Nutrients and Energy Digestibility of Microalgal Biomass for Fish Feed Applications

: Aquafeed accounts for at least 75–90% of aquaculture’s operating costs. Traditional aquafeed ingredients such as ﬁshmeal, ﬁsh oil, and soybean meal are unsustainable; further, their increasing cost necessities developing alternative feed ingredients. Microalgae-based aquafeed is not only environmentally friendly, but it can also be cost-effective with proper optimization. In addition, the nutrition proﬁle of microalgae is similar to that of many ﬁshes. The digestibility of a feed is one of the most important factors to consider in feed formulation. A highly digestible feed can lower production costs, reduce feed waste, and reduce the risk of eutrophication. This review discusses the digestibility of various nutrients such as protein, lipid, carbohydrate, amino acids, and fatty acids (including omega-3 fatty acids), dry matter, and energy of various microalgae in ﬁsh. Other commonly used aquafeed ingredients were also compared to microalgae in terms of nutrient and energy digestibility in ﬁsh. The intrinsic characteristics of microalgae, biomass pretreatment, and feed preparation methods are all discussed as factors that contribute to the nutrient and energy digestibility of microalgae in ﬁsh. Furthermore, methods for increasing the digestibility of microalgal biomass in ﬁsh are suggested. Finally, the review concludes with the challenges and prospects of using microalgae as a ﬁsh feed in terms of digestibility.


Introduction
Microalgae biomass is a promising feed ingredient in aquaculture. Various studies have reported success in the partial replacement of fishmeal, soybean meal, and the complete replacement of fish oil in the fish diet by microalgal biomass [1,2]. The inclusion of microalgae in feed improves weight gain, increases the protein and lipid content in the fish muscle, improves the disease resistance and stress tolerance in fish, and enhances the texture and taste of fish fillet [3].
Microalgae are a nutrition-rich ingredient that could be used in fish feed. However, the nutritional content of microalgae is of limited use in feed formulation because it does not provide information on the hydrolytic products available for growth after digestion [4]. This could be known through digestibility studies. The digestibility study provides information on the amount of digestible nutrients and energy in ingredients, including microalgae. The digestibility of nutrients in the diet indicates the proportion of food consumed to be absorbed, with the undigested portion eliminated as feces [5]. Therefore, the portion of undigested food is a waste of resources.
The addition of highly digestible ingredients to feed can help to improve feed conversion ratios, lower production costs, and reduce environmental impact by lowering eutrophication potential [6][7][8]. Enhanced digestibility would ultimately result in higher growth and nutrient retention rates in muscle, thus increasing productivity [9]. Therefore,

Methods to Improve the Digestibility of Microalgae
The microalgal cells harvested from the growth reactor are usually spray dried to preserve the biomass quality. In addition, on a large scale, microalgal biomass is sun, drum, or oven-dried. However, in these cases, the microalgae cell wall remains intact, which is related to poor digestibility [9,22,34]. Nutrient digestibility can be improved by the pretreatment/processing of biomass, including bead milling, pasteurization, freeze-drying, high pressure homogenization, pulse electric field, ultrasound, microwave, chemical and enzymatic treatment [21,52]. This is possible since biomass processing/pre-treatment disrupts the rigid cell wall of microalgae and thus releases intracellular nutrients for the fish digestive system and subsequent absorption. Literature reports suggest that biomass processing of selected microalgae is attributed to higher digestibility for various fishes [1,34,53].
In several studies, bead milling was found to be efficient among pre-treatments in improving microalgae digestibility in fish. For example, in juvenile Nile tilapia (Oreochromis niloticus), the diet containing bead milled Nannochloropsis gaditana had the highest protein, lipid, and energy digestibility when compared to other diets that contained the same algae but were treated with pasteurization, freezing, or freeze-drying methods [18]. Physical treatment of Nannochloropsis sp. and Chlorella sp. using bead milling improved protein digestibility by 3-8% compared to that of unprocessed ones in European seabass [34]. In particular, bead milling of Tetraselmis sp. improved protein digestibility by 20% compared to unprocessed cells in European seabass [34]. The digestibility of amino acids like phenylalanine, and aspartic acid in European seabass was improved by bead milled microalgae inclusion in diet rather than the whole biomass inclusion; however, essential amino acid digestibility was not improved by the pre-treatment of microalgae [34]. It was observed that 10-39% of the cell wall of Nannochloropsis sp. and Chlorella sp. could be broken by 10 min of bead milling and, in turn, may liberate nutrients and thus improve digestibility [1,17,54,55]. In some cases, the enzymatic processing also significantly improved the protein digestibility of Nannochloropsis sp. and Chlorella sp. and energy digestibility of Nannochloropsis sp., Chlorella sp., and Tetraselmis sp. by 14%, 11%, and 40% for European seabass [34]. The extent of cell disruption differs from one species to another; thus, nutrient accessibility could differ despite the same processing conditions [56].
The extrusion process is preferred for feed making since it produces pellets that could reduce feed loss and water pollution. It involves high temperature, high pressure, long processing time, and shear force. Under these conditions, the recalcitrant microalgal cell walls can be disrupted, which in turn increases nutrient bioavailability and digestibility [21,[57][58][59]. Furthermore, the extrusion process can denature the enzymes like trypsin inhibitors, which affects digestibility [38]. In a study, extruded feed containing defatted biomass of Nannochloropsis sp. and Desmodesmus sp. had higher digestibility of ash, dry matter, and protein than that of non-extruded diet [21]. Similarly, in Gibel carp fed with a microalgae-based diet, the digestibility of dry matter, protein, starch, and energy was higher than that of pellets prepared by cold pelletization technique [52]. In the cold pelletization technique, all ingredients are mixed with cold water and cold-pressed, usually through a meat mincer, then dried, crushed, and sieved to obtain pellets. Cold pelletization, unlike extrusion, does not use high temperatures or pressure, so the cell wall of microalgae is largely unaffected during the process.
The digestibility of feed ingredients, including microalgae, can be improved by the addition of enzymes in diet or by enzymatic processing of microalgal biomass [60,61]. For example, the addition of enzyme-like protease improved protein digestibility and degraded the anti-nutritional factors, including lectins, trypsin inhibitors, and antigenic proteins in diet [62,63]. Cellulase enzyme hydrolyzed 62% of the cellulose in Chlorella pyrenoidosa, resulting in a 75% increase in lipid extraction, indicating that enzymes have the potential to improve microalgae digestibility [64]. The addition of non-starch polysaccharide (NSP) enzymes to the diet containing defatted Nannochloropsis sp. biomass increased the protein digestibility in Nile tilapia [35]. Besides enzymes, the addition of organic minerals rather than inorganic minerals in the diet containing microalgae also improved the digestibility of fatty acids, especially polyunsaturated fatty acids in Atlantic salmon [60].

Dry Matter Digestibility
The dry matter contains all the components of biomass except for water. Microalgae's dry matter consists of lipids, proteins, carbohydrates, chlorophyll, vitamins, carotenoids, and ash. Dry matter content varies between 91% and 98% in microalgae [24]. Tables 1 and 2 show the digestibility of the microalgae dry matter in fish. All the values shown in Tables 1 and 2 are the digestibility values reported for microalgae and not for the diet in fish. The digestibility of microalgae dry matter in fishes evaluated ranged from 13% to 97%. In particular, digestibility of dry matter of Schizochytrium sp. in rainbow trout (Oncorhynchus mykiss) was in the range of 91-97% [65]. Similarly, for Nile tilapia (Oreochromis niloticus), the Schizochytrium sp. had dry matter digestibility of 82% [6]. Dry matter digestibility higher than 70% was observed in rainbow trout for Isochrysis sp., Nile tilapia for Spirulina sp., Chlorella sp., Spirulina maxima and Chlorella vulgaris, and African catfish (Clarus gariepinus) for Spirulina maxima and Chlorella vulgaris ( Table 1). The in vitro studies showed that Spirulina platensis, Chlorella sorokiniana, and Chlorella vulgaris had dry matter digestibility greater than 70% (Table 2). These microalgal species may have high digestible dry matter content due to their high amount of easily digestible nutrients and low anti-nutritional factors, as well as less rigid cell wall [66,67].
Microalgae-based diets typically include microalgae as well as other ingredients such as fish meal, plant-based ingredients, minerals, and vitamins. Studies have reported no significant difference in dry matter digestibility between a microalgae-based diet and a control diet lacking microalgae in fish. For instance, a diet containing Chlorella sp., and a diet containing Nannochloropsis oceanica had similar dry matter digestibility to control diet lacking microalgae in European seabass (Dicentrarchus labrax) juveniles [34]. Similarly, in another study, there was no difference in dry matter digestibility between the control and a diet containing 30% defatted Nannochloropsis sp. in Atlantic salmon (Salmo salar) [21]. However, studies also have reported that increasing the algal content in diet negatively affected the dry matter digestibility in fish. For instance, increasing Phaeodactylum tricornutum content in the diet, the dry matter digestibility decreased in Atlantic salmon (S. salar) [12]. In another study, with increased content of whole-cell Nannochloropsis gaditana in diet, the dry matter digestibility decreased in African catfish and Nile tilapia [18]. Similarly, other studies revealed that higher inclusion levels of algae led to a decrease in dry matter digestibility in fish [8]. In some cases, a microalgae-based diet's decreased dry matter digestibility was compensated by a higher intake of feed by fishes [8]. However, in such cases it could result in an increase in the feed conversion ratio.
The disruption of algal cells using bead miller led to an increase in dry matter digestibility of Nannochloropsis oceanica, Chlorella vulgaris, and Tetraselmis sp. for juvenile European seabass (D. labrax) by nearly 21%, 14%, and 50%, respectively [34]. Bead milling of Nannochloropsis gaditana increased the dry matter digestibility for juvenile African catfish (C. gariepinus) and juvenile Nile tilapia by 16% and 18%, respectively [68]. Enzymatic treatment increased the dry matter digestibility of Nannochloropsis oceanica, Chlorella vulgaris, and Tetraselmis sp. for juvenile European seabass (D. labrax) by 27%, 14%, and 12%, respectively [34]. The above results suggest that efficient cell disruption techniques to improve dry matter digestibility differ from one algal species to another. Defatting of the microalgae Nannochloropsis oculata decreased the dry matter digestibility for juvenile Nile tilapia by 7% [38]. This is most likely due to a decrease in the content of easily digestible lipids, such as polyunsaturated fatty acid (PUFA), in fishes, which has a negative impact on lipid digestibility and, as a result, on dry matter digestibility [69]. The starvation of Tetraselmis Suecica increased the dry matter digestibility (estimated by in vitro method) by nearly 5% [24]. This could be due to the fact that nutrient deprivation can increase the amount of highly digestible lipid-like PUFA and starch in microalgae [69]. The pelletization technique used to prepare feed has an impact on dry matter digestibility. This was demonstrated in a study in which extruded pellets had higher dry matter digestibility than pellets made by cold pelletization for Atlantic salmon diets containing Nannochloropsis sp. [21]. The above results suggest that the starvation of microalgae, cell disruption technique, and pelletization method can be applied to improve dry matter digestibility of microalgae.

Protein and Amino Acid Digestibility
Fish require diets comprising of 30% to 55% crude protein and an amino acid supply focused on specific requirements for maximum growth [70]. If the diet includes easily digestible ingredients, the high protein demand of fish can be met. The protein digestibility of microalgae in different fishes ranged from 50% to 94% (Tables 1 and 2). The digestibility of protein for Schizochytrium sp., Isochrysis sp., Spirulina sp., Chlorella vulgaris, Nannochloropsis oceanica, and Nannochloropsis oculata was found to be higher than 80% in fish such as rainbow trout (Oncorhynchus mykiss), Nile tilapia (Oreochromis niloticus), European seabass (Dicentrarchus labrax), and African catfish (Clarus gariepinus) ( Table 1). In comparison, the commonly used ingredient fish meal has protein digestibility ranging from 72% to 92% for salmonids and tilapia [8,11,21,[71][72][73][74][75][76][77]. Other popular ingredients in the fish diet include soybean meal and corn which have a protein digestibility of 91-96% and 53-83%, respectively, for salmonids and tilapia [70]. The wheat middling has a protein digestibility of 20-76% for tilapia [70]. The protein digestibility of seaweed in rainbow trout ranged from 66% to 80% [77]. Unlike seaweed, the microalgal biomass has lower total phenolic content (TPC) (<20 mg GAE g −1 DW) [46][47][48]78,79]. Protein digestibility is indirectly correlated to TPC content [80]. Thus, in general, microalgal biomass has higher protein digestibility than seaweed. Overall, the protein digestibility of selected microalgal species was comparable to that of fishmeal and plant-based ingredients and was higher than that of seaweed.
In the case of microalgae-based diets, a wide range of protein digestibility values has been reported for different fishes. The wide range of results could be explained by differences in feed processing technique, the type of ingredients used in combination with microalgae in the diet, and the digestive systems of fishes. In general, studies have shown a linear decrease in protein digestibility relative to the increase in the content of microalgae in the diet [8,12]. Protein digestibility is negatively affected by the fiber content of the diet, and microalgal biomass has a considerable amount of total fiber [61,81]. The fiber inhibits pepsin activity, which catalyses protein hydrolysis [24,81]. Furthermore, microalgal biomass has relatively high levels of soluble polysaccharide fibers that can entrap proteins in the cellular matrix, rendering them less bioavailable to enzymatic hydrolysis [82]. However, decreased protein digestibility values were compensated by high feed intake for sustaining the fish growth [8]. Overall, at an appropriate concentration of microalgae (less than 15%) in diet, the protein digestibility was not found to be affected [12].    Physical and enzymatic processing of microalgae was found to increase the protein digestibility of microalgal species. In comparison to whole-cell Tetraselmis sp., the bead milling processed biomass had 14% higher protein digestibility for European seabass [34]. Similarly, bead milling improved the digestibility of Nannochloropsis gaditana protein by 16% and 17% in African catfish (C. gariepinus) and Nile tilapia (O. niloticus), respectively [17,68]. Compared to whole-cell Nannochloropsis oceanica, the enzymatically processed microalgae had 6% higher protein digestibility [34]. The addition of organic minerals improved the protein digestibility of a diet containing Schizochytrium sp. [60].
Amino acid digestibility of various microalgae ingredients is shown in Table 3. The amino acid digestibility of N. oceanica and C. vulgaris, for European seabass (D. labrax), and C. vulgaris for Atlantic salmon (S. salar) were higher than 90% [34,83]. Tetraselmis sp. had relatively lower amino acid digestibility for European seabass (D. labrax) juveniles (Table 3). Among individual amino acids of microalgae, arginine, isoleucine, and lysine were generally more digestible than other amino acids. Processing of biomass by physical and enzymatic methods did not increase the essential amino acid digestibility except in cases of threonine and phenylalanine in physically processed Nannochloropsis sp.; however, the digestibility of protein for physically processed Chlorella sp., and Tetraselmis sp. was enhanced by 11−19% [34]. The reason for the increase in amino acid digestibility is that larger size protein would have been cleaved into peptides and individual amino acid during the pretreatment process.

Lipid and Fatty Acid Digestibility
Lipids are an excellent source of energy for fish [14,85,86]. Fish use the beta-oxidation process to break down lipids in the mitochondria of the cell to generate energy. The lipid content of microalgae varies from 1 to 70% of their dry weight. Table 1 shows the digestibility of microalgae lipids in different fish. In Nile tilapia (Oreochromis niloticus), a digestibility of Schizochytrium sp. lipids as high as 98% has been reported [6]. Lipid digestibility higher than 80% was observed in juvenile European seabass (Dicentrarchus labrax) for Chlorella vulgaris, Nile tilapia (O. niloticus) for Chlorella vulgaris, Schizochytrium sp., Spirulina sp., and Chlorella sp., and African catfish (Clarus gariepinus) for Chlorella vulgaris and Spirulina maxima [6,17,34]. However, in some cases, lower lipid digestibility values were reported, as in the case of juvenile African catfish (C. gariepinus) for Nannochloropsis gaditana, which was only 40% [68].
The wide range of lipid digestibility by fishes could be due to the type of lipid and fatty acid present in the microalgae, as well as the fish to which algae is fed. Various studies have reported that an increase in algal content in the diet decreased the lipid digestibility in fish [12,29,32,34,83]. In addition, the presence of lipase inhibitors in microalgae at higher concentrations can decrease lipid digestibility [32]. Many marine microalgae are known to contain lipase inhibitors [32]. Lipase inhibitors inhibit lipase, which is an enzyme responsible for lipid digestion. An example of a lipase inhibitor present in microalgae is terpene caulerpenyene [33]. In addition, the form of lipid could also affect lipid digestibility. For example, polar lipids such as phospholipids were found to be better digested by Nile tilapia than neutral lipids such as triglycerides [6,7].
The digestibility of fatty acids varies with the melting point. Fatty acids with higher melting point have lower digestibility [87][88][89]. The digestibility of fatty acid for fish is also dependent on carbon length and degree of saturation of fatty acid [90]. With an increase in the carbon length of fatty acids, the digestibility of fatty acids decreased [91]. However, an increase in the degree of unsaturation increased the digestibility of fatty acids [87,92,93]. Typically, polyunsaturated fatty acid (PUFA) is highly digestible compared to monounsaturated fatty acid (MUFA) and saturated fatty acid (SFA), as shown for several fishes listed in Table 4. Studies involving other biomass also showed PUFA to be more digestible and absorbed than MUFA and SFA for rainbow trout, cod, and Atlantic salmon [6,12,35,88,[93][94][95][96]. One of the most important fatty acids in fish nutrition is omega-3 fatty acid. Higher digestibility of omega-3 fatty acids, including docosahexaenoic acid (DHA), were observed in Schizochytrium sp., a species rich in omega-3 fatty acid, particularly DHA [6,7,65].

Carbohydrate Digestibility
Digestibility of microalgal carbohydrates depends on the type of carbohydrates present in microalgal species, carbohydrate content in the biomass, and fish species [41]. Therefore, there is a wide variation in the digestibility values reported for carbohydrates of microalgae. The carbohydrate digestibility of individual microalgal species for different fishes ranged from 22% to 83% in Tables 1 and 2. Higher carbohydrate digestibility (greater than 70%) was observed for species including Spirulina maxima and Chlorella vulgaris for Nile tilapia (Oreochromis niloticus) [17]. This can be attributed to the presence of starchlike, easily digestible carbohydrates in these microalgae. Moreover, species including Chlorella sorokiniana, Klamath, and Nannochloropsis sphaeroides displayed higher carbohydrate digestibility as estimated by in vitro studies [24]. Chlorella vulgaris had a higher carbohydrate digestibility value than other algal species in Nile tilapia, African Catfish, and in vitro studies (Table 1).

Ash (Mineral) Digestibility
The ash represents the mineral matter in the feed, which typically contains phosphorus, calcium, potassium, magnesium, and other micronutrients required for fish survival and growth. The ash content of microalgae ranges from 3% to 30%, depending on microalgae habitat [12,22,56]. Fresh water habitat species have less ash content than sea water species. Ash digestibility of microalgae in fish has been reported only in a few studies [12,22,56]. Ash digestibility of individual microalgae ranged from 23% to 83% for different fishes (Figure 1). An increase from 3% to 6% in the inclusion level of Phaeodactylum tricornutum in the diet of Atlantic salmon did not affect the digestibility of ash [12]. The digestibility of ash in the diet fed to seawater fishes has sometimes been reported to be a negative value [99]. This is due to the consumption of seawater by fishes [56,100]. As mentioned earlier, the microalgae-based diet also contains plant-based ingredients. In plant-based ingredients, phytate stores 80% of the total phosphorus content; the phytate chelates minerals and amino acids and is poorly digested by fish [101]. Supplementing the diet with phytases can help to achieve optimal fish growth. In a recent report, a phytase-expressing cellwall deficient Chlamydomonas reinhardtii strain was developed to solve poor phosphorus digestibility and decreased mineral availability [101]. Since some microalgae have welldeveloped genetic tools, methods similar to those described above can be used to over express digestion-related enzymes in microalgae.
One of the important minerals for fish growth and survival is phosphorus. However, data is scarce for the phosphorous digestibility of microalgae. Based on limited reports, the digestibility of microalgal phosphorus in fish was found to range from 38% to 100% [17,18]. The phosphorus digestibility of Nannochloropsis gaditana for juvenile Nile tilapia (Oreochromis niloticus) and juvenile African catfish (Clarias gariepinus) was reported to be 92% and 77%, respectively [18]. The phosphorus digestibility of Spirulina maxima, Chlorella vulgaris, and Scenedesmus dimorphus for Nile tilapia was 93%, 86%, and 39%, respectively [17]. Similarly, phosphorus digestibility values of Spirulina maxima, Chlorella vulgaris, Scenedesmus dimorphus for African catfish (Clarus gariepinus) was 92%, 84%, and 45%, respectively [17]. The phosphorus digestibility of selected microalgae is higher than that of soy meal in different fishes, including European seabass, Senegalese sole, and rainbow trout [102][103][104][105]. The form of phosphorus found in plants is organic phytic acid, which is indigestible and considered anti-nutritional for fish, whereas the form of phosphorus found in algae is not clearly established [105][106][107]. But some studies suggest that phosphorous in microalgae is found in the polyphosphate granules, whose digestibility is yet to be determined [106].
Scenedesmus dimorphus for African catfish (Clarus gariepinus) was 92%, 84%, and 45%, respectively [17]. The phosphorus digestibility of selected microalgae is higher than that of soy meal in different fishes, including European seabass, Senegalese sole, and rainbow trout [102][103][104][105]. The form of phosphorus found in plants is organic phytic acid, which is indigestible and considered anti-nutritional for fish, whereas the form of phosphorus found in algae is not clearly established [105][106][107]. But some studies suggest that phosphorous in microalgae is found in the polyphosphate granules, whose digestibility is yet to be determined [106].

Energy Digestibility
Energy is required by fish to sustain normal body functions, as well as to grow and reproduce. The energy digestibility of microalgae in fishes evaluated ranged from 44% to 90% (Table 1). Energy digestibility higher than 80% was reported in rainbow trout and Nile tilapia for Schizochytrium sp. [6,65], juvenile European seabass for Chlorella vulgaris [34], juvenile Nile tilapia for Nannochloropsis oculata, Spirulina sp., and Chlorella sp. [7,38]. The reported energy digestibility values of microalgae were similar to that of plant-based ones, fishmeal, animal by-products, seaweed for fishes [8,11,72,77,104,[108][109][110]. Energy digestibility in juvenile European seabass (Dicentrarchus labrax) was improved by 5% upon bead milling of the Chlorella vulgaris cell [34]. Bead milling of Nannochloropsis gaditana was also found to be the most efficient technique compared to other cell disruption methods to improve energy digestibility in juvenile African catfish (Clarias gariepinus) and juvenile Nile tilapia, as it improved the value by nearly 17% and 18%, respectively [68]. However, in another study, the enzymatically processed Nannochloropsis oceanica had 11% higher energy digestibility than the same microalgae that had been treated with bead milling for European seabass juveniles (Dicentrarchus labrax) [34]. A significant increase in dry matter

Energy Digestibility
Energy is required by fish to sustain normal body functions, as well as to grow and reproduce. The energy digestibility of microalgae in fishes evaluated ranged from 44% to 90% (Table 1). Energy digestibility higher than 80% was reported in rainbow trout and Nile tilapia for Schizochytrium sp. [6,65], juvenile European seabass for Chlorella vulgaris [34], juvenile Nile tilapia for Nannochloropsis oculata, Spirulina sp., and Chlorella sp. [7,38]. The reported energy digestibility values of microalgae were similar to that of plant-based ones, fishmeal, animal by-products, seaweed for fishes [8,11,72,77,104,[108][109][110]. Energy digestibility in juvenile European seabass (Dicentrarchus labrax) was improved by 5% upon bead milling of the Chlorella vulgaris cell [34]. Bead milling of Nannochloropsis gaditana was also found to be the most efficient technique compared to other cell disruption methods to improve energy digestibility in juvenile African catfish (Clarias gariepinus) and juvenile Nile tilapia, as it improved the value by nearly 17% and 18%, respectively [68]. However, in another study, the enzymatically processed Nannochloropsis oceanica had 11% higher energy digestibility than the same microalgae that had been treated with bead milling for European seabass juveniles (Dicentrarchus labrax) [34]. A significant increase in dry matter digestibility of over 40% was observed for Tetraselmis sp. by both bead milling and enzymatic methods in European seabass juveniles (D. labrax) [34]. High-pressure homogenization improved the energy digestibility of Chlorella vulgaris from 60% to 77% for juvenile Atlantic salmon (Salmo salar L.) [83]. Other than the cell disruption technique, the removal of lipid from the microalgae was found to negatively affect the energy digestibility. For instance, in juvenile Nile tilapia, the energy digestibility of defatted Nannochloropsis oculata was 7% less than that of the whole cell [38]. The removal of energy-rich lipid from biomass may have resulted in a reduction in energy digestibility. The above results suggest that the energy digestibility of microalgae in fish can be improved by careful selection of cell disruption techniques and avoiding lipid extraction of microalgae.

Digestibility of Individual Microalgal Species
Among all the microalgae, the nutrients and energy of Spirulina sp. are the most easily digested by fishes evaluated in the study (Tables 1 and 2). For instance, Spirulina sp.' nutrients and energy were well digested by Nile tilapia [7]. Similarly, the protein digestibility of Spirulina platensis and Spirulina pacifica estimated by in vitro (pepsin-pancreatic system) method was reported to be 94% and 86%, respectively [61]. The cell wall of Spirulina sp. lacks complex polysaccharides, which attributes to its higher digestibility. The dry matter, protein, lipid, and energy digestibility of Schizochytrium sp. were higher in rainbow trout (Oncorhynchus mykiss) and Nile tilapia (Oreochromis niloticus; 82-91%) when compared to many other microalgae [6,65]. Despite the presence of complex carbohydrates such as pectin, Schizochytrium sp. biomass was found to be more digestible by fish, indicating the need for further research in the future. Also, Isochrysis sp. in rainbow trout had higher protein, moderate lipid, and moderate energy digestibility, with values of 87%, 63%, and 73%, respectively [35]. Isochrysis sp. is a diatom that lacks a rigid cell wall and offers better digestibility. Although dry matter digestibility was lower, the protein digestibility was found to be higher (82%) for Chlorella vulgaris and Nannochloropsis oceanica in juvenile European seabass (Dicentrarchus labrax) [34]. Furthermore, in spite of poorer dry matter digestibility, the Nannochloropsis oculata for Nile tilapia and Nannochloropsis sphaeroides F&M-C117 (estimated by in vitro method) had a higher protein digestibility of 80% and 81%, respectively [24,38]. The studies mentioned above show that certain microalgae are an excellent source of digestible nutrients and readily available energy for fish.

Perspective and Future Direction
The composition and structure of microalgae cell walls affect digestibility. This underlines the importance of screening for potential commercial strains that could be easily processed for cell disruption by conventional methods. So far, only a handful of microalgal species have been tested for digestibility. Still, numerous microalgae, including Dunaliella sp., Botryococcus sp., and many cyanobacteria like Anabaena sp. and Nostoc sp.
, are yet to be tested for digestibility in fish and other aquatic species. It is worth mentioning that some strains (e.g., Dunaliella salina) lack a cell wall, which could improve the digestibility of the cellular metabolites in fish. Moreover, the environmental conditions and stress application that can change the biochemical composition of microalgae need to be explored. For instance, nitrogen starvation can lead to the accumulation of PUFA and starch, which are easily digestible by fishes. However, the biochemical response to stress like nitrogen starvation by microalgae varies from one species to another. For instance, nitrogen starvation can suppress the protein content while enhancing the carbohydrate content in several strains while no such difference can be observed for other species [69]. Therefore, optimization of stress conditions and selection of the right species are essential to obtain a suitable biochemical profile with desired digestibility.
So far, screening of microalgal species for digestibility has been tested in limited fishes including salmonids, tilapia, sea-bass, and African catfish. As the aquaculture industry expands rapidly, more novel fish species are introduced, for which the digestibility of microalgae must be tested. The feed screening studies should include the determination of anti-nutritional factors like digestive enzyme inhibitors (eg., terpene caulerpenyene) and factors that decrease nutrient bioavailability in microalgae. Until now, microalgae digestibility studies have focused on determining the digestibility of macronutrients, including lipids, carbohydrates, protein, and energy. However, the individual classes of these macromolecules have been demonstrated to affect digestibility. Therefore, the digestibility and content of fiber, starch, non-starch polysaccharide, amino acids, fatty acids, polar lipids like phospholipids, and neutral lipids like triglycerides in microalgae must be estimated for targeted fishes.
Microalgae biomass generation involves high production cost, which is due to the energy-intensive harvesting step. Microalgal biorefinery routes could be adopted to reduce the production cost of microalgal feed ingredients. In the biorefinery route, the high-value biochemical components like lipids, carotenoids, etc., are separated from biomass, and left-over biomass is used as fish/animal feed. So far, only a few studies have reported the effect of defatted biomass on digestibility in fishes. These studies have shown mixed results. In the biorefinery routes, supercritical fluid extraction and organic solvent extraction are typically employed to produce defatted biomass. Although digestibility studies on defatted biomass generated via the above-mentioned biorefinery routes have been published, no studies on biomass generated via the saponification-based biorefinery route have been reported. The saponification-based biorefinery is primarily used for the production of carotenoids and other value-added products from microalgae. In this approach, free fatty acids, sterols, squalene, and carotenoids are separated from the biomass, while the leftover biomass can be used as fish feed. Therefore, the left-over biomasses from such biorefinery routes also need to be tested for digestibility in fishes.
The use of cell disruption technique can be very useful in improving the digestibility of microalga species. So far, cell disruption-related studies on microalgal digestibility are minimal, therefore, necessitating further exploration. At the same time, there are some side effects of pre-treatment/processing. One of the side effects of processing the biomass to disrupt the cell wall is that it may increase the content of anti-nutrient factor-like fiber, hemicellulose, trypsin inhibitor, and lectin, as demonstrated in a study involving lipid extracted biomass of Nannochloropsis oculata [38]. Hence, the level of these anti-nutrient substances must be measured before and after pretreatment of microalgae to ensure the high digestibility of nutrients. Although cell disruption processes are useful to improve nutrient and energy digestibility of microalgae, these techniques utilize considerable energy, thus increasing the production cost of feed [111]. Therefore, cost-effective methods need to be identified to prepare algal feed and disrupt the cell wall of microalgae. A promising cost-effective route may be the utilization of cell-wall-less microalgae such as Isochrysis sp. and Dunaliella salina as promising candidates in future digestibility studies.
The selection of a high-pressure and high-temperature extrusion process to make microalgae-based feed pellets can further improve nutrient and energy digestibility. However, microalgal digestibility studies employed mostly cold pelletization or room-temperature extrusion processes. Future studies should focus on the effect of pellet-making processes (i.e., extrusion, cold pelletization, etc.) on the digestibility of different algae. The highpressure-high-temperature process of extrusion can damage the digestive enzymes present in microalgae, but at the same time degrade anti-digestive factors. Therefore, it would be interesting to study the effect of the extrusion process on digestibility inducing and inhibiting factors in microalgae.
On the methodology aspect, the processing conditions of pellets are not appropriately described in many studies. For instance, in several studies, the information on temperature and pressure at which the extrusion process is carried out and the type of pellet generated is missing. As mentioned earlier, these factors could significantly affect the digestibility of nutrients and energy of microalgae. Therefore, future studies should include such data. As microalgae are included only as an ingredient in the fish diet, many studies have determined the digestibility of the fish diet rather than individual microalgae. Therefore, more data on microalgae digestibility in fishes by direct and in-direct assessment is required in the future. Nevertheless, from the limited studies, the nutrient and energy digestibility of microalgae in fishes seems promising.

Conclusions
Microalgae are a promising aquafeed ingredient with the right blend of nutrients and functional components. Microalgae-based feed displays a wide variation in nutrient and energy digestibility, depending on several factors. This stresses the importance of digestibility estimation in microalgae-based feed formulation. Spirulina sp., Isochrysis sp., Chlorella vulgaris, and Schizochytrium sp. are some of the microalgae, which showed very high nutrient and digestibility values for the fishes. So far, the use of cell disruption techniques, pellet processing methods, selection of appropriate species, and optimization of environmental conditions to generate a suitable biochemical profile have been useful in improving the digestibility of microalgae for fishes. More screening of microalgae for digestibility in different fishes will further reveal its true potential.