High Lipid Accumulation Research Articles

247 EFFECTS OF SILDENAFIL OR SNAP SUPPLEMENTATION, OR BOTH, DURING IN VITRO MATURATION ON LIPID AND REACTIVE OXYGEN SPECIES ACCUMULATION IN BOVINE OOCYTES AND EMBRYOS

Previous studies have demonstrated that a nitric oxide (NO) donor (S-nitroso-N-acetylpenicillmaine, SNAP) and a phosphodiesterase inhibitor (Sildenafil, SILD) delay the meiotic resumption of oocytes removed from the follicular environment, and therefore could be used to improve the quality of in vitro-matured (IVM) oocytes. However, it has been reported that SILD-treated cells have increased lipid metabolism and that NO supplementation can modulate the oxidative stress. This study aims to determine the effects of SNAP or SILD supplementation, or both, during IVM on embryo developmental rates, on lipid accumulation of IVM oocytes and on reactive oxygen species (ROS) and lipid accumulation of embryos derived from IVM oocytes. Bovine oocytes were cultured in TCM199 containing 1.0 μg mL–1 of FSH, 50 μg mL–1 of hCG, 1.0 μg mL–1 of oestradiol, 0.2 mM pyruvate, 83.4 μg mL–1 of amikacin, 10% FBS (control group; GCONT), supplemented with 10 µM SILD (GSILD), 0.1 µM SNAP (GSNAP) or both (GS+S). After 24 h of IVM, matured oocytes were assessed for lipid quantification (approximately 49 per group) or used for in vitro embryo production (IVP; approximately 340 oocytes per group). For lipid quantification, denuded oocytes were fixed with 5% triton in 4% paraformaldehyde (PFA) for 30 min and stained with 1 ng mL–1 of Nile Red for 30 min. Embryo lipid analyses (approximately 55 per group) were performed as described for oocytes. For ROS assessment (approximately 58 per group), IVP embryos were stained with 10 µM of H2DFFDA for 1 h and fixed for 30 min in 4% PFA. Stained oocyte and embryo assessments were performed on epifluorescence microscopy, and captured images were analysed on ImageJ (NIH, Bethesda, MD, USA) to quantify the fluorescence intensity (f.i). Statistical analyses were performed with data from 3 replicates for oocytes and 4 for embryos: statistical differences were assessed for lipid and ROS quantity and development rates by split-plot ANOVA. Variables considered in the model were SNAP (presence/absence) and SILD (presence/absence). Means were compared by Student's t at P < 0.05. Regarding oocyte lipid accumulation, groups with SILD (GSILD and GS+S) presented higher lipid quantity (f.i: 52.11 and 47.24, respectively) compared with GCONT and GSNAP (f.i: 38.86 and 41.86, respectively). Supplementation during IVM did not affect development rates (cleavage of 88.1, 88.2, 88.8, and 89.5% and blastocyst rates of 41.2, 38.6, 40, and 41.2% for GCONT, GSNAP, GSILD, and GS+S, respectively). Regarding embryo lipid quantity, similar to oocyte results, SILD groups (GSILD and GS+S) presented higher lipid accumulation (f.i: 68.9 and 68.5, respectively) compared with GCONT (f.i: 55.8) and GSNAP (f.i: 58.9). Considering embryo ROS quantity, GCONT (f.i: 35.9) and GS+S (f.i: 34.2) had the highest levels; however, GS+S did not differ from GSNAP (f.i: 32.85), which was similar to GSILD (f.i: 30.4). In conclusion, SILD had a negative effect on lipid accumulation, which could be due to increased lipid synthesis without increasing lipid oxidation because no increase of embryo ROS levels was observed.

Nitrogen starvation for lipid accumulation in the microalga species Desmodesmus sp.

Recently, to obtain lipids from microalgae has been the object of extensive research, since it is viewed as a promising feedstock for biodiesel production, especially when compared with crops such as soybean and sunflower, in terms of theoretical performance. The reduction of nutrient availability in culture media, especially nitrogen, stresses the microorganisms and affects cell growth, thus inducing lipid accumulation. This is an interesting step in biodiesel feedstock obtention from microalgae and should be better understood. In this study, four levels of nitrogen concentration in the BG-11 culture medium were evaluated in the growth of the chlorophycean microalga Desmodesmus sp. Both cell growth and lipid content were monitored over 7 days of cultivation, which yielded a final cell density of 33 × 10(6) cells mL(-1) with an initial NaNO3 concentration of 750 mg L(-1) in the medium and a maximum lipid content of 23 % with total nitrogen starvation. It was observed that the microalgae presented high lipid accumulation in the fourth day of cultivation with nitrogen starvation, although with moderate cell growth.

Metabolism of n-C10:0 and n-C11:0 fatty acids by Trichoderma koningii, Penicillium janthinellum and their mixed culture: I. Biomass and CO2 production, and allocation of intracellular lipids

The capacity of two soil fungi, Trichoderma koningii and Penicillium janthinellum, to oxidize n-C10:0 and n-C11:0 fatty acids to CO2 and store intracellular lipids during growth is unknown. This article reports for the first time the metabolism of decanoic acid (DA, C10:0), undecanoic acid (UDA, n-C11:0), a mixture of the acids (UDA+DA) and a mixture of UDA+ potato dextrose broth (PDB) by T. koningii and P. janthinellum and their mixed culture. A control PDB complex substrate was used as a substrate control treatment. The fungal cultures were assayed for their capacity to: (1) oxidize n-C10:0 and n-C11:0 fatty acids to CO2 and (2) store lipids intracellularly during growth. On all four fatty acid substrates, the mixed T. koningii and P. janthinellum culture produced more biomass and CO2 than the individual fungal cultures. Per 150 mL culture, the mixed species culture grown on UDA+PDB and on PDB alone produced the most biomass (7,567 mg and 11,425 mg, respectively). When grown in DA, the mixed species culture produced the least amount of biomass (6,400 mg), a quantity that was lower than those obtained in UDA (7,550 mg) or UDA+DA (7,270 mg). Amounts of CO2 produced ranged from 210 mg under DA to 618 mg under PDB, and these amounts were highly correlated with biomass (r2 = 0.99). Fluorescence microscopy of stained lipids in the mixed fungal cell cultures growing during the exponential phase demonstrated larger fungal cells and higher accumulation of lipids in membranes and storage bodies than those observed during the lag and stationary phases. T. koningii and P. janthinellum grown on n-C10:0 and n-C11:0 fatty acids produced lower amounts of biomass and CO2, but stored higher amounts of intracellular lipids, than when grown on PDB alone.

Abstract 4114: Silibinin inhibits hypoxia-induced proliferation, angiogenesis and lipogenesis in prostate cancer cells both in vitro and in vivo

Abstract Hypoxia (low oxygen conditions) in prostate tumors is associated with aggressive phenotype and poor prognosis. Also, hypoxia is considered the first major challenge faced by growing mass of neoplastic cells, as cells in hypoxic areas are surrounded by metabolic waste, acidic pH and necrotic cells. To overcome these hostile conditions, tumor cells activate transcriptional machinery leading to neo-angiogenesis, altered metabolism and selection of resistant clones. This suggests that prostate cancer (PCa) growth and progression could be prevented via targeting hypoxia-induced signaling. Here, we analyzed silibinin effect on hypoxia-induced proliferation, angiogenesis and metabolic changes in human PCa cells both in vitro and in vivo. Silibinin inhibited LNCaP cells clone formation (exposed to 1% O2 for 48 hrs and then returned to normoxic culture conditions) by 90-100% at 25-50 µM doses. Under similar hypoxic conditions in 22Rv1 cells, silibinin inhibited the clone formation by 63-77% at 25-50 µM doses. Importantly, conditioned media collected from PCa cells under hypoxic conditions (1% O2 for 24 hrs) induced tube formation by human umbilical vein endothelial cells (HUVEC) on matrigel, whereas conditioned media from silibinin-treated hypoxic PCa cells or silibinin addition in hypoxic-conditioned media abrogated HUVEC tube formation. Interestingly, we observed higher lipid accumulation in LNCaP cells under hypoxia, which was decreased (∼70%) by silibinin. Molecular analyses showed that silibinin decreases hypoxia-induced HIF-1α expression without affecting HIF-1β in PCa cells. Furthermore, silibinin increased the level of FIH (factor inhibiting HIF) and prolyl-hydroxylase 2, involved in HIF-1α degradation. Also, silibinin decreased the level of phosphorylated mTOR and ERK1/2 in PCa cells under hypoxia, and strongly inhibited lipid synthesis via activating AMP-activated protein kinase and decreasing acetyl Co A carboxylase, fatty acid synthase, acetyl Co A synthetase, fatty-acyl Co A synthetase and lipin expression. In vivo, silibinin effect on hypoxia-induced proliferation, angiogenesis and metabolism was analyzed in 22Rv1 xenograft model. Mice with established 22Rv1 tumors were imaged at base-line as well as 6 and 15 days following silibinin administration (200 mg/kg body weight) by diffusion weighted (DWI), dynamic contrast enhanced (DCE)-MRI and 18FDG-PET. At the study end, endogenous tumor metabolites were assed by NMR and various biomarkers by IHC. In vivo results showed that silibinin feeding reduced the tumor volume (by 47%), inhibited HIF-1α signaling, decreased proliferation and angiogenesis (DCE-MRI), induced apoptotic death and modulated tumor metabolism (PET and NMR). Together, these findings further support silibinin usefulness in PCa prevention through inhibiting hypoxia-induced proliferation, angiogenesis and lipogenesis. Citation Format: Gagan Deep, Anand M. Ramteke, Dhanya K. Nambiar, Anil K. Jain, Natalie J. Serkova, Chapla Agarwal, Rajesh Agarwal. Silibinin inhibits hypoxia-induced proliferation, angiogenesis and lipogenesis in prostate cancer cells both in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4114. doi:10.1158/1538-7445.AM2014-4114

English

Developments in the Biofuel production has created more awareness in Research due to the rapid increase in oil price and growing environmental issues. Microalgae have gathered attention towards them in the Biofuel production. Transesterification process converts algal oil or lipids to fatty acids. Because of their high Biomass and Lipid accumulation, the microalgae suited for the production of bio oil in industrial level. The lipid content was 28.22±1.38%, carbohydrate 16.8% and protein 58.2% for Chlorella pyrenoidosa. The amount of oil extracted was found to be 25.8 ml for (Alternate Media) AM1 and 20.6 ml for AM2. The pH was found to be 7.8 for AM1 and 7.6 for AM2. In this study, scaling up of Chlorella pyrenoidosa in poultry waste water was done with fertilizers and analysingparameters for its growth and production of Bio oil from Chlorella pyrenoidosa has been examined from the scaling up in poultry waste water

<i>Olea europaea</i> Linn (Oleaceae) Fruit Pulp Extract Suppresses Sterol Regulatory Element-Binding Proteins-1c via AMP-Activated Protein Kinase Activation in Human Hepatic Cells

Purpose: To investigate the molecular mechanisms underlying the role of Olea europaea Linn (Oleaceae) fruit pulp extract (OPF) in the prevention of high glucose-induced lipid accumulation in human HepG2 hepatocytes.Methods: HepG2 cells were pretreated with various concentration of OPF (0, 10, 20, 40 and 80 µg/ml) and then treated with serum-free medium with normal glucose (5 mM) for 1 h, followed by exposure to high glucose (25 mM D-glucose) for 24 h. Cell viability and cytotoxicity parameters were measured using LDH and MTT assay while triglyceride and total cholesterol levels were evaluated using respective enzymatic reagent kits. Protein expressional levels were analyzed by Western blotting.Results: OPF did not influence the cell viability (up to 200 µg/ml) and showed any signs of cytotoxicity (up to 80 µg/ml). OPF significantly attenuated lipid accumulation in human HepG2 hepatocytes whenexposed to high glucose (25 mM D-glucose) in a dose-dependently (p < 0.05, 0.01 and 0.001 at 20, 40 and 80 µg/ml concentrations, respectively). Nile red staining showed that 10, 20, 40 and 80 µg/ml concentrations of OPF reduced lipid accumulation by 25, 33.7 (p< 0.05), 43.7 (p attenuated by OPF (p < 0.001 at 80 µg/ml). Further, OPF attenuated the expression of fatty acid synthase and sterol regulatory element-binding protein-1. Adenosine monophosphate-activated protein kinase was also activated by OPF treatment when exposed to high glucose (25 mM D-glucose) inhuman HepG2 hepatocytes.Conclusion: This study indicates that OPF has hypolipidemic effects by inhibiting lipid biosynthesis mediated via AMPK signaling. Thus, this extract can be potentially developed into an anti-obesity agent.Keywords: Olea europaea, Fruit pulp extract, Lipogenesis, AMPK SREBP-1, Anti-obesity; HepG2 cells

Systematically programmed adaptive evolution reveals potential role of carbon and nitrogen pathways during lipid accumulation in Chlamydomonas reinhardtii.

BackgroundThe concept of adaptive evolution implies underlying genetic mutations conferring a selective advantage to an organism under particular environmental conditions. Thus, a flow cytometry-based strategy was used to study the adaptive evolution in Chlamydomonas reinhardtii wild-type strain CC124 and starchless mutant sta6-1 cells, with respect to lipid metabolism under nitrogen-(N) depleted and -replete conditions.ResultsThe successive sorting and regeneration of the top 25,000 high-lipid content cells of CC124 and sta6-1, combined with nitrogen starvation, led to the generation of a new population with an improved lipid content when compared to the original populations (approximately 175% and 50% lipid increase in sta6-1 and CC124, respectively). During the adaptive evolution period, the major fatty acid components observed in cells were C16:0, C16:1, C18:0, and C18:1-3, and elemental analysis revealed that cellular carbon to nitrogen ratio increased at the end of adaptive evolution period In order to gain an insight into highly stimulated intracellular lipid accumulation in CC124 and sta6-1 resulting from the adaptive evolution, proteomics analyses of newly generated artificial high-lipid content populations were performed. Functional classifications showed the heightened regulation of the major chlorophyll enzymes, and the enzymes involved in carbon fixation and uptake, including chlorophyll-ab-binding proteins and Rubisco activase. The key control protein (periplasmic L-amino acid oxidase (LAO1)) of carbon-nitrogen integration was specifically overexpressed. Glutathione-S-transferases and esterase, the enzymes involved in lipid-metabolism and lipid-body associated proteins, were also induced during adaptive evolution.ConclusionsAdaptive evolution results demonstrate the potential role of photosynthesis in terms of carbon partitioning, flux, and fixation and carbon-nitrogen metabolism during lipid accumulation in microalgae. This strategy can be used as a new tool to develop C. reinhardtii strains and other microalgal strains with desired phenotypes such as high lipid accumulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0117-7) contains supplementary material, which is available to authorized users.

Life cycle assessment on microalgal biodiesel production using a hybrid cultivation system.

A life cycle assessment (LCA) was performed on a putative biodiesel production plant in which the freshwater alga Chlorella vulgaris, was grown using an existing system similar to a published commercial-scale hybrid cultivation. The hybrid system couples airlift tubular photobioreactors with raceway ponds in a two-stage process for high biomass growth and lipid accumulation. The results show that microalgal biodiesel production would have a significantly lower environmental impact than fossil-derived diesel. Based on the functional unit of 1 ton of biodiesel produced, the hybrid cultivation system and hypothetical downstream process (base case) would have 42% and 38% savings in global warming potential (GWP) and fossil-energy requirements (FER) when compared to fossil-derived diesel, respectively. Sensitivity analysis was performed to identify the most influential process parameters on the LCA results. The maximum reduction in GWP and FER was observed under mixotrophic growth conditions with savings of 76% and 75% when compared to conventional diesel, respectively.

Smoking and lipid‐related indices in patients with diabetes mellitus

Lipid-related indices, including the ratio of LDL cholesterol to HDL cholesterol, the ratio of triglycerides to HDL cholesterol and lipid accumulation product, are known to be good discriminators for cardiovascular disease. The aim of this study was to clarify the relationships between smoking and the lipid indices in patients with diabetes. Subjects were those who had been diagnosed as having diabetes mellitus at annual health check-ups at their places of work (n=2563). The subjects were divided into three groups of non-smokers, light smokers (≤20 cigarettes/day) and heavy smokers (>20 cigarettes/day). The relationships between smoking and the lipid indices were investigated. Both in all subjects and in the subjects without a habit of alcohol drinking, the LDL cholesterol:HDL cholesterol ratio and the log-transformed triglyerides:HDL cholesterol ratio tended to be higher with an increase in the amount of smoking, and the log-transformed lipid accumulation product was significantly higher in heavy smokers than in non-smokers. In the non-alcohol drinking subjects, the odds ratios of heavy smokers vs. non-smokers for high LDL cholesterol:HDL cholesterol ratio [2.32 (95%CI 1.40-3.84)], for high triglycerides:HDL cholesterol ratio [1.69 (95%CI 1.06-2.69)] and for high lipid accumulation product [1.65 (95%CI 1.02-2.67)] were significantly higher than a reference level of 1.00. The associations between smoking and the lipid indices were weaker in alcohol drinkers than in non-drinkers. In patients with diabetes, the levels of lipid-related indices were higher in smokers than in non-smokers, and cardiometabolic disorders, reflected by high lipid indices, are thought to be involved in the proneness of smokers to develop atherosclerotic cardiovascular disease.

Systematically programmed adaptive evolution reveals potential role of carbon and nitrogen pathways during lipid accumulation in

BackgroundThe concept of adaptive evolution implies underlying genetic mutations conferring a selective advantage to an organism under particular environmental conditions. Thus, a flow cytometry-based strategy was used to study the adaptive evolution in Chlamydomonas reinhardtii wild-type strain CC124 and starchless mutant sta6-1 cells, with respect to lipid metabolism under nitrogen-(N) depleted and -replete conditions.ResultsThe successive sorting and regeneration of the top 25,000 high-lipid content cells of CC124 and sta6-1, combined with nitrogen starvation, led to the generation of a new population with an improved lipid content when compared to the original populations (approximately 175% and 50% lipid increase in sta6-1 and CC124, respectively). During the adaptive evolution period, the major fatty acid components observed in cells were C16:0, C16:1, C18:0, and C18:1-3, and elemental analysis revealed that cellular carbon to nitrogen ratio increased at the end of adaptive evolution period In order to gain an insight into highly stimulated intracellular lipid accumulation in CC124 and sta6-1 resulting from the adaptive evolution, proteomics analyses of newly generated artificial high-lipid content populations were performed. Functional classifications showed the heightened regulation of the major chlorophyll enzymes, and the enzymes involved in carbon fixation and uptake, including chlorophyll-ab-binding proteins and Rubisco activase. The key control protein (periplasmic L-amino acid oxidase (LAO1)) of carbon-nitrogen integration was specifically overexpressed. Glutathione-S-transferases and esterase, the enzymes involved in lipid-metabolism and lipid-body associated proteins, were also induced during adaptive evolution.ConclusionsAdaptive evolution results demonstrate the potential role of photosynthesis in terms of carbon partitioning, flux, and fixation and carbon-nitrogen metabolism during lipid accumulation in microalgae. This strategy can be used as a new tool to develop C. reinhardtii strains and other microalgal strains with desired phenotypes such as high lipid accumulation.

Houttuynia cordata attenuates lipid accumulation via activation of AMP-activated protein kinase signaling pathway in HepG2 cells.

Houttuynia cordata (H. cordata) from the family Saururaceae is a perennial herb native to Southeast Asia. It possesses a range of medicinal properties to treat several disease symptoms including allergic inflammation and anaphylaxis. In the present investigation, we provided the molecular mechanisms underlying the role of H. cordata extract (HCE) in the prevention of high glucose-induced lipid accumulation in human HepG2 hepatocytes. HepG2 cells were pre-treated with various concentrations of HCE (0, 10, 20, 40, and 80 μg/mL) and treated with serum-free medium with normal glucose (5 mM) for 1 h, followed by exposure to high glucose (25 mM D-glucose) for 24 h. HCE significantly and dose-dependently attenuated lipid accumulation in human HepG2 hepatocytes when exposed to high glucose (25 mM D-glucose) (p < 0.05, p < 0.01 and p < 0.001 at 20, 40, and 80 μg/mL concentrations, respectively). Further, HCE attenuated the expression of fatty acid synthase (FAS), sterol regulatory element-binding protein-1 and glycerol 3-phosphate acyltransferases (GPATs). The adenosine monophosphate-activated protein kinase (AMPK) was also activated by HCE treatment when exposed to high glucose (25 mM D-glucose) in human HepG2 hepatocytes. This study suggests the hypolipidemic effects of HCE by the inhibition of lipid biosynthesis mediated through AMPK signaling, which may play an active role and can be developed as an anti-obesity agent.

Heterotrophic Microorganisms: A Promising Source for Biodiesel Production

The authors discuss occurrence of heterotrophic microbes, specifically bacteria, yeast, and molds, in biodiesel production is well discussed. Besides, these oleaginous microorganisms are reported to accumulate triacylglycerols (TAGs) that contain long-chain fatty acids. Most of the evidence suggests that TAGs accumulated are suitable for biodiesel production. Lipid accumulation (de novo or ex novo) required a set of enzymes and expression of various genes that are under complex regulatory system. The authors take a step in this direction and provides a summary of the actual knowledge on heterotrophic oleaginous species, their fatty acids, TAGs, and sterol biosynthesis with a biochemical and genetic approaches to better understand their regulation. Likewise, the impact of abiotic factors that can contribute to higher lipid accumulation is summarized. This review also highlights various issues concerning genetic engineering that may contribute to an advanced path forward for microbial feedstock-based biodiesel.

Comparative Proteomics Profile of Lipid-Cumulating Oleaginous Yeast: An iTRAQ-Coupled 2-D LC-MS/MS Analysis

Accumulation of intracellular lipid in oleaginous yeast cells has been studied for providing an alternative supply for energy, biofuel. Numerous studies have been conducted on increasing lipid content in oleaginous yeasts. However, few explore the mechanism of the high lipid accumulation ability of oleaginous yeast strains at the proteomics level. In this study, a time-course comparative proteomics analysis was introduced to compare the non-oleaginous yeast Saccharomyces cerevisiae, with two oleaginous yeast strains, Cryptococcus albidus and Rhodosporidium toruloides at different lipid accumulation stages. Two dimensional LC-MS/MS approach has been applied for protein profiling together with isobaric tag for relative and absolute quantitation (iTRAQ) labelling method. 132 proteins were identified when three yeast strains were all at early lipid accumulation stage; 122 and 116 proteins were found respectively within cells of three strains collected at middle and late lipid accumulation stages. Significantly up-regulation or down-regulation of proteins were experienced among comparison. Essential proteins correlated to lipid synthesis and regulation were detected. Our approach provides valuable indication and better understanding for lipid accumulation mechanism from proteomics level and would further contribute to genetic engineering of oleaginous yeasts.

Detection of intracellular neutral lipid content in the marine microalgae Prorocentrum micans and Phaeodactylum tricornutum using Nile red and BODIPY 505/515

Diatoms and dinoflagellates not only have extensive distribution and a huge biomass in marine ecosystems, but also have high lipid accumulation in nature or after physiological and genetic modification, which indicates that these organisms may be optimal candidate algal strains for biodiesel production. In this study, we determined the content of intracellular neutral lipids (triacylglycerol [TAG]) in the dinoflagellate Prorocentrum micans and in the diatom Phaeodactylum tricornutum using NR and BODIPY 505/515 staining. The freshwater green alga Scenedesmus obliquus was used as a control. Optimum concentrations of 1.000 and 1.500 μg mL−1 were determined for neutral lipid Nile red (NR) staining in P. micans and P. tricornutum. Unlike NR staining, the optimal concentrations of BODIPY 505/515 staining in P. micans and P. tricornutum were lower, at 0.100 and 0.075 μg mL−1, respectively. High correlation coefficients of R2 = 0.990 and R2 = 0.989 were obtained for P. micans and P. tricornutum intracellular neutral lipid content and the relative fluorescence intensity with NR staining, while the reference alga, S. obliquus, had a relatively low correlation coefficient of R2 = 0.908 when stained with NR. The neutral lipid content determined by thin-layer chromatography-flame ionization detector matched the analytical data from fluorescence measurements. These results indicated that NR and BODIPY 505/515 staining can be used as an excellent high-throughput approach to screen marine diatoms and dinoflagellates.

Bioethanol production from the nutrient stress-induced microalga Chlorella vulgaris by enzymatic hydrolysis and immobilized yeast fermentation

The microalga Chlorella vulgaris is a potential feedstock for bioenergy due to its rapid growth, carbon dioxide fixation efficiency, and high accumulation of lipids and carbohydrates. In particular, the carbohydrates in microalgae make them a candidate for bioethanol feedstock. In this study, nutrient stress cultivation was employed to enhance the carbohydrate content of C. vulgaris. Nitrogen limitation increased the carbohydrate content to 22.4% from the normal content of 16.0% on dry weight basis. In addition, several pretreatment methods and enzymes were investigated to increase saccharification yields. Bead-beating pretreatment increased hydrolysis by 25% compared with the processes lacking pretreatment. In the enzymatic hydrolysis process, the pectinase enzyme group was superior for releasing fermentable sugars from carbohydrates in microalgae. In particular, pectinase from Aspergillus aculeatus displayed a 79% saccharification yield after 72h at 50°C. Using continuous immobilized yeast fermentation, microalgal hydrolysate was converted into ethanol at a yield of 89%.

PH effects on growth and lipid accumulation of the biofuel microalgae Nannochloropsis salina and invading organisms

Biofuels derived from non-crop sources, such as microalgae, offer their own advantages and limitations. Despite high growth rates and lipid accumulation, microalgae cultivation still requires more energy than it produces. Furthermore, invading organisms can lower efficiency of algae production. Simple environmental changes might be able to increase algae productivity while minimizing undesired organisms like competitive algae or predatory algae grazers. Microalgae are susceptible to pH changes. In many production systems, pH is kept below 8 by CO2 addition. Here, we uncouple the effects of pH and CO2 input, by using chemical pH buffers and investigate how pH influences Nannochloropsis salina growth and lipid accumulation as well as invading organisms. We used a wide range of pH levels (5, 6, 7, 8, 9, and 10). N. salina showed highest growth rates at pH 8 and 9 (0.19 ± 0.008 and 0.19 ± 0.011, respectively; mean ± SD). Maximum cell densities in these treatments were reached around 21 days into the experiment (95.6 × 106 ± 9 × 106 cells mL−1 for pH 8 and 92.8 × 106 ± 24 × 106 cells mL−1 for pH 9). Lipid accumulation of unbuffered controls were 21.8 ± 5.8 % fatty acid methyl esters content by mass, and we were unable to trigger additional significant lipid accumulation by manipulating pH levels at the beginning of stationary phase. Ciliates (grazing predators) occurred in significant higher densities at pH 6 (56.9 ± 39.6 × 104 organisms mL−1) than higher pH treatments (0.1–6.8 × 104 organisms mL−1). Furthermore, the addition of buffers themselves seemed to negatively impact diatoms (algal competitors). They were more abundant in an unbuffered control (12.7 ± 5.1 × 104 organisms mL−1) than any of the pH treatments (3.6–4.7 × 104 organisms mL−1). In general, pH values of 8 to 9 might be most conducive to increasing algae production and minimizing invading organisms. CO2 addition seems more valuable to algae as an inorganic carbon source and not as an essential mechanism to reduce pH.

Nicotinamide, a glucose-6-phosphate dehydrogenase non-competitive mixed inhibitor, modifies redox balance and lipid accumulation in 3T3-L1 cells

AimsExcessive energy uptake of dietary carbohydrates results in their storage as fat and requires glucose-6-phosphate dehydrogenase (G6PD)-mediated NADPH production. We sought to assess whether the nicotinamide-induced reduction of G6PD activity might modulate redox balance and lipid accumulation in 3T3-L1 cells. Main methods3T3-L1 preadipocytes (days 4 and 6 of differentiation) and adipocytes were cultured in the presence of 5 or 25mM glucose. The cells cultured in 25mM glucose were supplemented with nicotinamide (5–15mM). Next, we evaluated the following parameters: cell viability, apoptosis, lipid accumulation, lipolysis, reducing power, reactive oxygen species (ROS), NAD(P)H and NAD(P)+, isocitrate dehydrogenase (IDP), malic enzyme and G6PD, as well as the protein and mRNA levels of G6PD. We also analysed the kinetics of the nicotinamide-induced inhibition of G6PD. Key findingsG6PD mRNA levels increased at day 4 of adipocyte differentiation, whereas G6PD activity progressively increased at days 4 and 6 of differentiation and was reduced in adipocytes. Concomitantly, ROS, reducing power and lipid accumulation increased gradually as the preadipocytes matured into adipocytes. High glucose increased the activity of G6PD, which coincided with an increase in ROS, reducing power and lipid accumulation. All of these changes are prevented by nicotinamide, with the exception of lipid accumulation in adipocytes. Nicotinamide increased IDP activity without affecting NADPH levels. Lastly, nicotinamide inhibited G6PD in a non-competitive mixed way. SignificanceNicotinamide modulates G6PD via a non-competitive mixed inhibition and decreases high glucose-dependent oxidative stress and lipid accumulation. Nicotinamide maintains NADPH levels by increasing the activity of IDP.

Carotenoid and Fatty Acid Compositions of an Indigenous Ettlia texensis Isolate (Chlorophyceae) Under Phototrophic and Mixotrophic Conditions

Ettlia oleoabundance (formerly known as Neochloris oleoabundance) is an attractive candidate for biodiesel production because of its high lipid accumulation, and it's taking the majority of the attention among the strains of Ettlia genus; however, potential of the other genus members is unknown. An indigenous strain from Salda Lake (South West Turkey) identified by 18S rDNA sequencing as Ettlia texensis (GenBank accession no: JQ038221), and its fatty acid and carotenoid compositions under phototrophic and mixotrophic conditions was investigated to evaluate the potential of the strain for commercial uses. A threefold increase was observed in total lipid content (total fatty acids; from 13% to 37%) in mixotrophic culture respect to the phototrophic growth conditions. The oleic acid (C18:1) and alpha-linolenic acid (18:3) were the major unsaturated fatty acids accounting for 40% and 13.2% of total fatty acids in mixotrophic culture, respectively. Carotenoid analyses of the mixotrophic culture revealed the metabolite canthaxanthin, a commercially valuable carotenoid used mainly for food coloring, was the major constituent among other pigments. The possible use of E. texensis in biotechnological applications is discussed.

An integrated microfluidic device for the high-throughput screening of microalgal cell culture conditions that induce high growth rate and lipid content

This study describes the development of a microfluidic device for the high-throughput screening of culture conditions, such as the optimum sodium acetate concentration for promoting rapid growth and high lipid accumulation of Chlamydomonas reinhardtii. An analysis of the microalgal growth on the microfluidic device revealed an optimum sodium acetate concentration of 5.72 g L(-1). The lipid content, determined by the 4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY® 505/515) staining method, increased with the sodium acetate concentration. The results were found to be statistically reproducible with respect to cell growth and lipid production. Other nutrient conditions, including the nitrogen and phosphorus concentrations, can also be optimized on the same microfluidic platform. The microfluidic device performance results agreed well with the results obtained from the flask-scale experiments, validating that the culture conditions were scalable. Finally, we, for the first time, established a method for the absolute quantification of the microalgal lipid content in the picoliter culture volumes by comparing the on-chip and off-chip data. In conclusion, we successfully demonstrated the high-throughput screening of sodium acetate concentrations that induced high growth rates and high lipid contents in C. reinhardtii cells on the microfluidic device.

Morin reduces hepatic inflammation-associated lipid accumulation in high fructose-fed rats via inhibiting sphingosine kinase 1/sphingosine 1-phosphate signaling pathway

SphK1/S1P signaling pathway is involved in the development of hepatic inflammation and injury. But its role in high fructose-induced NAFLD has not yet been reported. The aim of this study was to elucidate the crucial role of SphK1/S1P signaling pathway in high fructose-induced hepatic inflammation and lipid accumulation in rats. Moreover, the hepatoprotective effects of morin, a flavonoid with anti-inflammatory and anti-hyperlipedimic activities, on these hepatic changes in rats were investigated. High fructose-fed rats were orally treated with morin (30 and 60mg/kg) and pioglitazone (4mg/kg) for 8 weeks, respectively. Fructose feeding induced hyperlipidemia, and activated SphK1/S1P signaling pathway characterized by the elevation of SphK1 activity, S1P production as well as SphK1, S1PR1 and S1PR3 protein levels, which in turn caused NF-κB signaling activation to produce IL-1β, IL-6 and TNF-α and inflammation in the liver of rats. Subsequently, hepatic insulin and leptin signaling impairment and lipid metabolic disorder were observed in this animal model, resulting in liver lipid accumulation. Morin restored high fructose-induced the activation of hepatic SphK1/S1P signaling pathway in rats. Subsequently, the reduced NF-κB signaling activation by morin decreased inflammatory cytokine production, recovered insulin and leptin signaling impairment to reduce lipid accumulation and injury in the rat liver. These effects of morin were confirmed in Buffalo rat liver (BRL3A) cell model stimulated with 5mM fructose. Thus, the inhibition of hepatic SphK1/S1P signaling pathway may be a novel mechanism by which morin exerts hepatoprotection in high fructose-fed rats, possibly involving liver inflammation inhibition and lipid accumulation recovery.