Nanobiotechnology as a novel paradigm for enzyme immobilisation and stabilisation with potential applications in biodiesel production

Nanobiotechnology is emerging as a new frontier of biotechnology. The potential applications of nanobiotechnology in bioenergy and biosensors have encouraged researchers in recent years to investigate new novel nanoscaffolds to build robust nanobiocatalyt

Exploring omega-3 fatty acids, enzymes and biodiesel producing thraustochytrids from Australian and Indian marine biodiversity

The marine environment harbours a vast diversity of microorganisms, many of which are unique, and have potential to produce commercially useful materials. Therefore, marine biodiversity from Australian and Indian habitat has been explored to produce novel bioactives, and enzymes. Among these, thraustochytrids collected from Indian habitats were shown to be rich in saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), together constituting 51-76 % of total fatty acids (TFA). Indian and Australian thraustochytrids occupy separate positions in the dendrogram, showing significant differences exist in the fatty acid profiles in these two sets of thraustochytrid strains. In general, Australian strains had a higher docosahexaenoic acid (DHA) content than Indian strains with DHA at 17-31 % of TFA. A range of enzyme activities were observed in the strains, with Australian strains showing overall higher levels of enzyme activity, with the exception of one Indian strain (DBTIOC-1). Comparative analysis of the fatty acid profile of 34 strains revealed that Indian thraustochytrids are more suitable for biodiesel production since these strains have higher fatty acids content for biodiesel (FAB, 76 %) production than Australian thraustochytrids, while the Australian strains are more suitable for omega-3 (40 %) production.

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%.

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.

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.

Evaluation of bread crumbs as a potential carbon source for the growth of Thraustochytrid species for oil and omega-3 production

The utilization of food waste by microorganisms to produce omega-3 fatty acids or biofuel is a potentially low cost method with positive environmental benefits. In the present study, the marine microorganisms Thraustochytrium sp. AH-2 and Schizochytrium sp. SR21 were used to evaluate the potential of breadcrumbs as an alternate carbon source for the production of lipids under static fermentation conditions. For the Thraustochytrium sp. AH-2, submerged liquid fermentation with 3% glucose produced 4.3 g/L of biomass and 44.16 mg/g of saturated fatty acids after seven days. Static fermentation with 0.5% and 1% breadcrumbs resulted in 2.5 and 4.7 g/L of biomass, and 42.4 and 33.6 mg/g of saturated fatty acids, respectively. Scanning electron microscopic (SEM) studies confirmed the growth of both strains on breadcrumbs. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy for both strains were consistent with the utilization of breadcrumbs for the production of unsaturated lipids, albeit at relatively low levels. The total lipid yield for static fermentation with bread crumbs was marginally lower than that of fermentation with glucose media, while the yield of unsaturated fatty acids was considerably lower, indicating that static fermentation may be more appropriate for the production of biodiesel than for the production of omega-3 rich oils in these strains.

Combination of calcium and magnesium ions prevents substrate inhibition and promotes biomass and lipid production in thraustochytrids under higher glycerol concentration

Effect of calcium and magnesium ions was studied in detail in batch mode in shake flask cultures of two fast growing strains of thraustochytrids (Aurantiochytrium sp. DBTIOC-18 and Schizochytrium sp. DBTIOC-1) for biomass and lipid production. These strains were previously isolated from Indian marine biodiversity. Screening of these two strains on different carbon and nitrogen sources revealed the suitability of glycerol over glucose and sodium nitrate over yeast extract for the cultivation of these strains. The presence of higher concentration of glycerol in the medium inhibited the glycerol utilization by the cell thus resulting in lower biomass and lipid production in both the strains. Supplementing media with calcium and magnesium ions promoted glycerol utilization thus resulted in a substantial rise in volumetric production of biomass (55.12 g L-1, 48.12 g L-1), fatty acid for biodiesel (27.14 g L-1, 22.15 g L-1) and docosahexaenoic acid (14.57 g L-1, 10.12 g L-1) with both strains Aurantiochytrium sp. DBTIOC-18 and Schizochytrium sp. DBTIOC-1, respectively. Growth profile study of these two strains showed further improvement in production of biomass, fatty acid for biodiesel and docosahexaenoic acid when cultures were extended up to 7 days. Finding of this work underlines the importance of calcium and magnesium salts in designing new fermentation strategies to prevent substrate inhibition and achieve high cell density culture under high nutrient concentration especially carbon sources.