiont only the nucleomorph and chloroplast. Four strains from three genera of this algal class were studied to identify a set of non‐phosphorous‐containing polar lipids and their associated fatty acids using the techniques of positive‐ion electrospray ionization/mass spectrometry (ESI/MS) and electrospray ionization/mass spectrometry/mass spectrometry (ESI/MS/MS). Fourteen non‐phosphorous‐containing polar lipids, classified as betaine lipids were primarily identified as forms of diacylglyceryl‐N,N,N‐trimethylhomoserine (DGTS) and its structural isomer diaclyglycerylhydroxymethyl‐N,N,N‐trimethyl‐β‐alanine (DGTA). Though the number of forms of DGTA and DGTA were roughly equal, DGTS composed more of the polar lipid portion present in three of the strains tested, while the fourth, Lotharella globosa, was dominated by forms of DGTA. In addition, a lipid tentatively identified as diacylglycerylcarboxyhydroxymethylcholine (DGCC) was observed twice in minor amounts. The polar lipid‐associated fatty acids of the aforementioned algal strains generally included dodecanoic acid (12:0), tetradecanoic acid (14:0), hexadecanoic acid (16:0), octadecanoic acid (18:0), octadecenoic acid (18:1), and eicosapentaenoic acid [20:5(n‐3)]. The differences in betaine lipid content among the species studied may allow for further conclusions to be drawn regarding the taxonomy of chlorarachniophytes.     

Lipid metabolism and Drosophila sperm development

Lipids are essential membrane structural components and important signal carriers. The major enzymatic metabolisms of various lipids (phospholipid, sphingolipid, cholesterol) are well studied. The developmental function of lipid metabolism has remained, for the most part, elusive. With the help of new techniques and model organisms, the important roles of lipid metabolism in development just start to emerge. Drosophila spermatogenesis is an ideal system for in vivo studies of cytokinesis and membrane remodeling during development. The metabolic regulators of many lipids, including phosphatidylinositol (PI) lipids, fatty acids and cholesterol, are reported to play critical roles in various steps during Drosophila spermatogenesis. In this mini-review, we summarized recent findings supporting a tight link between lipids metabolism and Drosophila sperm development.     

Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production

Global petroleum reserves are shrinking at a fast pace, increasing the demand for alternate fuels. Microalgae have the ability to grow rapidly, and synthesize and accumulate large amounts (approximately 20-50% of dry weight) of neutral lipid stored in cytosolic lipid bodies. A successful and economically viable algae based biofuel industry mainly depends on the selection of appropriate algal strains. The main focus of bioprospecting for microalgae is to identify unique high lipid producing microalgae from different habitats. Indigenous species of microalgae with high lipid yields are especially valuable in the biofuel industry. Isolation, purification and identification of natural microalgal assemblages using conventional techniques is generally time consuming. However, the recent use of micromanipulation as a rapid isolating tool allows for a higher screening throughput. The appropriate media and growth conditions are also important for successful microalgal proliferation. Environmental parameters recorded at the sampling site are necessary to optimize in vitro growth. Identification of species generally requires a combination of morphological and genetic characterization. The selected microalgal strains are grown in upscale systems such as raceway ponds or photobireactors for biomass and lipid production. This paper reviews the recent methodologies adopted for site selection, sampling, strain selection and identification, optimization of cultural conditions for superior lipid yield for biofuel production. Energy generation routes of microalgal lipids and biomass are discussed in detail.     

Accumulation of Free Fatty Acids during Hardening of Chlorella ellipsoidea

Chlorella ellipsoidea Gerneck (IAM C-27) was synchronously grown, and cells at an intermediate stage in the ripening phase of the cell cycle were hardened at 3°C for 48 hours. A nonpolar lipid which increased greatly during hardening was analyzed by gas-liquid chromatography. Palmitic, oleic, linoleic, and linolenic acids were the main components of the lipid. Electron micrographs revealed the appearance of lipid bodies in hardened cells. When formation of free fatty acids and lipid bodies was inhibited with cycloheximide, oligomycin, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the development of a high level of hardiness was always inhibited. However, the converse results were not always realized. Cells hardened in the dark in the absence of glucose developed a measurable hardiness in spite of their failure to form free fatty acids. The appearance of lipid bodies was invariably accompanied by the formation of the fatty acids. In pulse-labeling with [¹⁴C]NaHCO₃ for 4 minutes at zero time and at the 12th hour of hardening, initial incorporation rates of ¹⁴C into total lipids of whole cells and the cellular membrane fraction were significantly higher than that into free fatty acids. These results suggest that, although fatty acids are inserted into membrane lipids during hardening, the accumulation of free fatty acids and the appearance of lipid bodies per se are not involved in the development of frost hardiness.     

Biotechnological conversion of waste cooking olive oil into lipid-rich biomass using Aspergillus and Penicillium strains

Aims: In this study, we have investigated the biochemical behaviour of Aspergillus sp. (five strains) and Penicillium expansum (one strain) fungi cultivated on waste cooking olive oil. The production of lipid‐rich biomass was the main target of the work. In parallel, the biosynthesis of other extracellular metabolites (organic acids) and enzyme (lipase) and the substrate fatty acid specificity of the strains were studied. Methods and Results: Carbon‐limited cultures were performed on waste oil, added in the growth medium at 15 g l^?1, and high biomass quantities were produced (up to c. 18 g l^?1, conversion yield of c. 1·0 g of dry biomass formed per g of fat consumed or higher). Cellular lipids were accumulated in notable quantities in almost all cultures. Aspergillus sp. ATHUM 3482 accumulated lipid up to 64·0% (w/w) in dry fungal mass. In parallel, extracellular lipase activity was quantified, and it was revealed to be strain and fermentation time dependent, with a maximum quantity of 645 U ml^?1 being obtained by Aspergillus niger NRRL 363. Storage lipid content significantly decreased at the stationary growth phase. Some differences in the fatty acid composition of both cellular and residual lipids when compared with the initial substrate fat used were observed; in various cases, cellular lipids more saturated and enriched with arachidic acid were produced. Aspergillus strains produced oxalic acid up to 5·0 g l^?1. Conclusions: Aspergillus and Penicillium strains are able to convert waste cooking olive oil into high‐added‐value products. Significance and Impact of the Study: Increasing fatty wastes amounts are annually produced. The current study provided an alternative way of biovalourization of these materials, by using them as substrates, to produce added‐value compounds.     

La flora e le associazioni algali della Sorgente di Salomone (Anterselva,Bolzano). Facies estiva

Abstract

The flora and algal associations of Salomon's spring (Anterselva, Bolzano). Summer facies. – Salomon's spring is situated in Anterselva valley (Bolzano), a characteristic valley of a glacial escavation, having a typical profile formed like a « U » and a wide peat-bog on the bottom. The source springs at 1100 mts above the sea-level from a zone of waste-layer formed by fragments of metamorphic rocks (ortogneiss). Then it gives birth to a small pool from which the water flows very slowly forming a little stream. The spring has a plentiful algal flora rich of species (66, including the varieties too) belonging to the following classes: Cyanophyceae (12), Xanthophyceae (4), Bacillariophyceae (38), Euchlorophyceae (8), Ulothricophyceae (3), Zygophyceae (1).

In the spring are described the following algal associations:

–Diatometum hiemalis, that forms many brown-reddish little flocks on the borders and in the anfractures of the big stones that occupy the source;

–Diatometum hiemalis subass. ceratoneietosum, formed by gelatinous little masses amoung the stalks of some phanerogams forming floating clods at the surface of the spring;

–Melosiretum italicae, the most developed association of the spring, looking like wide islets floating on the surface and filamentous drapings fixed at the stones;

–Homoeothriceto-Chamaesiphonetum, which forms wide blue-coloured casings on many submerged stones;

–Phormidietum membranaceum (Fjerdingstad, 1950, Douglas, 1958; Phormidium community, Fritsch, 1929; Fjerdingstad, 1964), that forms many thick mucous blue-green coloured coats on some of the submerged stones.