1. Introduction
Due to the high growth population ratio and the subsequent fish protein demand, aquaculture is one of the fastest-growing food-producing sectors [
1]. This growth must be in consonance with the Sustainable Development Goals (SDGs) established by the UN in the Agenda 2030, especially in aquaculture nutrition [
2]. Traditional ingredients used in the design of aquafeeds for cultured fish, i.e., fishmeal and fish oil, are considered unsustainable due to being derived primarily from fish obtained through extractive fishing, generating wild fish population reliance. In this way, many scientific efforts have focused on exploring alternative protein sources suitable for aquafeed formulation [
3]. A nutritional alternative for aquafeed design could be the use of plant protein meals, as they have shown successful application as fish feed ingredients in various studies’ assays [
4]. However, vegetal protein sources can contain a broad variety of anti-nutritional factors. High levels of these components in feeds can lead to adverse effects on the growth [
5] and digestive enzyme activities [
6] of cultured fish.
In this context, microalgae could constitute an optimal complement for aquafeed design. They are usually the main feed of some farmed species, such as some mollusks, and critical ingredients in the early stages of some teleost fish species [
7]. The recent industrial-scale microalgae production has sparked interest in their application in aquafeeds [
8]. Due to their high protein content, good amino acid profile, and high quantity of essential fatty acids, marine microalgae are considered suitable substitutes for fishmeal in several fish species [
9]. Thus, the incorporation of marine microalgae into aquafeeds has been assessed in several fish species, demonstrating favorable results in terms of growth performance, physiology, and metabolism [
10,
11], even at low or very low levels of inclusion [
12]. Moreover, including microalgae has promoted positive effects on fish pigmentation [
13] or product quality [
14], which are particularly important for consumers.
However, some microalgae have a complex cell wall, which may contain anti-nutritional factors, making it difficult for carnivorous fish such as gilthead seabream (
Sparus aurata) to extract nutrients from them efficiently [
15]. Furthermore, other microalgae species possess high recalcitrant cell walls, such as
Chlorella species [
16], or high carbohydrate content that impair the digestive enzymatic activity [
17,
18]. This impairment can reduce feed digestion and absorption, negatively affecting growth performance [
19]. In this sense, the role of microorganisms in the hydrolysis of complex compounds through degradative processes is well known [
20], and the reduction in levels of anti-nutritional factors such as phytate, tannins, or protease inhibitors in plant substrates has been verified due to the action of microbial activities [
21,
22].
Functional diets that include probiotic microorganisms have enormous potential in addressing the present nutritional problems in aquaculture. In this sense, probiotics can aid in the breakdown of various components of aquafeeds, such as carbohydrates, lipids, and anti-nutritional factors, thereby enhancing growth and feed conversion. It is important to note that the enzymatic activity of microorganisms can be modulated by the composition of the medium from which they were isolated or the medium in which they grow. For example, the synthesis of microbial chitinase is controlled through a receptor-inducer system, susceptible to significant effects from cultivation conditions and media components in diverse
Bacillus strains [
23]. Similarly, it was observed that maltose and beef extract served as the most effective carbon and nitrogen sources, respectively, significantly influencing the production of protease enzymes in
Bacillus aryabhattai Ab15-ES [
24]. This approach could provide a valuable strategy for identifying novel enzymes with unique catalytic properties and applications in various biotechnological fields, including aquaculture, food, and pharmaceutical industries.
Gilthead seabream (
S. aurata) is one of the top-cultured marine fish species in the Mediterranean, including Spain [
1], being a perfect model of carnivorous teleost in European aquaculture. Another group of fish species, which are acquiring a special role in the diversification of Mediterranean aquaculture, is the mullets. They have been described as an easily cultured species, making them a promising candidate for aquaculture diversification at a low-trophic level [
25,
26]. Grey mullet (
Mugil cephalus) is a cosmopolitan fish strongly ligated to estuary water with herbivorous feeding behavior, mostly phytoplankton and detritus, having a digestive system specially adapted to that feeding regime [
27].
Feeding fish with microalgae-supplemented diets involves a selection pressure on their intestinal microbiota that can be used to achieve enrichment in bacteria with a set of enzymatic activities capable of metabolizing and mobilizing the components, particularly those enriched with microalgae while also having antibacterial activity against fish pathogens. That way, this piece of research was aimed at assessing the potential of microalgae blend as a dietary ingredient for feeding carnivorous fish, such as gilthead seabream, and herbivorous fish, such as mullet juveniles, and to isolate and characterize probiotic microorganisms from the intestinal microbiota of fish fed with microalgae diets. The microorganisms will be isolated from different media and characterized based on their enzymatic activities, as well as their antagonism against important fish pathogens.
4. Discussion
Microalgae are often used as food sources in aquaculture for bivalves and other filter-feeding species as rotifers or artemia, commonly employed as a first feed for larval fish culture due to their nutritional content and their capacity to synthesize and store essential polyunsaturated fatty acids (PUFA) [
40]. Although its use as an aquafeed ingredient for the fattening phase of fish is not widespread, in recent years, there has been a growing interest in exploring the potential of microalgae as functional components in aquafeed design for fish, replacing traditional ingredients such as fish and plant protein. These microalgae offer various health benefits, enhance growth, and improve the quality of fish products [
11,
12,
13,
14,
41].
In the present study, we employed a blend of microalgae (25%
Chlorella sp., 25%
Arthrospira sp., 25%
Tisochrysis sp., and 25%
Nannochloropsis sp.) in aquafeeds. These species have been previously evaluated for their possible use as dietary ingredients because they are a good source of proteins, amino acids, essential fatty acids, vitamins, and minerals [
42,
43,
44,
45].
During the assay period, no mortality was registered for fish fed with different diets. This could indicate that designed diets cover energy demands and structural components for both fish’ correct development and growth. In terms of growth performances, this study shows fish species differences. It is worth noting the lower growth potential of
M. cephalus with respect to
S. aurata, as showing lower values of Weight Gain (WG), Specific Growth Rate (SGR), and Feeding Efficiency (FE). This would represent different growth rhythms and productive performances that carnivorous species [
12] showed compared to omnivorous/herbivorous fish species [
45].
For
M. cephalus, there were no significant differences observed in growth parameters (K, WG, SGR, FE), somatic index (HSI and ILI), and plasma biochemical parameters analyzed. These findings align with the results presented by García-Márquez et al. [
45], demonstrating that the incorporation of microalgae in the diet did not negatively impact mullet growth performance, feed efficiency, and plasma metabolites. The lack of biochemical and physiological modifications would suppose that the use of microalgae supplemented diet (M25-MC) does not imply a significant change in the nutrient assimilation structures (intestinal absorption surface), energy storage (liver), and transport (plasma) in mullet. This assumption is further reinforced by the lack of significant differences in cortisol plasma levels observed in fish fed both diets (CT-MC and M25-MC). Elevated cortisol levels are associated with increased energy expenditure and reduced growth rate [
46]. Consequently, in the absence of relevant results, it could be interesting to analyze quality product parameters to evaluate the suitability of a supplement microalgae diet for the culture of this fish species.
For
S. aurata, this assay shows that microalgae addition significantly promoted growth performance. Fish fed the M25-SA diet showed higher WG, SGR, and FE values than those fed the CT-SA diet. These results are in accordance with other studies where
S. aurata fed supplemented microalgae extract diets also showed higher growth values without adverse effects in allometric growth (K) of fish [
11,
12,
41]. This fact could be the cause or the consequence of the significant increase in the intestinal length, with improved feeding efficiency of fish fed diet supplemented with microalgae ingredients compared to those fed a control diet, given that as denoted by Molina-Roque [
11] and Perera et al. [
12], a higher intake of diets formulated with a high content of vegetable ingredients is associated with a significant increase in the intestine length index, as well as the absorption area of the intestinal microvilli, with improved feeding efficiency of fish fed diets supplemented with plant ingredients compared to those fed without plant ingredients. This would demonstrate the phenotypic plasticity of carnivorous fish, such as
S. aurata, in adapting to a diet with a higher proportion of plant protein, resulting in improved nutrient processing and assimilation [
47]. Furthermore, these observations may suggest the positive impact of microalgae inclusion in the aquafeed formulation on product performance. The lack of significant differences in the MSI and lower HSI values observed in fish fed the M25-MC diet, as compared to the control diet, suggests that there is no additional accumulation of hepatic or perivisceral fat. This finding, in line with the observations of Molina-Roque et al. [
11], indicates that the achieved growth is likely associated with fillet yield rather than an increase in perivisceral fat accumulation, which could potentially have adverse effects on consumers.
Regarding intermediary metabolism, a significant increase in plasmatic glucose and triglyceride levels was observed in seabream fed the M25-SA diet compared with fish fed control diet. This suggests that the M25-SA diet significantly enhanced carbohydrate and lipid metabolism, which could suggest a good metabolic condition, as aerobic glycolysis is predominant in obtaining high-energy biomolecules such as ATP and NADH Krebs’s cycle [
48]. Similarly, triglycerides are associated with mitochondrial energy mobilization, aerobically, next to carbohydrates [
49]. The increase in plasma triglycerides in fish fed the M25-SA diet may be linked to higher intestinal bioaccessibility of nutrients, particularly fatty acids from microalgae [
11]. Although no significant differences were shown in plasma circulating proteins, the
p-values (
p = 0.053) were close to the significance limit of
p < 0.05. Proteins serve as an energy source and structural component for tissue development and growth [
50,
51]. Although fish fed the M25-SA diet exhibited the highest mean value, the lack of significant differences in plasma protein concentration between the experimental groups indicates a homeostatic balance at circulating levels. However, it could be interesting to highlight that higher levels of plasmatic proteins could be associated with higher growth parameters shown by fish fed M25-SA diet, resulting in high protein quality of microalgae. The analysis of growth and metabolic results, together with the lower values of cortisol, a hormone associated with situations of stress prolonged [
52] and whose low levels can stimulate protein synthesis resulting in better growth [
53], shown by the fish fed M25-SA diet, could denote the suitability of microalgae inclusion in aquafeed for carnivorous cultured teleost, as
S. aurata, in terms of nutrition and animal welfare.
Finally, it is important to address the impact of the COVID-19 pandemic during the execution of our feeding experiment, which took place between March and June 2020. The pandemic significantly affected our research operations, leading to a reduced workforce and limited access to facilities, making it challenging to include additional biological replicates in this study. While we fully acknowledge the importance of replicates for robust statistical analysis, the unique circumstances imposed by the pandemic made it impractical to incorporate them in this specific study. Despite the absence of biological replicates, we proceeded with this study using sufficient sample size and statistical power to maintain a level of confidence in the results, considering the limitations brought about by the pandemic. We carefully evaluated the experimental conditions and the available resources, and the decision to proceed without biological replicates was made in the interest of conducting this study under the prevailing circumstances. Although the results obtained are promising and provide valuable insights, we are fully aware that biological replicates are critical in scientific research to enhance data reliability and generalizability. The absence of biological replicates may potentially impact the robustness of our conclusions. Therefore, we emphasize the need for further trials with increased replicates to gain a more comprehensive understanding of the beneficial effects of microalgae inclusion on fish.
The gastrointestinal tract’s microbial community in fish plays important functions, including vitamin production, nutrient distribution, regulation of innate immunity, and maintenance of intestinal tissue integrity [
54]. These functions can be influenced and modulated by the fish’s diet [
55]. The isolation and characterization of native potential probiotics would enable the detection of positive benefits, e.g., in vitro antagonistic activity towards pathogens or extracellular enzyme production [
56]. Probiotics are used in aquaculture as additives to enhance host health by improving feed utilization and digestion, enhancing digestive enzymatic activity, modulating intestinal microbiota, improving the immune system, and controlling fish diseases [
57]. Moreover, probiotic bacteria originating from fish (autochthonous) are expected to exhibit superior performance compared to those obtained from terrestrial hosts when used in fish applications [
58].
In this way, Chivotiya et al. [
59] demonstrated that precise optimization of various components (e.g., urea, peptone, calcium chloride, magnesium sulfate, trace elements) in a specific medium can significantly enhance cellulase production from gut bacteria of tilapia fish. This optimization was achieved using the Plackett Burman design and carboxymethyl cellulose (CMC) as the substrate. Similarly, Yang et al. [
60] determined the optimal growth conditions (culture media, temperature, and pH) for lactic acid bacteria (LAB) to optimize the production of bacteriocins, which were significantly different in MRS versus BHI. Therefore, the importance of isolating microorganisms from different culture media to develop an efficient technology for the optimized production of desired enzymatic and antagonistic activities is clear since native probiotics would establish within the original host more efficiently [
61].
In this study, we screened 117 bacterial strains isolated from the gastrointestinal tract of seabream fed the M25-SA diet, aiming to identify indigenous candidate probiotics. Probiotics can produce different enzymes of biotechnological interest, in particular in the aquafeed industry as a feed additive for improving the digestion and absorption of different energy sources (e.g., carbon, lipids, proteins), and hydrolyzing different anti-nutritional factors (e.g., phytate, tannins, cellulase) present in the feeds [
22,
62]. Therefore, including enzyme-producing probiotics in feeds could enhance feed utilization in farmed species. That is the reason for assessing the enzymatic activity of the bacterial isolates.
Out of 117 isolates, 50, 26, and 11 were isolated on TSAs, MMA, and MRS media, respectively. Moreover, 30 isolates were spore formers that grew on TSAs media after heat treatment. We found that 48% of the strains could hydrolyze proteins, 41% lipids, 77% collagen, and 30% starch, respectively. The hydrolysis of casein and collagen demonstrated the capacity to degrade proteins, in accordance with the reported ability of probiotics to enhance protease activity in various fish species [
63,
64]. Additionally, amylases, which are enzymes that break down carbohydrates into smaller molecules, such as glucose or maltose, were detected, potentially providing readily available nutrients for the host [
65]. Tween-80 is an unsaturated fatty acid employed as a food additive (up to 1%) [
66], and its hydrolyzation depends on lipase activity. The lipase activity has been increased by the application of probiotics [
67,
68]. Furthermore, the ability of the strains to metabolize some anti-nutritional factors was also investigated. The results showed that 46%, 8%, and 57% of isolates were able to metabolize phytate, tannins, and cellulose as only carbon and energy sources, respectively. Therefore, the respective isolates may likely improve the degradation of those anti-nutritional factors in the host’s intestine, enhancing fish nutrition. In addition, in relation to the medium used to isolate microorganisms, we found that the percentage of isolates with enzymatic activities was higher in microorganisms isolated from TSAs and MMA. Thus, although the isolated strains in different media had an interesting enzymatic profile, the use of TSAs and MMA as isolation mediums could be the best option to isolate microorganisms with different enzymatic activities.
A relevant criterion in probiotic selection for aquaculture is their antimicrobial activity against fish pathogens [
69]. Unlike chemotherapeutics, which have been utilized for disease treatment in aquaculture but often come with undesirable side effects and promote antibiotic-resistant bacteria, probiotics offer an alternative approach. Probiotics can interact with or antagonize other bacteria, resisting colonization and directly suppressing opportunistic infections, thus reducing their incidence [
70]. Our study found that 38% of the isolates inhibited
V. anguillarum; 47% inhibited
P. damselae subsp.
piscicida, and 56% of the isolates inhibited
T. maritimum. The antimicrobial activity of the strains against these pathogens was evaluated due to the incidence of these pathogens in aquaculture and the economic losses they produce [
71,
72,
73]. Thus, it is suggestive to think that using these isolates in aquaculture may limit chemotherapeutics against those pathogens, avoiding the impacts produced by the overuse of these products. Furthermore, although the number of microorganisms isolated on the MRS medium selected for antimicrobial activity was the lowest (only 3 isolates out of 32), it had the highest percentage of isolates with antimicrobial activity against the three fish pathogens. MRS is a selective growth medium for LAB species [
74]. In this sense, some LAB species produce antimicrobial compounds (e.g., lactic acid, bacteriocins, etc.), which have been found to inhibit the growth of important aquaculture pathogens [
75].
Considering the extracellular enzyme activity (hydrolysis of ≥four substrates) and the antagonism against the three pathogens tested, four bacterial isolates (UMA-140, UMA-143, UMA-169, isolated in TSAs; and UMA-216, isolated in MMA) were characterized as putative probiotics (
Table 5). The four bacterial strains were identified as the
Bacillus genus. The UMA-143 strain showed similarity to
Bacillus cereus, while UMA-140, UMA-169, and UMA-216 showed homology with different strains of
Bacillus pumilus. In this sense, the isolation of
B. cereus and
B. pumilus from different fish species has been previously reported [
75,
76,
77,
78,
79].
Bacillus species are selected as probiotics to enhance digestive enzyme activities, given their effective metabolism of a diverse range of lipids, proteins, and carbohydrates [
80,
81]. The improvement in fish digestive enzyme activity following
Bacillus administration might be associated with enzymes generated by the bacteria, or
Bacillus could promote the development of indigenous enzymes in the fish [
82]. In this sense,
Bacillus species often modify the digestive enzymes protease, amylase, trypsin, and lipase [
83], which is in agreement with the in vitro enzymatic activities exhibited by the four isolates from our study and is consistent with findings from other studies on
B. cereus and
B. pumilus strains [
84,
85]. Furthermore, the isolated strains could hydrolyze the phytate, and two of them the cellulose, which is in line with other
Bacillus species [
86], including
B. pumilus [
87] and
B. cereus [
76]. Thus, it could be suggestive to think that including those strains in aquafeeds could enhance the feed digestibility and absorption by hydrolyzing those substrates present in the feeds and increasing the digestive enzymatic activity of the fish. Nevertheless, further in vivo experiments are necessary to assess this hypothesis.
Some
Bacillus species can produce bacteriocins or other bioactive compounds with antagonistic effects against fish pathogens [
88]. In this regard, the four
Bacillus isolates were effective against the three tested fish pathogens. Previous studies found that fish-gut-isolated
Bacillus species inhibited several fish pathogens, including
Tenacibaculum,
Photobacterium, and
Vibrio species [
89,
90,
91]. Thus, the antagonism reported in our study expands the potentiality of
Bacillus species against vibriosis, photobacteriosis, and tenacibaculosis diseases, protecting the host against opportunistic bacteria.
Finally, aside from their functional characteristics, probiotics must undergo a safety examination, such as blood hemolytic activity, before being used in human or animal feeding [
92]. Therefore, the hemolytic activity of the four selected strains was assessed on Columbia blood agar plates. Cells from UMA-140 and UMA-143 showed β-hemolytic activity, while cells from UMA-169 and UMA-216 had γ hemolytic, indicating negative or no hemolytic activity. The results are consistent with Cui et al. [
93] and Bottone and Peluso [
94], who reported hemolytic activity of
B. cereus and
B. pumilus strains, respectively. On the other hand, several studies have reported candidate probiotics with no hemolytic activity [
95,
96,
97], which is crucial in ensuring the safety of probiotic selection. Thus, to ensure the safety of any potential application of the microorganisms, we would not recommend using strains UMA-140 and UMA-143 in any activity involving human or animal feeding.