Enhancing Digestibility of Chlorella vulgaris Biomass in Monogastric Diets: Strategies and Insights

Simple Summary This study aimed to review the potential of the microalgae Chlorella vulgaris (CV) as an animal feed source especially for monogastric animal diets, due to their high content of essential nutrients. The findings of a systematic literature review showed that adding CV to poultry and swine diets had different results in terms of nutrient digestibility, although pre-treatments increased nutrient accessibility and digestibility. Cost-effectively produced CV biomass has the potential to be a supplement or substitute for expensive feed ingredients and improve animal health and immunity. Variations in results may be due to differences in microalgal strain, cultivation conditions and dietary inclusion levels. This study provides new insights into the use of CV biomass in animal diets. Abstract Microalgae, such as Chlorella vulgaris (CV), have been identified as promising animal feed sources due to their high content of essential nutrients, including proteins, total lipids, n-3 polyunsaturated fatty acids, and pigments. This study aimed to review the digestibility, bioaccessibility, and bioavailability of nutrients from CV biomass, and to analyse strategies to enhance their digestibility in monogastric animal diets. The study conducted a systematic review of the literature from databases such as PubMed, Scopus, Google Scholar, and Web of Science, up until the end of January 2023. The results of adding CV to poultry and swine diets were diverse and depended on a number of variables. However, pre-treatments applied to CV biomass improved nutrient digestibility and accessibility. CV biomass, produced in a cost-effective manner, has the potential to serve as a supplement or substitute for expensive feed ingredients and improve animal health, physiology, and immune status. Variations in results may be due to differences in microalgal strain, cultivation conditions, and dietary inclusion levels, among other factors. This study provides new insights and perspectives into the utilization of CV biomass in animal diets, highlighting its potential as a valuable ingredient to improve nutrient utilization.


Introduction of Chlorella vulgaris
Microalgae, such as Chlorella vulgaris, have garnered interest as food and feed sources due to their high growth rate and rich content of essential nutrients, particularly protein [1][2][3]. Unlike traditional food crops, microalgae can grow in a variety of environments without requiring arable land, and, therefore, avoid competition with crops, such as soybean and cereal grains [4]. In addition, the ability of microalgae to fix carbon from the atmosphere makes them a valuable tool in reducing greenhouse gas emissions [5]. Therefore, the reasons for choosing microalgae as an ingredient or supplement for animal feed are, for instance, their high nutritional value, if cultured under optimal conditions and fed to adapted animals [6], and their ability to act as feed additives. Particularly, extracted Figure 1. Bioaccessibility, bioavailability, and digestibility of Chlorella vulgaris fed to poultry and swine.

Nutritional Composition of Chlorella vulgaris
The chemical composition of CV is rich in various nutrients including protein, which can reach levels of up to 65.5% dry matter (DM) [23]. However, its protein content is highly dependent on the cultivation conditions and can be as low as 13.6% DM [24] under nitrogen-limiting conditions [25]. The protein quality of CV is high, as it contains all

Nutritional Composition of Chlorella vulgaris
The chemical composition of CV is rich in various nutrients including protein, which can reach levels of up to 65.5% dry matter (DM) [23]. However, its protein content is highly dependent on the cultivation conditions and can be as low as 13.6% DM [24] under nitrogenlimiting conditions [25]. The protein quality of CV is high, as it contains all essential amino acids, including a significant amount of leucine and lysine (average of 9 to 10% of total amino acids) [26,27]. Lysine, in particular, is a limiting amino acid for poultry and swine, and can be found in C. vulgaris at levels as high as 10.4 to 13.2% [26,27].
The dried biomass of CV also contains significant amounts of ash, reaching up to 27.3% [28]. The ash is rich in essential minerals such as phosphorus, potassium, iron, manganese, and zinc [24,26]. While there may be some concern about heavy metal toxicity in Chlorella spp., studies have shown that such toxic elements, including arsenic, cadmium, and mercury, are present in concentrations as low as 0.59-1.1 mg/kg, 0.01-0.10 mg/kg, and 0.02-0.10 mg/kg, respectively, and no lead was detected [25]. These low levels of heavy metals ensure the safety of CV as a dietary supplement.
CV contains a variable amount of carbohydrates, which can range from 8.08 [29] to 65.0% [23], with an average of 23.4% DM ( Table 1). The cell wall of CV is composed of hemicellulose (22-25%) and chitin-like polysaccharides (60-66%) [16], and also contains a small amount of starch (up to 4.41% DM). The variability in carbohydrate content is largely influenced by growth conditions and the stage of algal growth [16,30]. In early stages of cell growth, the cell wall of CV is composed of a single microfibrillar layer, while in later stages, a two-layer structure appears with a thick outermost layer and a thinner inner layer separated by an electron translucent interspace [16].
The total lipid content of CV is significant, with an average of 12.1% DM. However, the lipid content can vary significantly, ranging from 5.10 [31] to 19.7% DM [32]. The variability is largely due to differences in nitrogen supply, as nitrogen limitation can increase lipid content while decreasing protein content [25]. Environmental stress, such as high light intensity and nitrogen deprivation, has also been shown to increase lipid production in other microalgae [33]. The essential PUFA 18:2n-6 and 18:3n-3 are the most predominant fatty acids in CV biomass, averaging 21.6% and 18.8%, respectively [23,26,32]. The 18:3n-3 and 18:2n-6 are converted into n-3 LC-PUFA and n-6 LC-PUFA, respectively, although the efficiency of these pathways is low [34]. Nevertheless, including CV in animal diets, such as piglet [15] and finishing pig [35], can increase the n-3 PUFA content in meat and improve its nutritional value.
CV is a rich source of pigments such as chlorophylls a and b and carotenoids (e.g., β-carotene and lutein) [20]. The total chlorophyll and carotenoid content in CV can vary and reach levels up to 24.0 [28] and 3.49 g/kg DM [28], respectively, depending on the drying process, culturing conditions, and harvest time [36]. These pigments have significant antioxidant and radical scavenging properties [36]. The accumulation of carotenoids in the longissimus lumborum muscle of finishing pigs was shown to enhance the nutritional value of meat without affecting its colour [35]. In addition, the accumulation of carotenoids in egg yolks [37] and broiler chicken meat [38] has been reported to cause a decrease in redness (a*) or an increase in yellowness (b*) parameters, which could potentially impact consumer perception of the egg or meat [6]. Additionally, CV is a valuable source of vitamin E (α-tocopherol) [35] and B-complex vitamins, with a prevalence of niacin (vitamin B3), with levels ranging from 145 [31] to 247 [39] mg/kg DM, as well as active forms of vitamin B12 (cyanocobalamin) [40].

Enhancing the Digestibility of Chlorella vulgaris Nutrients
Disruption of algal cell wall is necessary to improve the digestibility and bioaccessibility of CV [21]. This can be achieved through mechanical/physical procedures, such as high-pressure homogenization and sonication [48], or enzymatic pre-treatments [49,50]. Previous studies showed that incorporating undisrupted microalgae in animal diets may require double the amount to have the same effect as disrupted microalgae [11]. The polysaccharide content should also be considered, since high levels of polysaccharides can negatively affect protein digestibility [51].
Using in vitro experiments, Gerken et al. [49] tested the enzymatic (chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase) degradation of Chlorella cell walls and reported that this microalga was more sensitive to chitinase and lysozyme than to other enzymes. Both enzymes drastically affected cell permeability, thus influencing nutrient digestibility. According to Canelli et al. [50], enzymatic pre-treatments are a good choice for improvement of nutrient bioaccessibility, expressed as the proportion between the amount of nutrient incorporated into the micellar phase and that in full digesta. The bioaccessibility of proteins was particularly improved from 49.2 to 58.7%, without lipid oxidation and preserving cell wall integrity, opposing the mechanical pre-treatments, like high-pressure homogenization, which drastically affected oxidation and provoked some off-flavour formations although enhancing lipid accessibility (36.9 to 61.8%) compared to controls. Gille et al. [48], using an in vitro digestion model, concluded that sonication before digestion of CV, improved bioaccessibility of lutein at 11% (7 to 18% after sonication) and β-carotene at 12.5% (0 to 12.5% after sonication). Specifically, Kose et al. [52] tested the influence of pancreatin before in vitro protein digestion with trypsin and α-chymotrypsin, and apparent digestibility improved from 35% (protein digestibility of untreated CV) up to 70%. According to Wild et al. [53], the disruption of CV's cell walls can enhance in vitro crude protein digestibility by 5% (79 to 84%), compared to non-cell-disrupted microalgae. An in vitro trial, where CV was digested with pepsin and pancreatin, reported some nutrient digestibility values. Digestibility was over 60% for dry matter, between 60 and 70% for carbohydrates and organic matter, and 76% for crude protein [14]. Table 2 summarises the main effects of in vitro pre-treatments on the hydrolysis and digestibility of CV. Pepsin and pancreatin Dry matter, carbohydrate and organic matter, and crude protein digestibility of 60%, 60-70%, and 76%, respectively Niccolai et al. [14] A study by Neumann et al. [54] tested the impact of various pre-treatments on the digestibility of CV biomass in mice. The study incorporated 5, 15, and 25% of ball-milled CV (phototrophic or mixotrophic cultured) into mouse diets and found that protein availability was not impacted by the inclusion of CV up to 25%. However, the mixotrophically cultured CV at 25% had the lowest values of apparent digestibility (AD at 76.4%) and protein net utilization (NPU at 45.9%). Bead milling was identified as an effective method for disrupting the cell wall, thereby improving protein bioavailability. Moreover, the study determined the fatty acid content in livers and calculated the absorption index to assess the bioavailability of fatty acids. Despite the fact that CV-containing diets had ten times higher polyunsaturated fatty acids (PUFAs) than the control diet, the bioavailability of fatty acids was not affected and did not differ from control values.
Tsiplakou et al. [55] investigated the effect of 1% lyophilized CV on the chemical composition and fatty acid profile of goat's milk and found no changes in the fatty acid profile. Meanwhile, Tibbetts et al. [56] studied the influence of cell-disrupted and non-disrupted CV on the diet of juvenile Atlantic salmon. The results showed that the inclusion of celldisrupted C. vulgaris from 6 to 30% feed did not impact dietary dry matter digestibility, and similar results were obtained for lipid and protein with algal levels up to 18% and 24%, respectively. However, all incorporation levels improved the apparent digestibility coefficient of carbohydrates. The moderate inclusion of whole-cell CV up to 18% did not affect the dietary ADC for most essential amino acids, but high inclusion (24 to 30%) of cell-disrupted C. vulgaris did not affect this parameter for any essential amino acid.
In a trial conducted by Kholif et al. [57], the effect of dietary supplementation with 10 g/day of CV was evaluated in goats. The study involved two groups of goats, one that received the CV supplement along with copper (ALCU) and another without copper supplementation (AL). The results showed that the inclusion of CV improved the nutrient digestibility of crude protein, ether extract, neutral detergent fibre, and acid detergent fibre. Additionally, CV-containing diets led to an increase in the concentrations of total unsaturated fatty acids (9.8% and 5.4% for ALCU and AL, respectively), monounsaturated fatty acids (9.8% and 5.2% for ALCU and AL, respectively), and total conjugated linoleic acid (7.4% and 9.3% for ALCU and AL, respectively). The concentrations of saturated fatty acids also decreased by 4% for ALCU and 2.4% for AL, respectively. The improvement in nutrient digestibility was attributed to the presence of Chlorella growth factor [58]. Table 3 summarizes the main effects of different pre-treatments on the hydrolysis and digestibility of CV when tested in animal trials.

Impact of Chlorella vulgaris Biomass Digestibility in Poultry
The impact of CV on poultry digestion has been an area of interest for many years, dating back to 1950. Although Alshelmani et al. [59] refers to a widespread use of CV in poultry nutrition, there is still a lack of information on its effects on nutrient digestibility, bioavailability, and accessibility when incorporated into poultry diets.
The inclusion of CV in poultry diets has been studied for its potential to provide essential amino acids, fatty acids, and antioxidants. According to Kang et al. [60], 1% CV inclusion may impact the palatability of the diets, leading to reduced feed intake and average daily gain. On the other hand, Zheng et al. [61] found that incorporating 0.1 or 0.2% fermented CV in laying hen diets for 42 days improved egg production and yolk colour. The improvement was attributed to the enhanced availability of CV compounds after the fermentation process, which also positively impacted the hens' digestive efficiency by altering the microflora profile in the ceca. The shift in microflora may have degraded algal polysaccharides and other components, contributing to more efficient digestion.
Alfaia et al. [38] recently evaluated the impact of 10% CV on broiler performance, meat quality, and lipid composition. The study found that the inclusion of CV, either alone or in combination with enzymes, led to an increase in the viscosity of the duodenum, jejunum, and ileum. The combination of CV with enzymes, Rovabio Excel AP (0.005%) and a mix of recombinant CAZymes (0.01%), resulted in higher viscosity compared to CV alone. Despite this, the study found that CV had a minor impact on the fatty acid composition in breast or thigh meat, but it did enhance some PUFAs, such as 18:3n-3, in the breast.
Roques et al. [62] also evaluated the effect of 0.8% dried powder CV in broiler diets on growth performance, immune response, and intestinal morphology. The study found that the inclusion of CV at this level had a positive impact on overall broiler performance and they maintained a strong immune response. These findings have led some animal nutritionists to consider the use of low doses of CV as a cost-effective alternative to traditional broiler feed formulations.
Kang et al. [63] and Mirzaie et al. [64] have studied the impact of Chlorella by-products on various aspects of poultry nutrition, such as broiler performance, meat quality, and gut health. These studies showed that incorporating 2.5, 5.0, or 7.5% Chlorella by-products into broiler diets increased villus height and crypt depth, which could enhance nutrient absorption and utilization [63]. Similarly, Mirzaie et al. [64] found that feeding 1 or 2% Chlorella by-products improved intestinal morphology by increasing villus height and crypt depth, and reducing the villus height to crypt depth ratio [64].
As described by Roques et al. [62] and Kang et al. [63], the small intestine, particularly the jejunum, plays a crucial role in digestive processes, such as enzyme digestion and nutrient uptake. The villus height reflects the surface area available for nutrient absorption, while the crypt depth indicates the rate of cell removal in the villi. A deeper crypt can suggest faster tissue turnover, which may be the organism's response to counteract the effects of harmful toxins [62,63]. A decreased villus height to crypt depth ratio is an indication of improved digestive efficiency in the small intestine [63]. The development of favourable intestinal morphology is a hallmark of a healthy gut, with improved nutrient absorption and bioavailability for the animals [63]. Table 4 summarizes the effects of CV inclusion in poultry feeding.
Overall, CV is also a good source of essential amino acids, fatty acids, and antioxidants, and, therefore, its incorporation into poultry diets can enhance polyunsaturated fatty acids in meat, improve egg production, have positive effects in broiler performance and digestive efficiency, and induce a good immune response. However, this microalga might influence the viscosity of the duodenum, jejunum, and ileum when added at an ingredient level, which can compromise nutrient digestibility. Table 4. Summary of the main effects of inclusion of Chlorella vulgaris (CV) biomass or Chlorella by-products in poultry diets.

Influence of Chlorella vulgaris Biomass Digestibility in Swine
The impact of incorporating CV biomass into swine diets on bioaccessibility, bioavailability, and digestibility has been scarcely reported in the literature. Yan et al. [65] conducted a study involving the inclusion of 0.1 and 0.2% fermented CV in the diets of growing pigs. The results showed that the apparent total tract digestibility (ATTD) of nitrogen and energy was not affected, though there was a tendency for a slight decrease in ATTD of nitrogen (78.87 to 78.37%) and an almost 1% increase in ATTD of energy (75.74 to 76.94%) in comparison to the control group. However, the inclusion of 0.1% fermented microalga had a significant effect on dry matter ATTD, improving it from 76.04 to 78.61%. The authors also found that the inclusion of fermented CV reduced the concentration of E. coli and increased the concentration of Lactobacillus in the gut microbiome. This shift in microbial populations and decrease in faecal noxious gas content improved gut health and likely contributed to the increased ATTD [65,66]. The effects of incorporating this microalga on the gut microbiome of swine must be considered, as the intestine is a major site for nutrient absorption and plays a critical role in altering production performance [66]. According to Furbeyre et al. [67], the inclusion of 1% spray-dried Chlorella spp. in the diets of weaned piglets improved the ATTD of crude energy and showed a tendency to enhance the ATTD of dry matter, organic matter, and neutral detergent fibre. The height of the jejunum villus was also increased with Chlorella inclusion, thereby improving nutrient digestibility. During the crucial post-weaning period, Chlorella can effectively manage mild digestive problems, as demonstrated by a decrease in diarrhoea incidence with its inclusion. Conversely, Furbeyre et al. [68] reported shorter ileal villus and a higher and earlier occurrence of diarrhoea but with a fast recovery in piglets fed bead-milled Chlorella at 385 mg/kg body weight per day. This may be due to the bead-milling pre-treatment, which increased the viscosity and concentration of E. coli populations, leading to looser faeces.
The inclusion of 5% CV into the diets of finishing pigs was previously shown to improve lipid, antioxidant, pigment, and n-3 PUFA meat content, resulting in a reduction of the n-6:n-3 PUFA ratio and an overall improvement in the nutritional value of pork [35]. The combination of CV with a mixture of enzymes (0.005% Rovabio Excel AP or 0.01% mix of recombinant CAZymes) further enhanced C22:5n-3 and C22:6n-3 contents in meat by 1.6 times compared to control, without affecting microalgal digestive utilization by pigs under these conditions. Similarly, Martins et al. [15] found that incorporating CV, with or without enzymes, in piglet diets improved the nutritional value of meat by increasing the total carotenoid content (a two-fold increase compared to control) and n-3 PUFA while reducing the n-6:n-3 ratio. This positive result demonstrates a good correlation between the compounds found in microalgae and those deposited in muscle.
The study by Martins et al. [69] investigated the impact of 5% CV incorporation on nutrient digestibility of weaned piglets, either alone or in combination with enzymes (0.005% Rovabio Excel AP or 0.01% mix of recombinant CAZymes). The results showed that CV incorporation had a negative effect on ATTD, particularly of fibre, due to decreased effectiveness in CV cell wall disruption in the intestine. The viscosity of the duodenum and the height of the jejunum tended to increase with the addition of the microalga, but the simultaneous increase of duodenum villus height may have contributed to a healthier microbiota and improved gut health by stimulating prebiotic populations. The combination of CV and Rovabio resulted in values that were close to the control, suggesting a better degradation of the cell wall and improved nutrient digestibility [69].
Lastly, Ribeiro et al. [70] studied the impact of 5% CV incorporation, either alone or in combination with enzymes (0.005% Rovabio Excel AP or 0.01% mix of recombinant CAZymes), on the livers of finishing pigs. CV inclusion influenced lipid metabolism and oxidative stress, while the addition of CAZymes improved liver metabolism of n-3 PUFAs compared to the control group, leading to enhanced PUFA digestibility and hepatic metabolism. The combination of CV and CAZymes also decreased oxidative stress, which was suggested to be related to an increase in carotenoid content in the liver. The effects of CV inclusion in swine feeding are summarized in Table 5.  Overall, the incorporation of CV in swine diets may improve ATTD of gross energy and dry matter with a tendency to enhance the ATTD of nitrogen and organic matter, although it can negatively affect fibre ATTD. This microalga can also improve lipid metabolism, and, thus, increase n-3 PUFAs and decrease the n-6:n-3 PUFA ratio in meat.

Conclusions and Future Perspectives
This review showed that the impact of incorporating CV in poultry and swine diets varies and is influenced by multiple factors, including microalga strain, cultivation conditions, and dietary inclusion levels. However, pre-treatments applied to microalgal biomass can improve nutrient digestibility and accessibility. CV biomass can serve as a feed supplement or partial substitute for common feed sources, providing valuable basic nutrients, pigments, antioxidants, vitamins, growth factors, and prebiotics. This can increase the nutritional value of animal products, promote animal physiology and health, and ultimately lead to a more sustainable and profitable animal production system. Further research is required to optimize the application of CV in monogastric diets, including the selection of appropriate strains, cultivation conditions, pre-treatment methods, and inclusion level. This can lead to a better understanding of the effects of CV on animal health, digestion, and overall performance, and, thus, to a more widespread and efficient use of this microalga in animal nutrition. Additionally, further work is needed to investigate the mechanisms behind the positive effects of CV on animal health, including the role of microalgae on gut microbiota and regulation of oxidative stress. These insights will be critical in the development of more effective animal feeding strategies that enhance animal health, welfare, and performance.