Extraction and Characterization of Bioactive Compounds from Diverse Marine Microalgae and Their Potential Antioxidant Activities.

This study compared free and bound phenolic compounds in various marine microalgae species. It assessed total phenolic content (TPC), total flavonoid content (TFC) and total condensed tannin content (TCT) and their antioxidant capacities using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⋅+ ) radical cation-based assay and ferric ion reducing antioxidant power assay. Liquid chromatography-mass spectrometry (LC-MS) was also employed to characterize the phenolic profiling. Results showed that free phenolic compounds ranged from 1.83-6.45 mg GAE/g d. w., while bound phenolic compounds ranged from 4.03-26.03 mg GAE/g d. w., indicating significant differences. These variations were consistent across assays, highlining unique profiles in different species. A total 10 phenolics were found in these seven microalgae, including 1 phenolic acid, 6 flavonoids, 1 other polyphenol and 2 lignans. 4'-O-methyl-(-)-epigallocatechin 7-O-glucuronide and chrysoeriol 7-O-glucoside in microalgae were firstly reported in microalgal samples. These findings have implications for future applications in industries.

Bioaccessibility and bioactivities of phenolic compounds from microalgae during in vitro digestion and colonic fermentation.

Microalgae are a developing novel source of carbohydrates, phenolic compounds, carotenoids and proteins. In this study, in vitro digestion and colonic fermentation were conducted to examine the total phenolic content and potential antioxidant activity of four microalgal species (Chlorella sp., Spirulina sp., Dunaliella sp., and Isochrysis sp.). The bioaccessibility of targeted phenolic compounds and the short-chain fatty acid (SCFA) production were also estimated. Particularly, Spirulina sp. exhibited the highest total phenolic content (TPC) and free radical scavenging (2,2'-diphenyl-1-picrylhydrazyl, DPPH) capacity after gastrointestinal digestion of 7.93 mg gallic acid equivalents (GAE) per g and 2.35 mg Trolox equivalents (TE) per g. Meanwhile, it had the highest total flavonoid content (TFC) of 1.07 quercetin equivalents (QE) per g after 8 h of colonic fermentation. Dunaliella sp. and Isochrysis sp. showed comparable ferric reducing antioxidant power (FRAP) of 4.96 and 4.45 mg QE per g after 4 h of faecal reaction, respectively. p-hydroxybenzoic and caffeic acid almost completely decomposed after the intestine and fermented in the colon with the gut microflora. In Dunaliella sp. and Isochrysis sp., these phenolic acids were found in the colonic fermented residual, probably due to the presence of dietary fibre and the interactions with other components. All four species reached the highest values of SCFA production after 16 h, except Spirulina sp., which displayed the most increased total SCFA production after 8 h of fermentation. It is proposed that Spirulina sp. could be more beneficial to gut health.

Interplay between interfacial behaviour, cell structure and shear enables biphasic lipid extraction from whole diatom cells (Navicula sp.).

HYPOTHESIS: Bacillariophyceae (i.e., diatoms) are an important class of algae with potential use in the production of proteins and lipids including long-chain ω-3 polyunsaturated fatty acids. Biphasic extraction of microalgae lipids using water-immiscible solvents such as hexane, can avoid the excessive energy required to distil solvents from water, but generally requires energy-intensive rupture of the cells. The unique cell structure and surface chemistry of diatoms compared to other microalgae species might allow biphasic lipid extraction without prior cell rupture. EXPERIMENTS: The kinetics of biphasic lipid extraction from intact Navicula sp. cells was investigated during low-shear and high-shear mixing, and with prior or simultaneous application of ultrasound (20 kHz at 0.57 W/mL). Dynamic interfacial tension measurements and electron microscopic analysis were used to investigate lipid extraction in relation to interfacial behaviour and cell structure. RESULTS: High yields (>80%) of intracellular lipids were extracted from intact cells over the course of hours upon low-shear contacting with hexane. The cells associated with and stabilised the hexane-water interface, allowing hexane to infiltrate pores in the frustule component of the cell walls and access the intracellular lipids. It was shown that mucilaginous extracellular polymeric substances (EPS) bound to the cell walls acted as a barrier to solvent penetration into the cells. This EPS could be removed by prior ultrasonication. Biphasic extraction was greatly accelerated by shear applied by rotor-stator mixing or ultrasound. High-shear could remove mucilaginous EPS from the cell surfaces to facilitate direct contact of the cell surface with hexane and produced smaller emulsion droplets with increased surface area. The combination of high-shear in the presence of hexane resulted in the in-situ rupture of the cells, which greatly accelerated lipid extraction and allowed high yields of neutral lipid (>95%) to be recovered from freshly harvested cells within less than 5 min. The study demonstrated the ability of shear to enable simultaneous cell rupture and lipid extraction from a diatom alga based on its cell structure and interfacial behaviour.

Thermally coupled dark-anoxia incubation: A platform technology to induce auto-fermentation and thus cell-wall thinning in both nitrogen-replete and nitrogen-deplete Nannochloropsis slurries.

Nitrogen-deprived Nannochloropsis cells invested their fixed carbon into the accumulation of triacylglycerol and cell wall cellulose (thickness of N-replete cell walls = 27.8 ±â€¯5.8, N-deplete cell walls = 51.0 ±â€¯10.2 nm). In this study, the effect of nitrogen depletion on the ability of the cells to weaken their own cell walls via autolysis was investigated. Autolytic cell wall thinning was achieved in both N-replete and N-deplete biomass by incubating highly concentrated slurries in darkness at 38 °C. The incubation forced cells to anaerobically ferment their intracellular cellulose and resulted in 30-40% reduction in cell wall thickness for both biomass types. This wall depletion weakened the cells and increased the extent of cell rupture by mechanical force (from 42 to 78% for N-replete biomass, from 36 to 62% for N-deplete biomass). Importantly, autolysis did not adversely impact the amino acid content of protein-rich N-replete biomass or the fatty acid content of lipid-rich N-deplete biomass.

Lipid profile remodeling in response to nitrogen deprivation in the microalgae Chlorella sp. (Trebouxiophyceae) and Nannochloropsis sp. (Eustigmatophyceae).

Many species of microalgae produce greatly enhanced amounts of triacylglycerides (TAGs), the key product for biodiesel production, in response to specific environmental stresses. Improvement of TAG production by microalgae through optimization of growth regimes is of great interest. This relies on understanding microalgal lipid metabolism in relation to stress response in particular the deprivation of nutrients that can induce enhanced TAG synthesis. In this study, a detailed investigation of changes in lipid composition in Chlorella sp. and Nannochloropsis sp. in response to nitrogen deprivation (N-deprivation) was performed to provide novel mechanistic insights into the lipidome during stress. As expected, an increase in TAGs and an overall decrease in polar lipids were observed. However, while most membrane lipid classes (phosphoglycerolipids and glycolipids) were found to decrease, the non-nitrogen containing phosphatidylglycerol levels increased considerably in both algae from initially low levels. Of particular significance, it was observed that the acyl composition of TAGs in Nannochloropsis sp. remain relatively constant, whereas Chlorella sp. showed greater variability following N-deprivation. In both algae the overall fatty acid profiles of the polar lipid classes were largely unaffected by N-deprivation, suggesting a specific FA profile for each compartment is maintained to enable continued function despite considerable reductions in the amount of these lipids. The changes observed in the overall fatty acid profile were due primarily to the decrease in proportion of polar lipids to TAGs. This study provides the most detailed lipidomic information on two different microalgae with utility in biodiesel production and nutraceutical industries and proposes the mechanisms for this rearrangement. This research also highlights the usefulness of the latest MS-based approaches for microalgae lipid research.

Quantitative evaluation of the ease of rupture of industrially promising microalgae by high pressure homogenization.

The susceptibility to rupture of the microalgae Nannochloropsis sp., Chlorella sp. and Tetraselmis suecica by high pressure homogenization was compared quantitatively to the yeast Saccharomyces cerevisiae. Methods for quantifying cell rupture were investigated including cell counting, turbidity, metabolite release and particle sizing. Cell counting was the only reliable method for quantitative comparisons of all microalgae, with turbidity complicated by agglomeration of cell debris for T. suecica, and measurement of metabolite release affected by degradation occurring for all microalgae after significant rupture. The rupture of all microalgae followed exponential decay as a function of number of passes. The pressure required to achieve rupture of 50% of the cells per pass was determined to be 170, 1070, 1380, and ca. 2000 bar for Tetraselmis sp., Chlorella sp., S. cerevisiae, and Nannochloropsis sp., respectively. These results extend the criteria for selecting microalgae for industrial applications beyond consideration of growth and compositional attributes.

A quantitative analysis of microalgal lipids for optimization of biodiesel and omega-3 production.

The lipid characteristics of microalgae are known to differ between species and change with growth conditions. This work provides a methodology for lipid characterization that enables selection of the optimal strain, cultivation conditions, and processing pathway for commercial biodiesel production from microalgae. Two different microalgal species, Nannochloropsis sp. and Chlorella sp., were cultivated under both nitrogen replete and nitrogen depleted conditions. Lipids were extracted and fractionated into three major classes and quantified gravimetrically. The fatty acid profile of each fraction was analyzed using GC-MS. The resulting quantitative lipid data for each of the cultures is discussed in the context of biodiesel and omega-3 production. This approach illustrates how the growth conditions greatly affect the distribution of fatty acid present in the major lipid classes and therefore the suitability of the lipid extracts for biodiesel and other secondary products.