Lipid Extraction Step Research Articles

A Lipidomic Approach to Identify Potential Biomarkers in Exosomes From Melanoma Cells With Different Metastatic Potential.

Melanoma, one of the most lethal cutaneous cancers, is characterized by its ability to metastasize to other distant sites, such as the bone. Melanoma cells revealed a variable in vitro propensity to be attracted toward bone fragments, and melanoma-derived exosomes play a role in regulating the osteotropism of these cells. We have here investigated the lipid profiles of melanoma cell lines (LCP and SK-Mel28) characterized by different metastatic propensities to colonize the bone. We have purified exosomes from cell supernatants by ultracentrifugation, and their lipid composition has been compared to identify potential lipid biomarkers for different migration and invasiveness of melanoma cells. Matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) lipid analysis has been performed on very small amounts of intact parental cells and exosomes by skipping lipid extraction and separation steps. Statistical analysis has been applied to MALDI mass spectra in order to discover significant differences in lipid profiles. Our results clearly show more saturated and shorter fatty acid tails in poorly metastatic (LCP) cells compared with highly metastatic (SK-Mel28) cells, particularly for some species of phosphatidylinositol. Sphingomyelin, lysophosphatidylcholine, and phosphatidic acid were enriched in exosome membranes compared to parental cells. In addition, we have clearly detected a peculiar phospholipid bis(monoacylglycero)phosphate as a specific lipid marker of exosomes. MALDI-TOF/MS lipid profiles of exosomes derived from the poorly and highly metastatic cells were not significantly different.

A novel methodology for direct esterification of olives optimized through design of experiments

A novel methodology was proposed, and optimized using the Design Expert software, aiming to enable characterization of the fatty acid profile of olives whilst abolishing the lipid extraction step. Furthermore, the proposed method was proven more efficient whereas requiring less time, and sample and solvent amounts, consequently improving process yield. Optimum conditions obtained after experimental design were as follows: sonication temperature and time of 60 ºC and 8 minutes, respectively, and concentrations for alkaline and acid reactions of 0.70 and 1.5 mol L-1, respectively. Total fatty acid content for olive sample was 172.0 mg g-1, the predicted value was and is in the coefficient of variation range of 11.52%

Life cycle assessment of industrial production of microalgal oil from heterotrophic fermentation

Heterotrophic fermentation has been proposed as an economically promising way to grow microalgae, a feedstock to produce microbial oils, which can substitute for fossil fuels and crop oil products. This study conducted a life cycle assessment (LCA) to evaluate the environmental impacts of microalgal oil produced from two microalgae Chlorella sp. and Schizochytrium sp., which were cultivated in commercial fermenters with a maximal volume of 60 m3. Algal oil produced from Chlorella sp. in autotrophic raceway ponds was also included as a base case this LCA study. The LCA results showed the oil produced by heterotrophic Schizochytrium sp. had generally lower impacts than autotrophic Chlorella sp. whereas oil produced from heterotrophic Chlorella sp. only had lower impacts in acidification, eutrophication, carcinogens, and ecotoxicity when compared to the autotrophic Chlorella sp. An in-depth analysis showed the algal fermentation stage accounted for over 86% of the total impacts in all categories, and nutrients and electricity use in fermentation caused the major impacts. Due to its high biomass density and high oil content properties, the harvest stage with a series of dewatering and lipid extraction steps for heterotrophic algae was processed with fewer impacts than autotrophic algae. This paper also compared four sugar sources used to grow algae in fermenters and found out that the sorghum stem was the best nutrient feedstock for fermentation. Finally, the authors determined the best fermentation time as around 131 h in algae cultivation to obtain a relatively high oil yield while keeping low environmental impacts.

Direct transesterification of wet microalgae to biodiesel using phosphonium carboxylate ionic liquid catalysts

In this study, four types of tetrabutylphosphonium carboxylate ionic liquids (ILs) were synthesized and used for a one-pot transesterification of wet Chlorella vulgaris (C. vulgaris) microalgae into fatty acid methyl esters (FAME) in the presence of methanol, as well as refined oils (sunflower, canola, and corn oil). The resulting process removed the need for complete drying and lipid extraction steps typically needed for biodiesel production from microalgae. The leading candidate ionic liquid catalyst, tetrabutylphosphonium formate ([P4444][For]), was further optimized using response surface methodology to minimize material consumption, increase water compatibility, reduce processing time while maximizing yield from wet microalgae biomass. The optimized conditions resulted in a yield of 98.0 ± 2.48% using biomass containing 40 wt% water. Finally, the reusability of the ionic liquid was confirmed and [P4444][For] could be recycled with less than 2% loss between cycles. Due to the simple and low cost of synthesizing this IL, [P4444][For] is a promising catalyst for a one-pot transesterification process for biodiesel production from wet microalgae biomass.

Development of one-step sample preparation methods for fatty acid profiling of milk fat

Determination of the fatty acid (FA) profile of milk fat generally involves total lipid extraction from liquid milk, transesterification and GC analysis. The lipid extraction step is time consuming and often employs toxic solvents such as chloroform. Two alternative methods are presented here that skip the lipid extraction step and allow the determination of FA composition via direct transesterification of dried milk and liquid milk respectively. We have shown that dried milk can be used directly in alkaline-catalysed methylation, whereas direct transesterification of both dried milk and fresh milk is feasible with acidic methanol. Both methods generate similar results as compared to the classical two-step method (i.e. lipid extraction and FA methylation) when optimised methylation parameters (temperature, time, milk and reagent volume) are followed. By omitting the lipid extraction step, these simplified one-step methods offer a much higher throughput and a reduced cost in FA composition analysis of milk samples.

Engineering a malic enzyme to enhance lipid accumulation in Chlorella protothecoides and direct production of biodiesel from the microalgal biomass

Genetic engineering of the eukaryotic microalga Chlorella protothecoides remains in the early stages of development. There is also an urgent need to develop cost-effective biodiesel production processes from wet and lipid-rich C. protothecoides. Malic enzyme is a potential key enzyme responsible for a rate-limiting step associated with lipid accumulation in some microorganisms. In this study, we genetically engineered C. protothecoides by overexpressing a key malic enzyme to improve lipid accumulation for direct biodiesel production from microalgal biomass. An open reading frame encoding 509 amino acid residues of a putative malic enzyme was obtained and identified. Cloning and overexpression of the malic enzyme gene allowed genetic engineering of C. protothecoides for lipid accumulation and further direct production of biodiesel without the need for lipid extraction steps. The total lipid accumulation in genetically engineered C. protothecoides increased 2.8-fold compared to the wild type. A dehydration step using methanol pretreatment coupled with H2SO4 or lipase catalyzed biodiesel production enabled 93–98% biodiesel yields directly from microalgal biomass without extraction of lipid under optimal conditions. The efficient direct dehydration and chemical and enzymatic transformation processes could significantly reduce the overall cost of biodiesel production from wet and lipid-rich microalgal feedstocks.

A simplified protocol for fatty acid profiling of milk fat without lipid extraction

Determination of the fatty acid profile of milk fat generally involves a protocol that comprises total lipid extraction, transesterification and GC analysis. The lipid extraction step is time-consuming and often employs toxic solvents such as chloroform. A novel protocol is presented here that skips the lipid extraction step and allows the determination of fatty acid composition via direct methylation of milk fat isolated after centrifugation of raw milk. This new method is reliable for relative quantification of fatty acids in raw milk fat and offers a much higher throughput compared with the classical methods.

Process conditions for a rapid in situ transesterification for biodiesel production from oleaginous yeasts

Abstract Background Microbial oils produced by diverse microorganisms are being considered as alternative sources of triglycerides for biodiesel production. However, the standalone production of biodiesel from microorganisms is not currently economically feasible. In case of yeasts, the use of low-value nutrient sources in microbial production and the implementation of cost-efficient downstream processes could reduce costs and make microbial lipids competitive with other commodity-type oils in biodiesel production. Industrial biodiesel synthesis from oleaginous seeds is currently based on a multistep process. However, a simple process called in situ transesterification (ISTE), which takes place within the biomass without a previous lipid extraction step, is receiving increasing interest. In this work, the optimal conditions for an ISTE process to obtain biodiesel from previously selected oleaginous yeast (Rhodotorula graminis S1/S2) were defined using the response surface methodology (RSM). Results Using the RSM approach, the optimal conditions for the maximum yield with minimum reaction time included a methanol-to-biomass ratio of 60:1, 0.4 M H2SO4, and incubation at 70°C for 3 h. The optimized in situ process yield was significantly higher (123%) than that obtained with a two-step method in which fatty acids from saponifiable lipids were first extracted and then esterified with methanol. The composition of the fatty acid methyl ester mixture obtained from R. graminis S1/S2 by ISTE met Uruguayan standards for biodiesel. Conclusion The characteristics achieved by the optimized method make microbial oil a potential alternative for biodiesel production from yeast at an industrial scale. How to cite: Martinez-Silveira A, Villarreal R, Garmendia G, et al. Process conditions for a rapid in situ transesterification for biodiesel production from oleaginous yeasts. Electron J Biotechnol 2018;37. https://doi.org/10.1016/j.ejbt.2018.11.006 .

Statistical analysis and optimization of biodiesel production from waste coffee grounds by a two-step process

Biodiesel was produced using waste coffee grounds (WCGs) via a two-step process comprising lipid extraction and subsequent transesterification steps. Each step was statistically analyzed, and optimum conditions for each step were suggested. WCGs were found to have 16.4% lipid content with 1.9% free fatty acid (FFA) content. The liquid-solid ratio (LSR) significantly influenced lipid extraction from WCGs, while extraction time and temperature did not; 92.7% of lipid extraction efficiency was achieved at 13.7 mL-hexane/g-WCGs, 30 min of extraction time, and 25°C. Owing to the relatively low FFA content, an alkaline catalyst (NaOH) reaction was used that requires less amount of catalyst, methanol, and shorter reaction time compared to an acid catalyst reaction. Reaction time and temperature were the major factors affecting biodiesel conversion, and 94.0% of biodiesel conversion was obtained at optimum conditions for transesterification: 0.5% catalyst, 1.5 mL-methanol/g-lipid, 45°C, and 9 h of reaction time. With the use of statistical analysis tools, high lipid extraction efficiency and biodiesel conversion were achieved at relatively mild conditions, which would reduce biodiesel production cost substantially.

Rapid biodiesel synthesis from waste pepper seeds without lipid isolation step

In situ transformation of lipid in waste pepper seeds into biodiesel (i.e., fatty acid methyl esters: FAMEs) via thermally-induced transmethylation on silica was mainly investigated in this study. This study reported that waste pepper seeds contained 26.9wt% of lipid and that 94.1% of the total lipid in waste pepper seeds could be converted into biodiesel without lipid extraction step for only∼1min reaction time. This study also suggested that the optimal temperature for in situ transmethylation was identified as 390°C. Moreover, comparison of in situ process via the conventional transmethylation catalyzed by H2SO4 showed that the introduced biodiesel conversion in this study had a higher tolerance against impurities, thereby being technically feasible. The in situ biodiesel production from other oil-bearing food wastes can be studied.

Lipid profiling of parkin-mutant human skin fibroblasts.

Parkin mutations are a major cause of early-onset Parkinson's disease (PD). The impairment of protein quality control system together with defects in mitochondria and autophagy process are consequences of the lack of parkin, which leads to neurodegeneration. Little is known about the role of lipids in these alterations of cell functions. In the present study, parkin-mutant human skin primary fibroblasts have been considered as cellular model of PD to investigate on possible lipid alterations associated with the lack of parkin protein. Dermal fibroblasts were obtained from two unrelated PD patients with different parkin mutations and their lipid compositions were compared with that of two control fibroblasts. The lipid extracts of fibroblasts have been analyzed by combined matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) and thin-layer chromatography (TLC). In parallel, we have performed direct MALDI-TOF/MS lipid analyses of intact fibroblasts by skipping lipid extraction steps. Results show that the proportions of some phospholipids and glycosphingolipids were altered in the lipid profiles of parkin-mutant fibroblasts. The detected higher level of gangliosides, phosphatidylinositol, and phosphatidylserine could be linked to dysfunction of autophagy and mitochondrial turnover; in addition, the lysophosphatidylcholine increase could represent the marker of neuroinflammatory state, a well-known component of PD.

Biodiesel Conversion via Thermal Assisted in-Situ Transesterification of Bovine Fat Using Dimethyl Carbonate as an Acyl Acceptor

This study experimentally evidenced that bovine fat could be directly converted into fatty acid methyl esters (FAMEs) without lipid extraction step via thermally assisted in-situ transesterification on a porous material such as SiO2 since providing thermal energy from an external heating source drove pseudocatalytic mechanisms caused by mobility difference between lipid in bovine fat and acyl acceptor. In particular, this study employed dimethyl carbonate (DMC) as an acyl acceptor due to its nontoxicity and economic viability. In order to validate thermal assisted in-situ transesterification, thermal degradation of bovine fat was characterized, which revealed that thermal behavior of lipid in bovine fat was nearly identical to refined lipid. The results also evidenced that bovine fat contains 12.51 wt % impurities. Fatty acid profiles were identical under different transesterification conditions, which provide evidence that the thermal assisted in-situ transesterification should be technically feasible. I...

Effects of lipid extraction and ultrafiltration on stable carbon and nitrogen isotopic compositions of fish bone collagen.

Fish bone collagen isotopic measurements are increasingly important in palaeodietary and paleoenvironmental studies yet differences in the chemical and physical properties of fish relative to other vertebrate bones are rarely considered. Lipid content in fish bone, which can exceed 50%, may underlie the poor collagen integrity criteria typically observed in archaeological studies. We compare stable carbon and nitrogen isotopic and elemental compositions of bone collagen prepared using four different methods from a wide range of modern fish species to: (1) assess the extent to which lipid content influences bone collagen δ(13) C and δ(15) N values, and (2) evaluate the relative efficacy of chemical (2:1 chloroform/methanol) and physical (30 kDa ultrafilters) methods for removing lipids from bones. Lower δ(13) C values were observed when the lipid content exceeded 5% of the initial bone mass. The lipid content did not influence the δ(15) N values. 30 kDa ultrafiltration, a common pretreatment for purifying archaeological collagen, removed fewer lipids and was associated with reduced collagen yields (37% loss) as well as altered amino acid compositions. In contrast, collagen prepared using a 2:1 chloroform/methanol lipid extraction step resulted in significantly improved collagen yields, elemental compositions, and isotopic measurements relative to a control treatment. The chemical lipid extraction method (2:1 chloroform/methanol) performed significantly better than the physical lipid extraction method (30 kDa ultrafilters). Given the high quantities of lipids in fish bones we recommend the inclusion of a chemical lipid extraction step when isolating collagen from modern and archaeological fish bones. Copyright © 2016 John Wiley & Sons, Ltd.

Direct transesterification of wet microalgal biomass for preparation of biodiesel

Most conventional processes for algal biodiesel production involve separate lipid extraction steps or require usage of dry biomass that incurs extra cost and an energy intensive drying step. A novel process that involves dehydration of wet biomass via pretreatment with ethanol followed by direct in situ transesterification into biodiesel was investigated in this study. Under mild esterification at 80°C for 30min, pretreating the wet biomass twice with 3 volumes of ethanol resulted in a nearly four-fold increase of fatty acid ethyl ester (FAEE) yield from 3.04mg to 11.78mg, while increasing the ethanol from 1 volume to 10 volumes resulted in a six fold increase of yield from 3.18 to 18.29mg. The FAEE yield further increased when the esterification reaction was run at higher temperature and longer durations of up to 120°C for 2h. The overall positive impact of the pretreatment step on the final yield was far greater for milder reaction conditions, which makes the process more attractive in terms of economics and energy savings. In addition, it was found that the yield is unaffected by the choice of alcohol, which means methanol and butanol can also be used for the process. Lastly, it was found that the low concentration of water in the FAEE containing spent ethanol meant that both the solvent and sulfuric acid could be reused to further concentrate the quantity of FAEE in the final product mixture.

Lipid extraction and esterification for microalgae-based biodiesel production using pyrite (FeS2)

In this study, pyrite (FeS2) was used for lipid extraction as well as esterification processes for microalgae-based biodiesel production. An iron-mediated oxidation reaction, Fenton-like reaction, produced an expected degree of lipid extraction, but pyrite was less effective than FeCl3 commercial powder. That low efficiency was improved by using oxidized pyrite, which showed an equivalent lipid extraction efficiency to FeCl3, about 90%, when 20mM of catalyst was used. Oxidized pyrite was also employed in the esterification step, and converted free fatty acids to fatty acid methyl esters under acidic conditions; thus, the fatal problem of saponification during esterification with alkaline catalysts was avoided, and esterification efficiency over 90% was obtained. This study clearly showed that pyrite could be utilized as a cheap catalyst in the lipid extraction and esterification steps for microalgae-based biodiesel production.

Improving the FAME Yield of in Situ Transesterification from Microalgal Biomass through Particle Size Reduction and Cosolvent Incorporation

In situ transesterification of biomass has the potential to simplify and reduce the cost of biodiesel production from microalgae. This route has the advantage of reducing the number of unit operations, by avoiding the lipid extraction step, prior to transesterification reaction. Thus, the biomass is directly contacted with the alcohol and the catalyst required to convert lipids into biodiesel. In this study, essential process parameters for acid in situ transesterification of microalgae biomass to FAME (fatty acid methyl esters) production were evaluated. Parameters such as the particle size of dry microalgae conglomerates and cosolvent use in the reaction mixture were studied, besides of the temperature and catalyst dosage. An experimental Box–Behnken design was used for the optimization of the FAME yield. The best FAME yield obtained under optimized conditions was of 80% wt, using a microalgae particle size fraction of <150 μm, a catalyst dosage of 125% wt, and a reaction temperature of 67 °C and adding...

A high-throughput scintillation proximity assay for sphingosine-1-phosphate lyase.

The emergence of sphingosine-1-phosphate lyase (SPL) as a promising therapeutic target for inflammatory diseases has heightened interest in the identification of small molecules that modulate its activity. The enzymatic activity of SPL is typically measured using radiometric or fluorescence-based assays that require a lipid extraction step, or by direct quantitation of reaction products using mass spectrometry (MS). To facilitate testing large numbers of compounds to identify SPL modulators, we developed a robust scintillation proximity assay (SPA) that is compatible with high-throughput screening (HTS). This assay employs recombinant human full-length SPL in insect cell membrane preparations to catalyze the conversion of biotinylated aminosphingosine-1-[(33)P]phosphate (S1(33)P-biotin) to trans-2-hexadecenal-biotin and ethanolamine [(33)P]phosphate. To validate the SPA and confirm the fidelity of its measurement of SPL enzyme activity, we developed a Rapid-Fire MS method that quantitates nonradiolabeled S1P-biotin. In addition, we developed a simple, scalable method to produce S1(33)P-biotin in quantities sufficient for HTS. The optimized SPA screen in 384-well microplates produced a mean plate-wise Z'-statistic of 0.58 across approximately 3,000 plates and identified several distinct structural classes of SPL inhibitor. Among the inhibitors that the screen identified was one compound with an IC50 of 1.6 μM in the SPA that induced dose-dependent lymphopenia in mice.

Changes in fatty acid composition of Chlorella vulgaris by hypochlorous acid.

Hypochlorous acid treatment of a microalga, Chlorella vulgaris, was investigated to improve the quality of microalgal lipid and to obtain high biodiesel-conversion yield. Because chlorophyll deactivates the catalyst for biodiesel conversion, its removal in the lipid-extraction step enhances biodiesel productivity. When microalgae contacted the hypochlorous acid, chlorophyll was removed, and resultant changes in fatty acid composition of microalgal lipid were observed. The lipid-extraction yield after activated clay treatment was 32.7 mg lipid/g cell; after NaClO treatment at 0.8% available chlorine concentration, it was 95.2 mg lipid/g cell; and after NaCl electrolysis treatment at the 1 g/L cell concentration, it was 102.4 mg lipid/g cell. While the contents of all of the unsaturated fatty acids except oleic acid, in the microalgal lipid, decreased as the result of NaClO treatment, the contents of all of the unsaturated fatty acids including oleic acid decreased as the result of NaCl electrolysis treatment.

Aminoclay-conjugated TiO2 synthesis for simultaneous harvesting and wet-disruption of oleaginous Chlorella sp.

Abstract In microalgae-based biorefinement, the integration of harvesting and lipid extraction steps could reduce the costs of downstream processes. One potential of integrated methods, microalgae harvesting by aminoclay-conjugated TiO2 has been considered. Therein, aminoclay plays an efficient role in microalgae flocculation and direct cell disruption by TiO2 photocatalytic performance under UV-light irradiation. In the present study, two TiO2 photocatalysts, a commercial anatase/rutile bicrystalline (of ∼5 nm diameter) and anatase/brookite bicrystalline (of ∼3.5 nm diameter) by sol–gel reaction at room temperature, were distributed uniformly onto an aminoclay matrix by ultrasound-irradiated TiO2 particles, resulting in aminoclay-conjugated TiO2 composites. Within 10 min, the injection of aminoclay-conjugated TiO2 into the prepared 1.5 g/L-concentration microalgal feedstocks produced an ∼85% harvesting efficiency for oleaginous Chlorella sp. KR-1. Subsequently, the harvested wet-microalgae biomass was UV-irradiated at 365 nm for 3 h, thereby effecting the disruption of ∼95% of cells.

Industrial wastes as a promising renewable source for production of microbial lipid and direct transesterification of the lipid into biodiesel

Two strategies of converting industrial wastes to microbial lipid and direct transesterification of obtained lipid into biodiesel were attempted. Several oleaginous yeasts were cultivated on industrial wastes. The yeasts grew well on the wastes with low C/N ratio (i.e. serum latex) but accumulated high lipid content only when the wastes had a high C/N ratio (i.e. palm oil mill effluent and crude glycerol). The yeast lipids have similar fatty acid composition to that of plant oil indicating their potential use as biodiesel feedstocks. The combination of these wastes and two-phase cultivation for cell growth and lipid accumulation improved lipid productivity of the selected yeast. The direct transesterification process that eliminates cell drying and lipid extraction steps, gave comparable yield of biodiesel (fatty acid methyl ester >70% within 1h) to that of conventional method. These two successful strategies may contribute greatly to industrializing oil production from microbes and industrial wastes.