The production of biostimulants from algal biomass and cyanobacteria may involve the use of various techniques mainly aimed at breaking down cells by making bioactive molecules contained in or bound to cell walls available to the plant. This disruption can be achieved through physical/mechanical, chemical, or enzymatic methods [61]. The choice of extraction method is mainly dictated by the type of biomass used and the target molecules. For example, the physical/mechanical methods most commonly used for research purposes today, which involve mechanical cell wall disruption or the use of high pressure, high temperatures, ultrasound, or combinations thereof, cannot guarantee high extraction yields for micro- and macroalgae, which may have thicker cell walls than cyanobacteria [59]. Cell disruption may be followed by a phase of separation from the extract of cell residues by centrifugation or filtration or with an extraction phase using solvents in order to obtain specific fractions of the crude extract [1]. For instance, in the production of biostimulant polysaccharide extracts, polysaccharides are usually precipitated with ethanol following the physical breakdown of cells. A rather recent technique is extraction that uses supercritical CO₂ as a solvent, i.e., with chemical–physical properties intermediately between those of a liquid and those of a gas, obtained at low temperatures (50 °C) and under high pressure (200–500 bars), ensuring the preservation of thermolabile bioactive compounds in biomass [61]. In the preparation of microalgal and cyanobacterial hydrolysates, the use of chemical agents, mainly acids or bases such as sulphuric acid, hydrochloric acid, and sodium hydroxide, generally results in the breakdown of the macromolecules contained in cells. However, these methods have been less and less used, as they may lead to the degradation and inactivation of some bioactive molecules contained in biomass and require the subsequent disposal of large quantities of chemical compounds [63]. Enzymatic methods use single enzymes capable of breaking cell walls and/or proteolytic enzymes that cleave peptide bonds to produce protein hydrolysates, i.e., products rich in free amino acids and soluble peptides. Extracts and hydrolysates can be applied directly to the foliar apparatus by spraying or nebulisation or to the growing medium by fertigation, in which active molecules are absorbed by the root system, or they can be used for pre-sowing treatments [58,61]. Foliar application is generally preferred to soil application, as it allows the use of lower doses of product, limits losses due to leaching, and prevents degradation by soil microorganisms. To avoid the costs associated with the extraction/hydrolysis processes, live cells can be applied directly into growth media or onto plant leaves or be used for seed treatment. Alternatively, culture media separated from microalgal biomass by filtration or centrifugation can be used directly for biostimulant treatment, exploiting the action of the compounds released by the microbial cultures [59].

The application of microalgae and cyanobacteria or formulations derived from them (biomass, extracts, hydrolysates) on plants has been shown to produce a wide range of often interconnected beneficial effects. These responses vary depending on the microalgal species used to produce each biostimulant but also in relation to the plant species treated and the growing conditions (Figure 2) [64,65]. The phenological stage of the plant and the environmental conditions can also directly influence the success of the biostimulant treatment. Among the most common effects observed is an increase in growth and, consequently, yield in leafy vegetables (lettuce, spinach, rocket) and herbs (mint, basil) [59,64]. These increases in plant growth and fresh weight have been associated with a stimulation of the nitrogen and carbon metabolism in plants treated with microalgal extracts, whereby increases in leaf, protein, carbohydrate, and photosynthetic pigment (chlorophyll and carotenoid) content were observed [66]. The stimulation effects of the primary metabolism can be attributed to an increase in the nutrient uptake of plants subjected to the biostimulant treatment. In this sense, biostimulants can act either directly by improving soil structure and nutrient availability in the soil when applied basally or by directly influencing plant physiology when applied basally or foliarly [67]. Indeed, it is known that the inoculation of cyanobacteria into soil can promote the uptake of zinc and iron by plants through the production of siderophores [68]. In addition, the extracellular polysaccharides produced by many cyanobacterial species can stimulate rhizosphere microbiota by providing organic carbon for microbial growth and can improve soil aggregation and water retention capacity, increasing the volume of soil that can be explored by roots and indirectly promoting root growth [69]. Stimulation of root growth and development has also been observed in several studies after treatment with extracts and hydrolysates of microalgae and cyanobacteria [1,59]. For example, the use of Chlorella vulgaris and Scenedesmus quadricauda on beetroot produced positive effects on root architecture, including increases in the root length and also in the number of lateral roots and thus the root surface area for nutrient uptake [49]. These stimulation effects occurred when the biostimulant both was applied to the basal part of the plant and was absorbed directly by the roots, as well as when it was applied to the leaves and induced a concomitant increase in the macro- and micronutrient content in the plant tissues. In addition to foliar and soil application, seedling growth can also be stimulated following seed treatment in the pre-sowing phase. Seed treatment can also have the effect of increasing germination rates [64]. Due to their ability to accelerate germination and seedling development, stimulate early flowering, and increase numbers of flowers, microalgal and cyanobacterial biostimulants may also have interesting applications found in floriculture [64,70]. For example, aqueous extracts and lyophilised biomass of Desmodesmus subspicatus increased germination in vitro and accelerated development in the subsequent transplanting and acclimatisation phase in a greenhouse of the orchid Cattleya warneri [71]. The effect of biostimulants on plants does not only result in improved vegetative growth. For example, treatment of vines has resulted in a significant increase in grape yield [64]. Furthermore, the application of biostimulants can trigger biochemical processes that lead to the accumulation of important metabolites that improve the quality characteristics and shelf life of a final product [72,73]. Among these, we can mention an increase in the essential oil content of leaves in peppermint treated with the extracts of Anabaena vaginicola and Cylindrospermum michailovskoense [74] and an increase in total soluble solid content and reduction in weight loss during the storage of onions treated with extracts of Arthrospira platensis [75]. Although the incidence of abiotic stresses, such as drought, salinity, and temperature extremes, is expected to increase in the coming years as climate change intensifies, few strategies are available to date to mitigate the negative effects of such stresses [76]. Many abiotic factors have manifested themselves in plants as osmotic stresses, leading to the accumulation of reactive oxygen species (ROSs) that can cause severe oxidative damage to DNA, lipids, carbohydrates, and proteins [77]. It has been shown that the application of microalgae, cyanobacteria, and formulations derived from them will promote the growth and yield of certain plant species, such as rice, wheat, and tomato, under abiotic stress conditions, inducing an enhancement of the antioxidant defences in the plant tissues [59,64]. It is important to remember that the concentration of a biostimulant and its number of applications are determining factors in the success of treatment and that an increase in dose does not always correspond to an increase in positive effects on a plant [78]. In fact, it has been found in some studies that intermediate dilutions of biostimulants may be more effective in promoting growth and flowering, while application of high doses will usually reduce or even neutralise the effect. Effective doses may vary considerably depending on the plant species treated and the method of application. In general, foliar application is effective at lower concentrations than seed or soil application is [59]. In rice plants inoculated with microalgae, accumulations of phenolic acids and flavonoids have been observed in leaf tissue [60]. In addition, according to recent studies, polysaccharide extracts obtained from different microalgal strains, including Chlamydomonas reinhardtii, Chlorella rokiniana, Porphirydium spp., and Dunaliella salina, can increase the activity of antioxidant enzymes such as catalase, peroxidase, and superoxide dismutase in tomato plants subjected to salt stress [72]. Another important function of exopolysaccharides released into the soil is to sequester metal ions and sodium ions, reducing their uptake by plants and stimulating their growth in saline or polluted soils [78,79]. For example, coating maize seeds with Arthrospira platensis has led to a reduction of more than 90% of the cadmium absorbed by the roots 12 days after sowing [80,81].