Comparison of cell disruption methods for improving lipid extraction from thraustochytrid strains

Lipid extraction is an integral part of biodiesel production, as it facilitates the release of fatty acids from algal cells. To utilise thraustochytrids as a potential source for lipid production. We evaluated the extraction efficiency of various solvents and solvent combinations for lipid extraction from Schizochytrium sp. S31 and Thraustochytrium sp. AMCQS5-5. The maximum lipid extraction yield was 22% using a chloroform:methanol ratio of 2:1. We compared various cell disruption methods to improve lipid extraction yields, including grinding with liquid nitrogen, bead vortexing, osmotic shock, water bath, sonication and shake mill. The highest lipid extraction yields were obtained using osmotic shock and 48.7% from Schizochytrium sp. S31 and 29.1% from Thraustochytrium sp. AMCQS5-5. Saturated and monounsaturated fatty acid contents were more than 60% in Schizochytrium sp. S31 which suggests their suitability for biodiesel production

Selective enrichment of omega-3 fatty acids in oils by phospholipase A1

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. 13C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids

Understanding the role of cell disruption methods in extracting lipids

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Bead milling for lipid recovery from thraustochytrid cells and selective hydrolysis of Schizochytrium DT3 oil using lipase

Marine microalgae present a renewable alternative source for sustainable production of omega-3 fatty acids, as compared to conventional sources such as krill oil and fish oil. In this study, we optimised a method for lipid extraction from marine thraustochytrids using a bead mill and enzymatic concentration of omega-3 fatty acids from the thraustochytrid oil. The optimised lipid extraction conditions were, bead size 0.4-0.6μm, 4500rpm, 4min of processing time at 5g biomass concentration. The maximum lipid yield (% dry weight basis) achieved at optimum conditions were 40.5% for Schizochytrium sp. S31 (ATCC) and 49.4% for Schizochytrium sp. DT3 (in-house isolate). DT3 oil contained 39.8% docosahexaenoic acid (DHA) as a percentage of lipid, a higher DHA percentage than S31. Partial hydrolysis of DT3 oil using Candida rugosa lipase was performed to enrich omega-3 polyunsaturated fatty acids (PUFAs) in the glyceride portion. Total omega-3 fatty acid content was increased to 88.7%

A quick colorimetric method for total lipid quantification in microalgae

Discovering microalgae with high lipid productivity are among the key milestones for achieving sustainable biodiesel production. Current methods of lipid quantification are time intensive and costly. A rapid colorimetric method based on sulpho-phospho-vanillin (SPV) reaction was developed for the quantification of microbial lipids to facilitate screening for lipid producing microalgae. This method was successfully tested on marine thraustochytrid strains and vegetable oils. The colorimetric method results correlated well with gravimetric method estimates. The new method was less time consuming than gravimetric analysis and is quantitative for lipid determination, even in the presence of carbohydrates, proteins and glycerol

Percent hydrolysis of various fatty acids (from Fig 3) at 32% hydrolysis of anchovy oil by PLA1 (light) and BSL (dark).

<p>The relative proportions of fatty acids at other time points were also similar.</p

Percent accumulation of different fatty acid classes in triglyceride fraction after 30% hydrolysis of anchovy oil by PLA1 (dark) and BSL (light).

<p>A, Percent accumulation of each fatty acid is calculated as (^UHFA_x-^TotalFA_x)/^TotalFA_x) X 100). Each of the fatty acid fractions were calculated based on peak area in the NMR spectra. B, Percent accumulation data plotted with positional information.</p

Time course of hydrolysis of anchovy oil and fatty acid distribution in the hydrolysate.

<p>Anchovy oil was subjected to hydrolysis by PLA1 (A) and BSL (B). The reaction product at various times was subjected to methylation and GC analysis. Percent hydrolysis of each fatty acid was calculated based on its hydrolysis at t_x as a fraction of t_o. Saturated (□), Monounsaturated (●), EPA (○), DHA (▲), Total hydrolysis (■).</p

Positional distribution of various fatty acids in anchovy oil.

<p>Positional distribution of various fatty acids in anchovy oil.</p