LASSO Regression with Multiple Imputations for the Selection of Key Variables Affecting the Fatty Acid Profile of Nannochloropsis oculata.

The marine microalga Nannochloropsis oculata has garnered significant interest as a potential source of lipids, both for biofuel and nutrition, containing significant amounts of C16:0, C16:1, and C20:5, n-3 (EPA) fatty acids (FA). Growth parameters such as temperature, pH, light intensity, and nutrient availability play a crucial role in the fatty acid profile of microalgae, with N. oculata being no exception. This study aims to identify key variables for the FA profile of N. oculata grown autotrophically. To that end, the most relevant literature data were gathered and combined with our previous work as well as with novel experimental data, with 121 observations in total. The examined variables were the percentages of C14:0, C16:0, C16:1, C18:1, C18:2, and C20:5, n-3 in total FAs, their respective ratios to C16:0, and the respective content of biomass in those fatty acids in terms of ash free dry weight. Many potential predictor variables were collected, while dummy variables were introduced to account for bias in the measured variables originating from different authors as well as for other parameters. The method of multiple imputations was chosen to handle missing data, with limits based on the literature and model-based estimation, such as using the software PHREEQC and residual modelling for the estimation of pH. To eliminate unimportant predictor variables, LASSO (Least Absolute Shrinkage and Selection Operator) regression analysis with a novel definition of optimal lambda was employed. LASSO regression identified the most relevant predictors while minimizing the risk of overfitting the model. Subsequently, stepwise linear regression with interaction terms was used to further study the effects of the selected predictors. After two rounds of regression, sparse refined models were acquired, and their coefficients were evaluated based on significance. Our analysis confirms well-known effects, such as that of temperature, and it uncovers novel unreported effects of aeration, calcium, magnesium, and manganese. Of special interest is the negative effect of aeration on polyunsaturated fatty acids (PUFAs), which is possibly related to the enzymatic kinetics of fatty acid desaturation under increased oxygen concentration. These findings contribute to the optimization of the fatty acid profile of N. oculata for different purposes, such as production of, high in PUFAs, food or feed, or production of, high in saturated and monounsaturated FA methyl esters (FAME), biofuels.

Production of Antioxidants and High Value Biomass from Nannochloropsis oculata: Effects of pH, Temperature and Light Period in Batch Photobioreactors

Nannochloropsis oculata is a marine microalgal species with a great potential as food or feed due to its high pigment, protein and eicosapentaenoic acid contents. However, for such an application to be realized on a large scale, a biorefinery approach is necessary due to the high cost of microalgal biomass production. For example, techno economic analyses have suggested the co-production of food or feed with antioxidants, which can be extracted and supplied separately to the market. The aim of this study was to investigate the effect of cultivation conditions on the antioxidant capacity of Nannochlosopsis oculata extracts, derived with ultrasound-assisted extraction at room temperature, as well as the proximate composition and fatty acid profile of the biomass. A fractional factorial approach was applied to examine the effects of temperature (20-35 °C), pH (6.5-9.5) and light period (24:0, 12:12). At the end of each run, biomass was collected, washed with 0.5M ammonium bicarbonate and freeze-dried. Antioxidant capacity as gallic acid equivalents as well as pigment content were measured in the ethanolic extracts. Optimal conditions were different for productivity and biomass composition. Interesting results regarding the effect of light period (LP) and pH require further investigation, whereas the effect of moisture on the extraction process was confounded with biomass composition. Finally, further data is provided regarding the relation between chlorophyll content and apparent phenolic content using the Folin-Ciocalteu assay, in agreement with our previous work.

Ultrasound-Assisted Extraction of Nannochloropsis oculata with Ethanol and Betaine: 1,2-Propanediol Eutectic Solvent for Antioxidant Pigment-Rich Extracts Retaining Nutritious the Residual Biomass.

The aim of this study was the development of an efficient "green" extraction method of Nannochloropsis oculata to produce antioxidant extracts and nutritious residual biomass. Twenty-one extraction methods were evaluated by measuring the reactivity with the Folin-Ciocalteu reagent: ultrasonication or maceration at different temperatures with different organic solvents, extraction at different pH values, enzyme-assisted extraction, encapsulation with ß-cyclodextrin, and the use of natural deep eutectic solvents. Ultrasound-assisted extraction with ethanol or betaine: 1,2-propanediol in a molar ratio of 2:5 (BP) had optimal extractive capacity. Both extracts were evaluated with antioxidant assays and the ethanol extract exhibited significantly higher (at least twofold) values. The determination of carotenoids by LC-MS and HPLC-DAD revealed the dominance of violaxanthin and antheraxanthin and their fourfold higher concentrations in the ethanol extract. The 1H-NMR characterization of the ethanol extract confirmed the results of the colorimetric and chromatographic assays. The microalgal biomass was characterized before and after the extraction in terms of humidity, ash, carbohydrates, proteins, chlorophyll-a, carotenoids, and lipids; the identity and content of the latter were determined with gas chromatography. BP caused a smaller depletion of the lipids from the biomass compared to ethanol, but proteins, carbohydrates, and ash were at a higher content in the biomass obtained after ethanol extraction, whereas the biomass was dry and easy to handle. Although further optimization may take place for the scale-up of those procedures, our study paves the way for a green strategy for the valorization of microalgae in cosmetics without generating waste, since the remaining biomass can be used for aquafeed.

Total Phenolic Content, Biomass Composition, and Antioxidant Activity of Selected Marine Microalgal Species with Potential as Aquaculture Feed.

There has been growing interest in microalgal biomolecules for health and cosmetics, as well as in the use of microalgae as aquaculture feed due to the need to replace fishmeal and fish oil with sustainable yet equally nutritious alternatives. Aim of this study is to evaluate the potential of five marine microalgal species, namely Chlorella minutissima, Dunaliella salina, Isochrysis galbana, Nannochloropsis oculata and Tisochrysis lutea, for the co-production of antioxidants and aquaculture feed. Batch cultivation was performed under saturating light intensity and continuous aeration. Freeze-dried biomass was extracted sequentially with water and methanol and evaluated for phenolic content and antioxidant activity, as well as proximate composition and fatty acid profile. Methanolic extracts of C. minutissima presented the highest phenolic content, measured with the Folin-Ciocalteu assay, and antioxidant activity. However, HPLC and LC-MS showed the presence of non-pigment compounds only in T. lutea. Total phenolic content and antioxidant activity were correlated to chlorophyll content. N. oculata and T. lutea were rich in eicosapentaenoic acid and docosahexaenoic acid, respectively, as well as in protein. In conclusion, N. oculata and T. lutea are suitable candidates for further optimization, while the data presented suggest that pigment effects on the Folin-Ciocalteu method require reconsideration.