Direct methods based on (1)H NMR spectroscopic techniques have been developed for the determination of neutral lipids (triglycerides and free fatty acids) and polar lipids (glyceroglycolipids/phospholipids) in the solvent extracts of oleaginous microalgal biomasses cultivated on a laboratory scale with two species in different media. The chemical shift assignments observed in the (1)H and (13)C NMR spectra corresponding to unsaturated (C18:N, N = 1-3, C20:3, C20:5, C22:6, epoxy) and saturated (C14-C18) fatty acid ester components in a complex matrix involving overlapped resonances have been unambiguously confirmed by the application of 2D NMR spectroscopy (total correlation spectroscopy and heteronuclear single quantum coherence-total correlation spectroscopy). The study of the effect of a polar lipid matrix on the determination of neutral lipids by an internal reference blending process by a systematic designed experimental protocol has provided absolute quantification. The fatty acid composition of algal extracts was found to be similar to that of vegetable oils containing saturated (C16-C18:0) and unsaturated (C18:N, N = 1-3, C20:N, N = 3-4, C22:6) fatty acids as confirmed by NMR spectroscopy and gas chromatography-mass spectrometry analyses. The NMR methods developed offer great potential for rapid screening of algal strains for generation of algal biomass with the desired lipid content, quality, and potential for biodiesel and value-added polyunsaturated fatty acids in view of the cost economics of the overall cost of generation of the biomass.

In the present investigation, the application of NMR spectroscopic techniques was extensively used with an objective to explore the biodiesel potential of biomass cultivated on a lab scale using strains of Chlorella vulgaris and Scenedesmus ecornis. The effect of variation in the composition of culturing medium on the neutral and polar lipids productivity, and fatty acid profile of solvent extracts of microalgae biomass was studied. Determination of unsaturated fatty acid composition (C18:N = 1-3, ω3 C20:5, ω3 C22:6), polyunsaturated fatty esters (PUFEs), saturated fatty acids (SFAs), unsaturated fatty acids (UFAs), free fatty acids (FFAs), and iodine value were achieved from a single (1)H NMR spectral analysis. The results were validated by (13)C NMR and GC-MS analyses. It was demonstrated that newly developed methods based on (1)H and (13)C NMR techniques are direct, rapid, and convenient for monitoring the microalgae cultivation process for enhancement of lipid productivity and their quality aspects in the solvent extracts of microalgal biomasses without any sample treatment and prior separation compared to other methods. The fatty acid composition of algae extracts was found to be similar to vegetable and fish oils, mostly rich in C16:0, C18:N (N = 0 to 3), and n-3 omega polyunsaturated fatty acids (PUFAs). The lipid content, particularly neutral lipids, as well as most of the quality parameters were found to be medium specific by both the strains. The newly developed methods based on NMR and ultrasonic procedure developed for efficient extraction of neutral lipids are cost economic and can be an effective aid for rapid screening of algae strains for modulation of lipid productivity with desired biodiesel quality and value-added products including fatty acid profile.

The use of lipids obtained from microalgae biomass has been described as a promising alternative for production of biodiesel to replace petro-diesel. It involves steps such as the cultivation of microalgae, biomass harvesting, extraction and transesterification of lipids. The purpose of the present study was to compare different methods of extracting total lipids. These methods were tested in biomass of Chlorella vulgaris with the solvents ethanol, hexane and a mixture of chloroform:methanol in ratios 1:2 and 2:1. The solvents were associated with other mechanisms of cell disruption such as use of a Potter homogenizer and ultrasound treatment. The percentage of triglycerides in the total lipids was determinated by the glycerol-3-phosphate oxidase-p-chlorophenol method (triglycerides monoreagent K117; Bioclin). Among the tested methods, the mixture of chloroform:methanol (2:1) assisted by ultrasound was most efficient, extracting an average of 19% of total lipids, of which 55% were triglycerides. The gas chromatographic analysis did not show differences in methyl ester profiles of oils extracted under the different methods.

Microalgal biofuel alternatives have been hindered by their cost and energy intensive production. In the microalgal harvesting process, the intermediate step of flocculation shows potential in drastically reducing the need for costly centrifugation processes. Moringa oleifera seeds, which have been used for water treatment due to their high flocculation potential, low cost and low toxicity, are presented in this paper as strong candidate for flocculating Chlorella vulgaris, a microalgae with high biodiesel production potential. Early results of our group showed a very high flocculation (around 85% of biomass recovery). The aim of this work was to investigate the influence of Moringa oleifera seed flour concentration, sedimentation time and pH on the flocculation efficiency. Cell suspensions treated with Moringa seed flour (1 g L -1 ) had their flocculation significantly increased with the rise of pH, reaching 89% of flocculation in 120 min at pH 9.2. Sedimentation time of 120 min and a concentration of 0.6 g L -1 proved to be ample for substantial flocculation efficiency. In spite of the need for more research to ensure the economic viability and sustainability of this process, these results corroborate Moringa oleifera seeds as a strong candidate as a bioflocculant for Chlorella vulgaris cells and indicate optimal pH range of its action.

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