NMR techniques for determination of lipid content in microalgal biomass and their use in monitoring the cultivation with biodiesel potential

Abstract

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.

comparison of oil extraction methods for algae by NMR and Chromatographic techniques

“…The composition and nature of these components including fatty acids are dependent upon the types of species/strains, culture media and feed used for their cultivation [1][2][3][4][5][6][7][8] . Stressed culture conditions such as nitrogen limitation or nutrient imbalance as well as variation in temperature, pH, light, carbon source and salinity, can be applied during cultivation period for stimulating increase in biomass productivity for high biodiesel potential [14] . Improvements in molecular biology through genetic modification methods have resulted in enhancement of lipid yield and bi-products as well as improvement in their quality [5,6] .…”

“…The type of photo bioreactor (open pond, closed or hybrid) used for the cultivation of algae also influence the yield and quality of algae biomass [1][2][3][4][5][6] . The major challenge faced by the technology developer involved in the production of biodiesel and by-products from mi-crobial biomass is the extraction of oil, particularly neutral lipids (TAG and FFA), as microbial oil extraction processes are energy extensive and expensive [14][15][16][17][18] . A successful and economically viable algae-based biofuels industry depends on the rapid screening of algae strains for lipid content (glycerides) and improved extraction methods [2,3] .…”

“…Instead, it uses low-wattage, frequency-tuned microwave/ultrasound bursts that break the rigid and complex algae cell walls. Quantum fracturing is then applied to the now precracked cells to complete the oil extraction with ease.Ultra-sonication and microwave-assisted extraction with appropriate solvents have many advantages such as reduced extraction time, reduced solvent consumption, and greater penetration of solvent into cellular materials causing high cell disruptive effects for quick release of oil [7,8,14,25] . Extractions using the subcritical or supercritical fluids (SFC), mainly supercritical CO 2 , are applied to extract lipids and are considered economically viable and green process as drying process is completely eliminated [23] .…”

“…Detailed chemical composition of algae biomass is essential to understand the chemistry of neutral and polar lipids to explore their biodiesel and high value product potential as well as help in the prediction of quality, yield and physio-chemical properties of the algae derived fuels. Therefore, analytical methods based on NMR [7,8,14,24,[29][30][31][32][33][34][35][36][37][38][39] , GC/GC-MS [7,8,40,41] , IR [24,42] and HPLC [43] techniques are used to characterize and determine the neutral lipids content and their fatty acids composition of the algal oils. The importance of solvent polarity in the extraction of lipids and their composition, fatty acid profile, and yield has been reported from critical analyses of extracted oils obtained by different solvents by GC-MS and gravimetric methods [21] .…”

“…These methods have multiple advantages such as direct determination without prior separation, requiring small amounts of sample (2 to 5 mg), being rapid and convenient, and having potential for online process monitoring of the production of biodiesel from algae as discussed in details in our earlier published work on algal oils from microalgae and vegetable oils [7,8,14,24,50] . The present research work is a continuation of our endeavor and passion for unraveling and understanding the chemistry and various analytical aspects of oils and fats from vegetable oils, and algal oils extracted from different species/strains of microalgae, which possess biodiesel potential as investigated by high field NMR spectroscopic and other analytical techniques [7,8,14,24,49,50] . Chlorella vulgaris, Spirulina and Scenedesmus dimorph and other potential species/strains have been widely investigated for their biodiesel, PUFA, and co-product potential in fresh and waste water cultivation [2,[6][7][8]14] .…”