Microwave-assisted Nile red method for in vivo quantification of neutral lipids in microalgae

In vivo determination of neutral lipids with Nile red fluorescence has been used as a rapid screening method for certain types of microalgae, but has been unsuccessful in others, particularly those with thick, rigid cell walls that prevent penetration of the fluorescence dye into the cell. To solve the problem, a microwave-assisted Nile red staining method for microalgal lipid determination was developed. In a two-step staining protocol, 50 and 60 s were selected as the optimal microwave times for the pretreatment and staining process, respectively. Moreover, several calibration methods for quantitative analysis of neutral lipids in microalgae were investigated and compared with conventional gravimetric methods. Factors that affected the in vivo quantification of cellular neutral lipids were also investigated. Application of the new method for detection and quantification of neutral lipids in a number of green microalgae was demonstrated.     

Methods of downstream processing for the production of biodiesel from microalgae

Despite receiving increasing attention during the last few decades, the production of microalgal biofuels is not yet sufficiently cost-effective to compete with that of petroleum-based conventional fuels. Among the steps required for the production of microalgal biofuels, the harvest of the microalgal biomass and the extraction of lipids from microalgae are two of the most expensive. In this review article, we surveyed a substantial amount of previous work in microalgal harvesting and lipid extraction to highlight recent progress in these areas. We also discuss new developments in the biodiesel conversion technology due to the importance of the connectivity of this step with the lipid extraction process. Furthermore, we propose possible future directions for technological or process improvements that will directly affect the final production costs of microalgal biomass-based biofuels.     

Comparative study of lipid extraction from microalgae by organic solvent and supercritical CO2

Pavlova sp. was employed to evaluate the efficiency of different lipid extraction methods. The microalgal crude lipids content determined using the mixed solvent with ultrasonic method was 44.7 wt.%. The triglyceride content obtained by the mixed solvent method was 15.6 wt.%. The extraction yield was the FAME yield divided by the maximum FAME (15.9 wt.%). The extraction yield was improved by cell disruption prior to extraction, and the highest triglyceride extraction yield of 98.7% was observed using the supercritical fluid extraction (SFE) method with bead-beating. The results indicate that the SFE method is effective and provides higher selectivity for triglyceride extraction though the total lipid extracted was less than that using solvent extraction.     

A rapid, small-scale procedure for the structural characterization of lipid A applied to Citrobacter and Bordetella strains: discovery of a new structural element

Endotoxins [lipopolysaccharides (LPSs)] are part of the outer cell membrane of Gram-negative bacteria. Their biological activities are associated mainly with the lipid component (lipid A) and even more specifically with discrete aspects of their fine structure. The need for a rapid and small-scale analysis of lipid A motivated us to develop a procedure that combines direct isolation of lipids A from bacterial cells with sequential release of their ester-linked fatty acids by a mild alkali treatment followed by MALDI-MS analysis. This method avoids the multiple-step LPS extraction procedure and lipid A isolation. The whole process can be performed in a working day and applied to lyophilized bacterial samples as small as 1 mg. We illustrate the method by applying it to the analysis of lipids A of three species of Citrobacter that were found to be identical. On the other hand, when applied to two batches of Bordetella bronchiseptica strain 4650, it highlighted the presence, in one of them, of hitherto unreported hexosamine residues substituting the lipid A phosphate groups, possibly a new camouflage opportunity to escape a host defense system.     

Fractionation and lipase-catalyzed conversion of microalgal lipids to biodiesel

This study was conducted to evaluate the lipid fractionation and purification procedures of lipase-catalyzed conversion of neutral lipids to microalgal biodiesel. Microalgae lipids were efficiently recovered and purified by a combined extraction method and crude lipid extracts were separated into neutral lipids, glycolipids, and phospholipids by solid-phase extraction. The high purity of the neutral lipids fraction was confirmed by its low concentration of phosphorous (< 2.0 ppm). Transesterification was catalyzed by immobilized Candida antarctica lipase for 72 h with stepwise addition of methanol. The reaction displayed Michaelis–Menten kinetics and produced high yields of microalgal biodiesel (91.2% in the case of Dunaliella salina) with a high content of unsaturated fatty acids (81.5%). Neutral lipids were converted to biodiesel by three-step transesterification, while the removal of polar lipids maintained the activity of the immobilized lipase by reducing both reaction mixture viscosity and contamination risk.     

Effect of microalgal diets and commercial wheatgerm flours on the lipid profile of Ruditapes decussatus spat

The influence of both the lipid composition of microalgal diets and commercial flours on the lipid classes and fatty acids of Ruditapes decussatus spat was studied. These aspects of the nutritional value of the diets were discussed in relation to the growth of the spat. Four diets were tested; Diet A, composed of 100% of the daily food ration of microalgae; Diet B, composed of 100% of wheatgerm; Diet C, composed of 50% of microalgae and 50% of wheatgerm; and Diet D, composed of 25% of microalgae and 75% of wheatgerm. The microalgal cells present a higher lipid content than that for wheatgerm. Tahitian Isochrysis cells have phospholipids and triacylglycerols as majority lipids, whereas in the wheatgerm particles, the lipids more abundant are triacylglycerols. Fatty acid content was higher in the microalgal cells than in the wheatgerm particles. The n-3 fatty acids were the most abundant acids in the microalgae, whereas the n-6 fatty acids were in the wheatgerm. The n-3 PUFA were not detected in wheatgerm. Phospholipids were the main lipids present in the clam spat, followed by triacylglycerols. Other lipid classes, detected in significantly lower amounts, included free fatty acids, sterols, and sterol ester + waxes. The composition of fatty acids in the spat was influenced by the fatty acid composition of the diet. Highest spat growth rates were observed with those diets that present a higher phospholipid/triacylglycerol relation. A negative correlation in the relation n-6/n-3 vs. growth has also been observed, with better growth rates in diets with a lower ratio. If the fatty acid 20:5n-3 and 22:6n-3 considered "essential" for marine animals were not present in the diet, they were not present in the spat either. Desaturation and elongation capabilities of R. decussatus spat were also discussed.     

Lipid content in 19 brackish and marine microalgae: influence of growth phase, salinity and temperature

Algal lipids provide essential fatty acids for higher trophic levels in the marine food web, and understanding the fatty acid composition in phytoplankton is critical for evaluating its value as a diet. Nineteen microalgal species, mainly originating from the Baltic Sea, covering major algal classes were grown in different growth conditions. Samples were taken during both the exponential and stationary growth phases and analysed regarding their fatty acid methyl esters and free fatty acids. Our results show that across all screened species, total fatty acids increased significantly from exponential to stationary growth phase. Furthermore, it was observed that warm-water species contained more lipids and differed in their lipid profile as compared with the cold-water species. Brackish water species also showed a slightly higher lipid content than the marine species, but their lipid profile was not significantly different. Plotting changes in lipids against changes in cell nitrogen revealed a significant dependency between decrease in cell nitrogen and increase in lipids across all tested species.     

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (ω-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.     

Extraction of oil from microalgae for biodiesel production: A review

The rapid increase of CO(2) concentration in the atmosphere combined with depleted supplies of fossil fuels has led to an increased commercial interest in renewable fuels. Due to their high biomass productivity, rapid lipid accumulation, and ability to survive in saline water, microalgae have been identified as promising feedstocks for industrial-scale production of carbon-neutral biodiesel. This study examines the principles involved in lipid extraction from microalgal cells, a crucial downstream processing step in the production of microalgal biodiesel. We analyze the different technological options currently available for laboratory-scale microalgal lipid extraction, with a primary focus on the prospect of organic solvent and supercritical fluid extraction. The study also provides an assessment of recent breakthroughs in this rapidly developing field and reports on the suitability of microalgal lipid compositions for biodiesel conversion.     

Rapid transesterification of micro-amount of lipids from microalgae via a micro-mixer reactor

Background

Fatty acid composition is an important physiological parameter of microalgae, which is taken as the third generation alternative resource of biodiesel. To boost microalgal research and applications, a convenient, rapid, and acid-catalyzed transesterification procedure that satisfies the demand for the analysis of the fatty acid composition of lipids with micro-scale samples in the high-throughput screening of microalgal strains is needed, along with the evaluation of the physiological status of microalgae in response to nutrient stress.

Results

The reaction conditions of transesterification via a micro-mixer reactor were optimized as follows: 90 °C reaction temperature, 20 min reaction time, 6:1 volume ratio of H₂SO₄-methanol to lipid-in-hexane, and a Y-type micro-mixer with a 20-m-long extended loop that has a 0.3 mm diameter. The minimum amount of sample was decreased to 30 µg lipids. The new approach was successfully applied to the fatty acid composition analysis of soybean oil and microalgal lipids. Definitely, it could be applied to acyl related oils from different sources.

Conclusion

Here, we have developed a simple and rapid method for the analysis of the fatty acid composition of lipids. The new method requires less than 20 min for transesterification and a minimum of only 30 µg lipid sample. Furthermore, a high-throughput process can be easily realized by numbering up the micro-mixer reactors. The micro-mixer reactor has great potential for applications not only in large-scale biodiesel production but also for the micro-scale analysis of microalgae fatty acid compositions.

     

微波法提取鼠尾藻多糖的工艺研究

研究采用了微波法提取鼠尾藻多糖,考查了微波辐照时间、微波功率、料液比、超声预处理时间四个因素的影响,并采用正交实验法确定微波提取的最佳工艺。结果表明微波法提取多糖产率为6.5%,其优化工艺条件为:微波辐照时间8 min,微波功率540 W,料液比1∶30,超声时间3 min。经红外光谱得知鼠尾藻多糖的主要组成单糖为吡喃糖。微波法提取鼠尾藻多糖可行,工艺简单,产率令人满意。     

Zero-waste biorefinery of oleaginous microalgae as promising sources of biofuels and biochemicals through direct transesterification and acid hydrolysis

Oleaginous microalgae are considered as promising sources of biofuels and biochemicals due to their high lipid content and other high-value components such as pigments, carbohydrate and protein. This study aimed to develop an efficient biorefinery process for utilizing all of the components in oleaginous microalgae. Acetone extraction was used to recover microalgal pigments prior to processes for the other products. Microalgal lipids were converted into biodiesel (fatty acid methyl ester, FAME) through a conventional two-step process of lipid extraction followed by transesterification, and alternatively a one-step direct transesterification. The comparable FAME yields from both methods indicate the effectiveness of direct transesterification. The operating parameters for direct transesterification were optimized through response surface methodology (RSM). The maximum FAME yield of 256 g/kg-biomass was achieved when using chloroform:methanol as co-solvents for extracting and reacting reagents at 1.35:1 volumetric ratio, 70 °C reaction temperature, and 120 min reaction time. The carbohydrate content in lipid-free microalgal biomass residues (LMBRs) was subsequently acid hydrolyzed into sugars under optimized conditions from RSM. The maximum sugar yield obtained was 44.8 g/kg-LMBRs and the protein residues were recovered after hydrolysis. This biorefinery process may contribute greatly to zero-waste industrialization of microalgae based biofuels and biochemicals.     

Optimization of Cell Disruption and Transesterification of Lipids from <Emphasis Type="Italic">Botryococcus braunii</Emphasis> LB572

Several methods including microwave, Frenchpress, autoclave, bead-beating, ultrasonication, and osmotic shock were compared to identify the most effective microalgal cell disruption method. Botryococcus braunii LB572 was cultured in 5 L flasks containing JM medium mixed with oceanic sediment extract for 13 days. Among the methods tested, enhanced lipid extraction was achieved through microwave treatment (2450MHz, 1250W at 150°C for 20 min). Oleic (C18:1), linolenic (C18:3), and palmitic acids (C16:0) were found to be the major fatty acids among the C14-C24 acids from extracted lipid. In addition, the optimal conditions of transesterification were as follows: 70 mL of methanol, 6 mL of sulfuric acid, 8 mL of chloroform, and boiling at 100°C for 30 min; 85.4% of C14-C24 FAME and 78.5% of C16-C18 FAME were esterified from transesterifiable lipids.     

Active extracellular substances of Bacillus thuringiensis ITRI‐G1 induce microalgae self‐disruption for microalgal biofuel

Microalgal cultures are a clean and sustainable means to use solar energy for CO₂ fixation and fuel production. Microalgae grow efficiently and are rich in oil, but recovering that oil is typically expensive and consumes much energy. Therefore, effective and low‐cost techniques for microalgal disruption and oil or lipid extraction are required by the algal biofuel industry. This study introduces a novel technique that uses active extracellular substances to induce microalgal cell disruption. A bacterium indigenous to Taiwan, Bacillus thuringiensis, was used to produce the active extracellular substances, which were volatile compounds with high thermal stability. Approximately 74% of fresh microalgal cells were disrupted after a 12‐h treatment with the active extracellular substances. Algal lipid extraction efficiency was improved and the oil extraction time was decreased by approximately 37.5% compared with the control treatment. The substances effectively disrupted fresh microalgal cells but not dehydrated microalgal cells. An analysis of microalgal DNA from fresh cells after disruption treatment demonstrated typical DNA laddering, indicating that disruption may have resulted from programmed cell death. This study revealed that biological treatments are environmentally friendly methods for increasing microalgal lipid extraction efficiency, and introduced a microalgal cell self‐disruption mechanism.     

低场核磁共振技术快速检测小球藻油脂含量及其在高通量选育中的应用

为了建立一个高效的高产油微藻诱变育种流程,微藻中油脂含量快速和准确的测定在其中具有重要作用。在本研究中,首先利用低场核磁共振技术,建立了直接检测干藻粉和培养液中小球藻油脂含量的方法,其信号强度与细胞中油脂含量存在特异的线性关系,干藻粉和藻液中油脂含量与信号值拟合的R2均高于0.99,说明该方法用于小球藻油脂含量的检测是准确和可行的。同时该方法与传统油脂测量方法相比,具有快速、简便和准确等优点。但其通量不及尼罗红染色法,因此,我们开发了将尼罗红染色法用于初筛,低场核磁共振技术用于复筛的新型高通量藻种复合筛选方法,并将此筛选方法应用于一种异养高产油原壳小球藻的诱变育种过程中。首先从3 098株诱变藻种中初筛得到108株具有较高油脂含量的藻株,然后利用低场核磁共振技术复筛得到9株高产油性能的藻株,其中一株甘油三酯含量超过20%,比原始藻株提高1倍,培养168 h后培养液油脂浓度达到5 g/L,证明此诱变育种流程不仅提高了筛选的效率还可靠且可行。     

Superoxide dismutase deficiency and the toxicity of the products of autooxidation of polyunsaturated fatty acids in yeast

Mutants of Saccharomyces cerevisiae, deficient in cytosolic superoxide dismutase and catalase activities were used to study the role of various oxygen species in the process of lipid peroxidation in yeast cells. Lipid peroxidation does not occur normally in yeast, because this organism is unable to form fatty acids with more than one double bond, whereas under physiological conditions, only fatty acids with at least two double bonds undergo this process. The fatty acid content of cellular lipids was modified by growing the cells in anoxia in the presence of oleic or linolenic acid. Toxic effects of oxygen were observed almost exclusively in those cells of yeast mutants deficient in superoxide dismutase, which contain linolenic acid in cellular lipids. Hypersensitivity of the mutant cells, however, results mainly from toxic effects of the products of autooxidation of extracellular fatty acids. These facts suggest that superoxide dismutases are in some way involved in preventing toxic effects of the products of lipid peroxidation and to some extent prevent the process of lipid peroxidation.     

Oil extraction from microalgae for biodiesel production

This study examines the performance of supercritical carbon dioxide (SCCO₂) extraction and hexane extraction of lipids from marine Chlorococcum sp. for lab-scale biodiesel production. Even though the strain of Chlorococcum sp. used in this study had a low maximum lipid yield (7.1 wt% to dry biomass), the extracted lipid displayed a suitable fatty acid profile for biodiesel [C18:1 (∼63 wt%), C16:0 (∼19 wt%), C18:2 (∼4 wt%), C16:1 (∼4 wt%), and C18:0 (∼3 wt%)]. For SCCO₂ extraction, decreasing temperature and increasing pressure resulted in increased lipid yields. The mass transfer coefficient (k) for lipid extraction under supercritical conditions was found to increase with fluid dielectric constant as well as fluid density. For hexane extraction, continuous operation with a Soxhlet apparatus and inclusion of isopropanol as a co-solvent enhanced lipid yields. Hexane extraction from either dried microalgal powder or wet microalgal paste obtained comparable lipid yields.     

Lipid Extraction Methods for Lipomyces starkeyi

Various lipid extraction methods were applied to Lipomyces starkeyi, freeze-dried, heat-dried and intact cells. It was found that the freeze-dried cells were usually more extractable than other types of cells. A high lipid recovery was obtained by a lytic enzyme (Corticium centrifugum) treatment, conc. HCI treatment and Pedersen’s method using chloroform-methanol (1: 1). The first two methods, however, hydrolyzed phospholipids and released free fatty acids during the extraction of lipids. From the results we have obtained, the best method for lipid extraction from L. starkeyi is that in which the freeze-dried cells are extracted by the Pedersen’s method. The results obtained from the application of these methods to a hydrocarbon-grown yeast are also described.     

Free and bound lipids of Neurospora crassa

Extraction of light and dark grown cells of Neurospora crassa with chloroform-methanol gave a free lipid extract in which the relative amounts and compositions of sterols, fatty acid and carotenoid fractions were determined. Further extraction of the cells with methanolic potassium hydroxide solution liberated a bound lipid fraction from the cells. The levels of the bound lipid fraction were much lower than those of the free lipids but analysis showed that the composition was similar to that of the free lipids.     

Replacement of retinyl esters by polyunsaturated triacylglycerol species in lipid droplets of hepatic stellate cells during activation

Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed to obtain more insight in the dynamics and mechanism of LD loss. We have investigated the LD degradation processes in rat hepatic stellate cells in vitro with a combined approach of confocal Raman microspectroscopy and mass spectrometric analysis of lipids (lipidomics). Upon activation of the hepatic stellate cells, LDs reduce in size, but increase in number during the first 7 days, but the total volume of neutral lipids did not decrease. The LDs also migrate to cellular extensions in the first 7 days, before they disappear. In individual hepatic stellate cells. all LDs have a similar Raman spectrum, suggesting a similar lipid profile. However, Raman studies also showed that the retinyl esters are degraded more rapidly than the triacylglycerols upon activation. Lipidomic analyses confirmed that after 7 days in culture hepatic stellate cells have lost most of their retinyl esters, but not their triacylglycerols and cholesterol esters. Furthermore, we specifically observed a large increase in triacylglycerol-species containing polyunsaturated fatty acids, partly caused by an enhanced incorporation of exogenous arachidonic acid. These results reveal that lipid droplet degradation in activated hepatic stellate cells is a highly dynamic and regulated process. The rapid replacement of retinyl esters by polyunsaturated fatty acids in LDs suggests a role for both lipids or their derivatives like eicosanoids during hepatic stellate cell activation.