2.1. Cultivation

There are significant differences in methods for cultivating macro- and microalgae, making the process specific to each of these organisms [24]. The first step for a variety of uses (including bioenergy production) is biomass generation [45]. In the case of macroalgae, Laminaria japonica and Undaria pinnatifida (brown algae), and Eucheuma spp., Kappaphycus alvarezii, and Gracilaria verrucosa (red algae) are the most frequently cultivated species [46].

Macroalgal cultivation can be carried out in seawater using two systems. Nearshore farms are the most widely used (about 90% of the seaweed production), as they can be built near shorelines with shallow depths. In the offshore method, farms are constructed in deep waters to avoid the influence of the land, which requires growth structures anchored to the ocean bottom or floating lines with positioning devices [45,47].

Macroalgae are commonly cultivated with farming technology, and grown using kelp systems aligned vertically or horizontally [45,46]. In the vertical method, the rope is held vertically by weights placed at one end, while in the horizontal method, floating lines are connected by horizontal ropes [45]. Since carbohydrate compounds are abundant in macroalgae biomass, these organisms are considered promising sources for bioenergy applications, especially for bioethanol production [46,48].

Microalgae biomass has also been reported as a promising feedstock for biofuel uses [49]. Different conditions in microalgae cultivation result in significant changes in the concentration, productivity, and biochemical composition of biomass [50]. Therefore, the selection of the correct cultivation method is essential to ensure the subsequent use of the biomass [51,52]. In addition, microalgae cultures must consider design, cost, contamination risks, and cleaning, especially for commercial purposes [49]. Strain type and nutrient source also impact the cultivation method selection [50].

To increase the feasibility of using microalgae for bioethanol production, the carbohydrate content and biomass productivity need to be improved, which can be achieved through changes in different cultivation parameters [53,54]. This is possible due to the ability of microalgae to modify their biochemical composition in response to culture manipulation, which includes environmental and nutritional changes in the cultivation [55]. Light intensity, pH, salinity, and temperature are the main environmental factors that influence the biochemical composition of microalgae, while nutritional factors include the availability and type of source of nitrogen, carbon, phosphorus, sulfur, and iron [56], [57], [58]. Microalgal engineering (e.g. genomic, transcriptomic, proteomic, metabolomic strategies) is another promising alternative to enhance biofuel production, especially by increasing the productivity of biomass and carbohydrate accumulation of several microalgae strains [59], [60], [61]. These techniques can improve stress tolerance and photosynthetic rate, which directly impact biofuel production viability [62].

Microalgal cultivation can be classified as autotrophic, mixotrophic, or heterotrophic, with autotrophic being the most used method [52,54]. In this type of cultivation, the carbon source is inorganic, and light serves as a source of energy. In mixotrophic cultivation, both organic and inorganic carbon sources can be used. In heterotrophic cultivations, organic carbon serves as energy and a carbon source [54]. There are three main operational modes for growing microalgae: batch, semicontinuous or continuous [63].

Regarding the accumulation of carbohydrates, de Farias Silva et al. [64]performed a comparison between batch and continuous modes. The batch system can be divided into two stages: in the first, the biomass grows in an environment with excess nutrients, while in the second, the carbon is converted into energy reserves, causing the accumulation of carbohydrates and lipids. In this second stage, biomass growth almost stopped, and although carbohydrate productivity increased, biomass productivity decreased from 40% to 60%. By using a lower nitrogen concentration in continuous mode, it is possible to achieve growth and accumulation of carbohydrates simultaneously, reaching a stationary phase with carbohydrate productivity two to three times higher [64].

Microalgal biomass production can occur through open systems or closed photobioreactors [65]. Open ponds are more widely applied on a commercial scale due to economic and operational aspects but have low biomass productivity compared to closed photobioreactors. This high productivity is due to this system's greater control of cultivation variables [66]. According to Suparmaniam et al. [49], a raceway pond is the best option to cultivate microalgae for commercial biofuel production, whereas closed photobioreactors are more suitable for producing microalgae to obtain biomass with higher-value products. While macroalgae are mainly cultivated in coastal regions, microalgae cultures have the advantage of not requiring arable land and can use wastewater as a nutrient source, reducing cultivation costs.

2.2. Harvesting

After algae cultivation, macro or microalgae biomasses are harvested for further processing and applications [50]. Macroalgae have characteristics similar to plants and, as they are larger than microalgae, their harvest is technologically and economically advantageous [67], [68], [69]. Macroalgae can be harvested manually or mechanically. In the manual method (the most common), algae are harvested through a sickle, fork, or net, while mechanized methods require harvesters in boats or ships, such as rotation blades, suction, or dredging cutters [45].

The primary issue in the harvesting stage is separating a small amount of microalgae biomass from a large volume of culture medium [70]. The size of the microalgae cells (generally <20 µm), combined with high colloidal stability and a density slightly higher than that of the water, results in the spontaneous non-sedimentation of the microalgae cells by gravity [70,71]. These characteristics make harvesting one of the costliest steps in producing microalgae [72]. Mata et al. [73] report that harvesting microalgae can represent 20-30% of total biomass production costs. Therefore, harvesting is identified as a bottleneck for microalgae commercialization [49].

Given this bottleneck, several alternative strategies have been developed to optimize microalgae harvest to achieve a better cost-benefit ratio [70,74]. Microalgal harvesting can be performed using mechanical, electrical, biological, and chemical methods, which are generally divided into thickening (coagulation/flocculation, bioflocculation, gravity sedimentation, flotation, and electricity-based methods) and dewatering (centrifugation and filtration) [75]. Each microalgae harvesting method presents advantages and disadvantages related to biomass recovery rate, costs, and execution parameters [72,[75], [76], [77], [78]]. Physical methods are commonly reported as demanding high energy costs while using chemical products to harvest microalgae can make the subsequent use of biomass unfeasible due to heavy metal contamination [79]. As an alternative, the interest in using bioflocculants to harvest microalgae has been growing, since they are considered non-toxic, more sustainable, safe-handling, and eco-friendly [49,80]. Therefore, the harvest of macroalgae biomass is easier than microalgae since they can be harvested manually because of their size.

2.3. Cell rupture

To transform the biomass of microalgae and macroalgae into bioethanol, it is necessary to first rupture the cell wall, in order to release the intracellular compounds of interest, which, in the case of bioethanol, are carbohydrates [81,82]. Each species has a specific cell wall composition, which determines cell viability in a wide range of environments, defending cells from biotic and abiotic stresses and providing plasticity, which allows different cells to expand and form [83,84].

The cell wall structures of microalgae may differ in the functions of the growing environments and between species. To extract more intracellular organic compounds from microalgae, it is necessary to destroy the protective barrier of the cell wall. A microalgae cell wall is composed of mannans, glucans, chitinpolysaccharides, arabinogalactans, and rhamnose, which can be found through different types of glycoside visualizations, and extracted and quantified as carbohydrates [26,85,86].

The cell wall of microalgae also has proteins and clusters of amino acids, such as valine, alanine, glutamic acid, and glycine. This protein matrix adds structural integrity to the cell wall. In addition, the cell wall of the microalgae has lipid contents, basically formed by palmitic acid, stearic acid, and 1,3-di-tert-butylbenzene. Other elements make up the structure of the cell wall and its layers; these layers are called microfibrillaries, which form the rigid part of the wall. It is noteworthy that the cell wall of microalgae can change significantly under different environmental conditions, such as nutrient depletion [26,86].

In general, macroalgae (primarily marine lineages) have cell walls formed by sulfated polysaccharides, rich in mannose, xylose, arabinose, and glucose, in addition to xylan, mannan, and glucan. They also have protein and lipid structures very similar to the microalgae wall. Macroalgae have a higher amount of these sulfated polysaccharides, which form macromolecules that promote flexibility and resilience. They act against physical forces exerted by waves and ocean currents, preventing their drying out when exposed to high solar radiation and stress when exposed to variations in salinity and pH [26,87,88].

Due to the similarities between the microalgae and macroalgae cell walls and the variability between species, there are no specific cell disruption methods for each alga; both have thick cell walls with very similar compositions. The most frequently used methods are physical (ultrasound, high-pressure homogenization), chemical (acid and base solutions), and biological processes(enzymes) [89], [90], [91].

However, it is worth mentioning that some macroalgal species may have more complex structures in their intracellular compounds, such as sulfated polysaccharides, which would require more complex pretreatments or even the use of high temperatures and pressure. These more complex pretreatments are a disadvantage to macroalgae cultivation because, in addition to requiring greater investments, they can lead to the formation of by-products that would alter the final ethanol yield [92], [93], [94].

2.4. Saccharification of polysaccharides

After releasing algal intracellular compounds, they must be hydrolyzed to be transformed into different monosaccharides for the later fermentation stage. Saccharification strategies must prioritize high efficiency and reduction of generation of by-products that can influence fermentation [95], [96], [97].

A series of methodologies are employed to transform intracellular carbohydrates from microalgae, using concentrated acids and commercial enzymes. According to the literature, these processes can be developed simultaneously to optimize the saccharification process. However, depending on the microalgae species, optimization can be achieved using only enzymatic hydrolysis methods. Enzymatic hydrolysis is widely used for providing milder pH and temperature conditions and less by-product formation. Two enzyme complexes are used to obtain complete hydrolysis of the microalgal polysaccharides: amylases (alpha-amylase and amyloglucosidase) and cellulases (endoglucanases, exoglucanases, beta-glucosidases). Thus, the hydrolysis of microalgae compounds can be expensive, in addition to the use of different processes, which makes large-scale implementation difficult [26,82,98].

During the hydrolysis of macroalgal polysaccharides, intracellular compounds such as starch and cellulose are transformed into fermentable sugars. The same difficulties of large-scale implementation apply to the production of macroalgal biomass, as they share a similar process. Like macroalgae, a set of specific enzymes must be used in the hydrolysis of microalgae, which can complicate the process, especially in large-scale implementation. However, macroalgae have an advantage over microalgae, as they can accumulate an average value of 60% of carbohydrates, which increases the yield of reducing sugar formation, making the process more advantageous [99,100].

Macroalgae contain particular polysaccharides and monosaccharides, such as ulvan, fucoidan, alginate, laminaran, floridian starch, mannitol (a sweetened alcohol), rhamnose, fucose, and uronic acids. In some algae, these compounds can be grouped in the form of agar, characterized as a polymer of galactose and galactopyranose, which is transformed into reducing sugars when hydrolyzed. Some of these polysaccharides are not present in microalgae so higher yields can be obtained after the hydrolysis of macroalgae [97,101]. Still, some species of macroalgae contain sulfated polysaccharides, generating compounds that can interfere with subsequent fermentation routes and impair ethanol production yields [92], [93], [94].

2.5. Fermentation

After hydrolysis, the monomers must be fermented to be transformed into bioethanol or another product of interest. Two methodological processes can produce ethanol from microalgae: fermentation using specific yeasts that use reducing sugars from microalgae biomass and direct production by genetically modified microalgae. The polysaccharides extracted from the microalgae can be fermented into ethanol through fermentation routes very similar to the consolidated production of starch cultures. This is an advantage of microalgae, since the fermentative routes are already established, helping implement these processes on large scales [26,81,96,102].

There are two types of yeast fermentation: simultaneous saccharification or separated saccharification and fermentation [103]. In the case of separated saccharification and fermentation, the pretreated microalgal biomass is hydrolyzed to glucose and subsequently fermented to bioethanol in separated units, while simultaneous saccharification and fermentation occur in a single step [104]. Saccharification and separate fermentation have the following advantages: low cost for chemicals, short duration, and simplicity for large-scale application. On the other hand, simultaneous saccharification and fermentation has fewer steps and higher bioethanol yields [104]. Yeasts of the genus Saccharomyces or bacteria of the genus Zymomonas are generally used in these processes [56]. Since ancient times, S. cerevisiae has been used in biotechnology to produce alcoholic beverages, as it has high efficiency in converting sugars, mainly glucose, into ethanol [104]. Per Costa and De Morais [105], microalgae are a potential source of fermentable substrates and may have high levels of carbon compounds in their composition, which can be fermented directly or after pretreatment. However, they also report that other compounds, such as CO2 and H2O, are generated during bioethanol production. Therefore, the maximum theoretical yield is 0.51 kg of ethanol and 0.49 kg of CO2 per kg of glucose.

The same processes can be used to ferment macroalgal monosaccharides. The difference is that macroalgae have particular monosaccharides that are fermented by specific routes, which require the use of specific biocatalysts, resulting in greater cost, which may render the process unfeasible. Many macroalgae have fractions of mannitol and laminaran, and these extracts are removed and fermented by specific microorganisms [46,106]. Such specific monosaccharide fractions are not present in microalgae, which adds an advantage to macroalgae. However, it is still necessary to optimize processes that carry out the total fermentation of macroalgal carbohydrate fractions (alginate, laminarin, and mannitol) to be converted into ethanol with high yields and productivity. This optimization of total fermentation can be achieved through the development of a microorganism capable of acting on all fractions of substrates. Nevertheless, such optimization still lacks technological advances, making it difficult to implement large-scale production and meet the biorefinery purpose, that is, the total use of the macroalgal fraction [29,94].

Such process optimization can lead to obtaining ethanol with higher yields or in high concentrations from the strategies listed here, such as changes in cultures, to increasingly obtain biomass of micro or macroalgae with high levels of carbohydrates. This provides the generation of hydrolysates with high levels of reducing sugar, through the use of a set of enzymes capable of acting on all the polysaccharides present in the biomass. After generating these fermentable sugars, the use of different yeasts can increase conversion yields (see the following section). In addition to this, purification strategies can be optimized through distillation to remove water and other impurities in the dilute alcohol product that can reach 10–15% ethanol [107]. Such processes and factors could thus optimize ethanol production (in terms of both concentration and quality) from algal biomasses.