Antioxidants (enzymatic or non-enzymatic) are classified depending on their mode of action as primary antioxidants (hydrogen or electrons donors) or secondary antioxidants (oxygen scavengers or chelating agents) [43,44]. Antioxidants can also be grouped according to size, solubility, or structure, Figure 1. Abundant enzymatic antioxidants in microalgae are superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione reductase (GR), glutathione transferase (GST). Non-enzymatic antioxidants consist of compounds such as Vitamin C (ascorbate), glutathione, carotenoid, phenolic compounds, proline, glycine, polyamine, PUFA, and some metals (Cu, Zu) [45,46,47]. Most enzymatic antioxidants and some non-enzymatic antioxidants (glutathione, ascorbate) are hydrophilic and mainly present in the cellular fluids (cytosol or cytoplasmic matrix), whereas the hydrophobic antioxidants (carotenoid, tocopherol) are primary located in the cell membranes [48].

Antioxidant enzymes prevent or delay the oxidation of other molecules by neutralising reactive oxygen species (ROS) [48]. These enzymes eliminate ROS by reducing the energy of free radicals or by donating electrons to free radicals [42], and as such, constitute the first level of defence in the cell's antioxidant network. Some molecules are not involved directly in the scavenging of free radicals but rather enhance other antioxidant molecules’ activity and may also be classified as antioxidants [41]. The majority of antioxidant enzymes are metalloenzymes and contain a metal ion in their catalytic site [45].

Antioxidants, or antioxidant-enriched extracts, are commercially used to prevent oxidative processes and to maintain the flavor, texture, and colour of food during storage. They also find uses as refining, bleaching, deodorising agents in the food processing industries [51,52,53,54,55], in extending the shelf life of lubricating oil and reducing vehicular emissions [56] and in stabilisation of synthetic fibre, rubber, thermoplastic, and adhesives by stopping autocatalytic reactions [57]. In cosmeceutical products, antioxidant compounds are used to prevent skin ageing and UV-induced skin damage and treat the appearance of wrinkles and erythema [58,59,60,61]. Industrial applications of antioxidants are listed in Table 1. The global demand for antioxidants was valued at ~USD 2.25 billion in 2014 and grew at a CAGR (compounds annual growth rate) of ~5.5% between 2015 and 2020 [62]. This increasing global demand is driving the search for synthetic and natural-derived antioxidants.

Commercially, synthetic antioxidants such as butylated hydroxyl anisole (BHA), butylated hydroxyl toluene (BHT), α-tocopherol and propyl gallate are used in foods, food packaging, cosmetics and pharmaceutical products [82,84]. However, the physical properties of BHT and BHA (high volatility and instability at elevated temperature), strict legislation on the use of synthetic food additives, and the carcinogenic nature of some synthetic antioxidants [85,86,87,88] have shifted the attention to finding antioxidants from natural sources that are pharmacologically potent and have low or no toxicity.

ROS is a collective term for oxygen-derived products (free radicals and non-radicals reactive derivatives of oxygen). They are produced in cellular compartments either exogenously (in response to environmental stress such as UV radiation or xenobiotics) or endogenously (from the intracellular metabolic pathway, enzymatic activities, mitochondrial respiration, or photosynthesis) [66]. Accumulation of ROS leads to oxidative stress in cells and causes damage to cellular macromolecules, including proteins, lipids, carbohydrates and DNA [89]. Various sources of ROS and corresponding modes of biochemical metabolism are summarised in Table 2.

The majority of ROS are generated when electrons leak from the chloroplastic electron transport systems during photosynthesis, from the mitochondrial electron transport chain during photorespiration, and from the peroxisomal membrane electron transport chain (Figure 2) [90]. Within plant cells, 1–2% of O₂ consumption leads to the formation of superoxide (O₂^•−), and 1–5% of mitochondrial O₂ consumption leads to the generation of H₂O₂ [45]. O₂^•− is generated during oxidisation of unsaturated fatty acids [91], from the activity of cytochrome P450 [92] and the cytochrome b5 family members [93,94]. Also, O₂^•− is produced in the peroxisome where xanthine oxidases catalyse the oxidation of xanthine and hypoxanthine to uric acid [45], and in the plasma membrane due to the reduction of O₂ by NADPH oxidases [49].

Superoxide ions (O₂^•−) are converted into hydrogen peroxide (H₂O₂) by the catalytic activity of SOD. H₂O₂ is also produced in peroxisomes when glycolate from the photorespiration is recycled. In addition, H₂O₂ can be formed by D-amino acid oxidase, urate oxidase, flavin oxidase, L-α-hydroxy acid oxidase, and fatty acyl-CoA oxidase, and by cell wall peroxidases. Reactive OH^• is produced from the reaction of O₂^•− and H₂O₂ at neutral pH and ambient temperature (Haber–Weiss reaction) or from H₂O₂ during the Fenton reaction [96]. In contrast, a proton (H⁺) addition to O₂^•− generates perhyroxyl radicals (HO₂^•). In some cases, intracellular ROS could also form during auto-oxidation of small molecules (epinephrine, flavins, and hydroquinones) [97,98]. The singlet oxygen (¹O₂) is generated from the reaction of oxygen (³O₂) with the triplet state of chlorophyll produced by the dissipation of insufficient energy during photosynthesis [45].

Under normal physiological conditions, ROS is neutralised by the cells’ antioxidant systems where antioxidant enzymes and antioxidant molecules maintain the delicate intracellular redox balance and mitigate undesirable cellular damage caused by ROS, Table 2 [48,50].

Different isozymes of SOD exist (Mn-SOD in mitochondria and peroxisome, Fe-SOD in the chloroplast, and Cu/Zn-SOD isozyme in cytosol) but they all participate in scavenging of O₂^•−. In addition to SOD, some antioxidants molecules (Vitamin C, glutathione, etc.) also eliminate O₂^•− [45]. Further, the individual or cumulative catalytic activity of catalase or peroxidases decomposes H₂O₂ into H₂O and O_2. CAT, peroxidases (GPX, APX), and SOD show a synergistic effect in the scavenging of O₂^•−. In addition to eliminating H₂O₂, GPX can protect cells by preventing intracellular lipid peroxidation [48]. APX may be more efficient compared to CAT or GPX in detoxification of H₂O₂ due to its higher affinity for H₂O₂. APX reduces H₂O₂ into H₂O in chloroplasts, cytosol, mitochondria, and peroxisomes, and in the apoplastic space using Ascorbic acid (ASc) as an electron donor [99].

Algal cells accumulate ASc with 30–40% remaining in the chloroplast. Ascorbic acid is water-soluble and acts as a potent antioxidant because of its ability to donate electrons in enzymatic and non-enzymatic reactions [100]. It protects cells by directly scavenging O₂^•−, HO₂^•− and regenerating the tocopherol from tocopheroxyl radicals [101]. All intracellular compartments generate the reduced form of glutathione, which plays a role as an excellent scavenger of many ROS such as O₂^•−, HO^•, O₃, NO₂, lipid hydroperoxides [50] due to the redox-active thiol group that becomes oxidised when GSH reduces ROS [102]. Carotenoid also protects cells from light-induced oxidative stress by quenching ¹O₂ or dissipating excess heat (excitation energy) or scavenging peroxy radicals [45,48].

As the accumulation of enzymatic and non-enzymatic antioxidants in the cell depends on the external environment, manipulating cultivation conditions could enhance the intracellular antioxidant levels.