Adarsha Gupta

Omega-3 biotechnology: Thraustochytrids as a novel source of omega-3 oils.

Thraustochytrids are large-celled marine heterokonts and classified as oleaginous microorganisms due to their production of docosahexaenoic (DHA) and eicosapentaenoic (EPA) ω-3-fatty acids. The applications of microbial DHA and EPA for human health are rapidly expanding, and a large number of clinical trials have been carried out to verify their efficacy. The development of refined isolation and identification techniques is important for the cultivation of thraustochytrids. With a high proportion of lipid biomass, thraustochytrids are also amenable to various production strategies which increase omega-3 oil output. Modifications to the existing lipid extraction methods and utilisation of sophisticated analytical instruments have increased extraction yields of DHA and EPA. Other metabolites such as enzymes, carotenoids and extracellular polysaccharides can also be obtained from these marine protists. Approaches such as the exploration for more diverse isolates having fast growth rates, metabolic engineering including gene cloning, and growing thraustochytrids on alternate low cost carbon source, will further enhance the biotechnological potential of thraustochytrids.

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.

Molecular identification of marine yeast and its spectroscopic analysis establishes unsaturated fatty acid accumulation

Marine microbes are competent organisms, some of which can accumulate large amounts of lipids. A yeast strain, Rhodotorula mucilaginosa AMCQ8A was isolated from the marine water of the Queenscliff region, Victoria, Australia. The yeast isolate was identified by sequencing 18s rDNA genes. Scanning electron microscopy images revealed scars on the surface of the yeast cells. The Fourier transform infrared spectroscopy microspectroscopy studies demonstrated the presence of unsaturated fatty acids by differential microscopic analysis. The sharp band at 1745 cm⁻¹ was represented by ν(C=O) stretches of ester functional groups from lipids and fats, and therefore indicated the presence of total lipids produced by the cells. Over 65% of the fatty acids from the yeast strain were analyzed as C₁₆ and C₁₈:₁ with omega-3 content from about 6% to 7%. Thus, this marine-derived yeast could be a potential source of lipids, including omega-3 fatty acids.

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.

FTIR microspectroscopy for rapid screening and monitoring of polyunsaturated fatty acid production in commercially valuable marine yeasts and protists

The increase in polyunsaturated fatty acid (PUFA) consumption has prompted research into alternative resources other than fish oil. In this study, a new approach based on focal-plane-array Fourier transform infrared (FPA-FTIR) microspectroscopy and multivariate data analysis was developed for the characterisation of some marine microorganisms. Cell and lipid compositions in lipid-rich marine yeasts collected from the Australian coast were characterised in comparison to a commercially available PUFA-producing marine fungoid protist, thraustochytrid. Multivariate classification methods provided good discriminative accuracy evidenced from (i) separation of the yeasts from thraustochytrids and distinct spectral clusters among the yeasts that conformed well to their biological identities, and (ii) correct classification of yeasts from a totally independent set using cross-validation testing. The findings further indicated additional capability of the developed FPA-FTIR methodology, when combined with partial least squares regression (PLSR) analysis, for rapid monitoring of lipid production in one of the yeasts during the growth period, which was achieved at a high accuracy compared to the results obtained from the traditional lipid analysis based on gas chromatography. The developed FTIR-based approach when coupled to programmable withdrawal devices and a cytocentrifugation module would have strong potential as a novel online monitoring technology suited for bioprocessing applications and large-scale production.

Omega-3 fatty acid production from enzyme saccharified hemp hydrolysate using a novel marine thraustochytrid strain.

In this work, a newly isolated marine thraustochytrid strain, Schizochytrium sp. DT3, was used for omega-3 fatty acid production by growing on lignocellulose biomass obtained from local hemp hurd (Cannabis sativa) biomass. Prior to enzymatic hydrolysis, hemp was pretreated with sodium hydroxide to open the biomass structure for the production of sugar hydrolysate. The thraustochytrid strain was able to grow on the sugar hydrolysate and accumulated polyunsaturated fatty acids (PUFAs). At the lowest carbon concentration of 2%, the PUFAs productivity was 71% in glucose and 59% in the sugars hydrolysate, as a percentage of total fatty acids. Saturated fatty acids (SFAs) levels were highest at about 49% of TFA using 6% glucose as the carbon source. SFAs of 41% were produced using 2% of SH. This study demonstrates that SH produced from lignocellulose biomass is a potentially useful carbon source for the production of omega-3 fatty acids in thraustochytrids, as demonstrated using the new strain, Schizochytrium sp. DT3.

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 sulfo-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.

Synchrotron-FTIR Microspectroscopy Enables the Distinction of Lipid Accumulation in Thraustochytrid Strains Through Analysis of Individual Live Cells

The superior characteristics of high photon flux and diffraction-limited spatial resolution achieved by synchrotron-FTIR microspectroscopy allowed molecular characterization of individual live thraustochytrids. Principal component analysis revealed distinct separation of the single live cell spectra into their corresponding strains, comprised of new Australasian thraustochytrids (AMCQS5-5 and S7) and standard cultures (AH-2 and S31). Unsupervised hierarchical cluster analysis (UHCA) indicated close similarities between S7 and AH-7 strains, with AMCQS5-5 being distinctly different. UHCA correlation conformed well to the fatty acid profiles, indicating the type of fatty acids as a critical factor in chemotaxonomic discrimination of these thraustochytrids and also revealing the distinctively high polyunsaturated fatty acid content as key identity of AMCQS5-5. Partial least squares discriminant analysis using cross-validation approach between two replicate datasets was demonstrated to be a powerful classification method leading to models of high robustness and 100% predictive accuracy for strain identification. The results emphasized the exceptional S-FTIR capability to perform real-time in vivo measurement of single live cells directly within their original medium, providing unique information on cell variability among the population of each isolate and evidence of spontaneous lipid peroxidation that could lead to deeper understanding of lipid production and oxidation in thraustochytrids for single-cell oil development.

Omega-3 Fatty Acids Produced from Microalgae

The applications of Omega-3 fatty acids for human health are rapidly expanding, which necessitates exploring alternative sources to fish. Many marine microorganisms across different kingdoms exhibit the ability to store a significant oil content, however are difficult to cultivate. Out of all marine microbes, thraustochytrids are considered a good source for the production of high value compounds such as polyunsaturated fatty acids (PUFA s). Optimization of culture conditions will be helpful in further enhancing cellular lipid content to suit fatty acid synthesis. This chapter describes some recent advances in the development of marine microbes for fatty acid production with a special emphasis upon thraustochytrids for biotechnological applications, focussing particularly on methods to enhanced docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA ) production.

Suitability of Novel Algal Biomass as Fish Feed: Accumulation and Distribution of Omega-3 Long-Chain Polyunsaturated Fatty Acid in Zebrafish

A 28-day feeding experiment with formulated feed using docosahexaenoic acid (DHA)-rich whole cells of freeze-dried marine microalgae Schizochytrium sp. to understand the distribution of fatty acids in a laboratory model zebrafish was conducted. Three feeds, commercial feed, 50:50 feed (50% commercial and 50% algae), and pure algae, were investigated. All feeds were consumed by zebrafish and showed optimal growth and weight gain with a survival rate of 100%. Lipids were extracted from four different tissues, brain, liver, muscle, and blood, to understand the distribution of fatty acids with respect to the feed. Maximum lipid was observed in zebrafish fed with 50:50 feed in all tissue samples. An increasing concentration of fatty acids was observed upon increasing the experimental time. Algae feed supported the DHA accumulation in all tissue samples compared to other feeds and resulted in an overall increment of polyunsaturated fatty acid content. To understand the role of fatty acids during zebrafish embryogenesis, eggs were collected at the end of the experiment and fatty acid content was analyzed. However, no significant difference was observed in fatty acid composition of embryos fed with algae. This provides a base for the understanding of fatty acid distribution in zebrafish with commercial and algae feeds and support the utilization of Schizochytrium biomass as a potential replacement for fishmeal.