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This work is focused on the effect of lethal and sub-lethal copper (Cu) concentrations on the free amino acid and polyphenol production by the marine diatom Phaeodactylum tricornutum (P. tricornutum) after 12, 18, and 21 days of exposure. The concentrations of 10 amino acids (arginine, aspartic acid, glutamic acid, histidine, lysine, methionine, proline, valine, isoleucine, and phenylalanine) and 10 polyphenols (gallic acid, protocatechuic acid, p-coumaric acid, ferulic acid, catechin, vanillic acid, epicatechin syringic acid, rutin, and gentisic acid) were measured by RP-HPLC. Under lethal doses of Cu, free amino acids reached levels significantly higher than those in the control cells (up to 21.9 times higher), where histidine and methionine showed the highest increases (up to 37.4 and 65.8 times higher, respectively). The total phenolic content also increased up to 11.3 and 5.59 times higher compared to the reference cells, showing gallic acid the highest increase (45.8 times greater). The antioxidant activities of cells exposed to Cu were also enhanced with increasing doses of Cu(II). They were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging ability (RSA), cupric ion reducing antioxidant capacity (CUPRAC), and ferric reducing antioxidant power (FRAP) assays. Malonaldehyde (MDA) exhibited the same tendency: cells grown at the highest lethal Cu concentration yielded the highest MDA level. These findings reflect the involvement of amino acids and polyphenols in protective mechanisms to overcome the toxicity of copper in marine microalgae. Graphical Abstract

The biochemical composition of three novel selected microalgae strains (Chlorophyta) was evaluated to confirm their potential possibilities as new sustainably produced biomass with nutritional, functional, and/or biomedical properties. Extracts from cultured Pseudopediastrum boryanum, Chloromonas cf. reticulata, and Chloroidium saccharophilum exhibited higher radical scavenging activity of DPPH (1,1-diphenyl-2-picrylhydrazyl) when compared to butylated hydroxytoluene (BHT), but lower than butylated hydroxyanisole (BHA). Total phenolic compounds and amino acids were determined by newly developed RP-HPLC methods. Total phenolic contents, as µg g−1 of dry biomass, reached 27.1 for C. cf. reticulata, 26.4 for P. boryanum, and 55.8 for C. saccharophilum. Percentages of total analysed amino acids were 24.3, 32.1, and 18.5% of dry biomass, respectively, presenting high values for essential amino acids reaching 54.1, 72.6, and 61.2%, respectively. Glutamic acid was the most abundant free amino acid in all microalgae samples, followed by proline and lysine in C. saccharophilum and P. boryanum, and methionine and lysine in C. reticulata. Soluble carbohydrates in aqueous extracts ranged from 39.6 for C. saccharophilum to 49.3% for C. reticulata, increasing values to 45.1 for C. saccharophilum and 52.7% for P. boryanum in acid hydrolysates of dried biomass. Results confirmed the potential possibilities of these microalgae strains.

Three freshwater microalgae (Spirogyra sp., Cosmarium sp., and Cosmarium blytii) collected from several locations in Gran Canaria have been studied to explore their potential as a novel source of bioactive compounds for biotechnological applications. Soluble carbohydrates were quantified after extraction with 3 M HCl at 100 °C, ranging from 35.8 to 43.3 %, and with water at room temperature, ranging from 19 to 22.8 %. Amino acids glutamic acid, proline and aspartic acid were quantified by RP-HPLC. Glutamic acid was the most abundant, ranging from 12.2 to 3.63 mg g À 1 of dry biomass. Cosmarium blytii was the richest sample in amino acids (24.02 mg g À 1 of dry weight). In addition, Cosmarium blytii and Spyrogira sp. exhibited higher radical scavenging activity (RSA) against 1,1-diphenyl-2-picrylhydrazyl (DPPH) than that of the synthetic antioxidant butylhydroxytoluene (BHT), commonly used as food additive. These results show a great potential of these microalgae for exploitation in the food, feed and pharmaceutical industries.