Flathead grey mullet (Mugil cephalus) is an omnivorous and detritivores fish species which has been selected as suitable for the diversification of Mediterranean aquaculture both in monoculture and polyculture systems due to its low trophic position (Vallainc et al., 2021; Solovyev and Gisbert, 2022). Grey mullet presents a rapid growth, a good adaptability to captivity and high market price of its salted and dried roe, known as “bottarga” (Whitfield et al., 2012). In addition, being a consumer of the lower trophic layers, its protein requirement is considered to be lower when compared to strict carnivorous farmed Mediterranean species. For instance, the protein content of standard aquafeed formulations for gilthead sea bream and European sea bass is normally around 45% (Busti et al., 2020a; Pelusio et al., 2022), while for flathead grey mullet can be as low as 30% (De Carvalho et al., 2010; Talukdar et al., 2020). Among the protein sources commonly used in aquafeeds, fishmeal (FM) is recognised as the most suitable in terms of nutritional value. However, the increased demand and the decline of the marine fishery stocks limit its application in aquafeeds (Glencross et al., 2016; Pereira et al., 2022). Alternatives to FM are conventionally represented by plant-based ingredients such as soybean meal (SBM), although with the presences of certain drawbacks (Colombo et al., 2022; Newton et al., 2023). As an example, SBM which is largely used into aquafeed formulation as a FM substitute, has a high carbon footprint if it is derived from crops grown on land converted from forest or savannah (Wilkinson and Young, 2020). In addition, its massive use into aquafeeds goes in direct competition with human consumption, as SBM is a key element in human diets.

In a circular economy, aquafeed ingredients derived from industry by-products such as single-cell ingredients are in the spotlight as a promising option for the sustainability of the aquaculture industry (Glencross et al., 2020; Colombo et al., 2022). Single-cell ingredients are a relatively broad class of materials including bacterial, fungal and microalgal-derived products, which have shown potential as a source of protein, lipids or specific nutritional components in aquafeed ingredients (Glencross et al., 2020; Pereira et al., 2022). Among such “circular” ingredients, bacterial single cell protein (SCP) presents a high percentage of crude protein with a favourable amino acid profile, along with vitamins and other functional compounds (Glencross et al., 2020; Jones et al., 2020). Bacteria may be produced using a wide range of substrates and nutrients ranging from industrial and agricultural residues to bioindustry by-products (Jones et al., 2020); thus, allowing the valorisation of waste streams and reducing the downstream costs related to industrial waste disposal (Pereira et al., 2022). Among different SCP sources, Corynebacterium glutamicum is a Gram-positive bacteria generally regarded as safe (GRAS organism) (Cheng et al., 2021; Liu et al., 2022), which is commonly used to produce amino acids (AA). After the AA production, filtered C. glutamicum cell mass, contains high protein content (>70%) and a suitable AA profile to be used as a replacement of conventional protein sources in livestock nutrition (Zhang et al., 2013). In aquaculture nutrition, its application has been successfully proven in gilthead seabream even at high inclusion level (Marchi et al., 2023). In general, digestibility and palatability of bacterial SCP is considered good; however, variable performances may appear between the tested bacteria genus (Glencross et al., 2020; Bratosin et al., 2021), the application of post-production cell lysis processes (e.g. autolysis or hydrolysis) (Schøyen et al., 2005; Teuling et al., 2019; Glencross et al., 2023) and the aquaculture species (Marit Berge et al., 2005; Hamidoghli et al., 2019; Zamani et al., 2020; Marchi et al., 2023).

To the best of our knowledge there is no literature to date concerning the application of a bacterial SCP in the diet of flathead grey mullet in captive conditions. In the present study, with the aim of providing a theoretical basis for the development of zero FM and SBM aquafeed for mullet, C. glutamicum was used to replace these conventional ingredients at three different inclusion levels. The effects of its inclusion in flathead grey mullet were evaluated on classical key performance indicators like growth performance, feed utilisation, and other variables like metabolic blood parameters, digestive enzyme activities and gut microbiota in order to evaluate holistically the effects of this new sustainable ingredient in fish nutrition.

The present study sought to define the capacity of flathead grey mullet, reared in captive conditions, in utilising a SCP ingredient derived from C. glutamicum in substitution of SBM, FM and PM. By using diets with equivalent levels of protein, lipid and digestible energy but different levels of bacterial SCP inclusion, a clear effect was seen at the end of the experiment. In particular, fish fed the SCP diets presented significantly lower growth performances, and feed and protein utilisation in comparison with fish fed control diet. To the best of our knowledge, there is no literature to date investigating the effects of such an ingredient in the diet of flathead grey mullet. Despite of this, the findings of this trial were quite unexpected, since representatives from the family mugilidae are able to utilise a variety of ingredients in captive conditions such as plant meals (Luzzana et al., 2005; Gisbert et al., 2016; Liu et al., 2021), algae (Rosas et al., 2019) and yeast (Wassef et al., 2001; Luzzana et al., 2005). In addition, considering the assumption that wild fish represent a reference for the development of formulated diets, and it has been reported previously that bacteria account for 15–30% of the organic carbon in the stomach content of this species (Moriarty, 1976; Crosetti and Blaber, 2015), the results from the current nutritional study were puzzling.

In our experiment, feed intake was higher in fish fed diets containing bacterial SCP inclusion. Published studies have revealed contradictory effects of SCP sources on feed intake in fish (Aas et al., 2006; Glencross et al., 2020). Those that found a positive correlation of SCP inclusion on feed intake, suggested that the high content of nucleotides (e.g. inosinic acid and inosine) presented in bacterial SCP may act as an attractant enhancing the palatability of the diet (Gamboa-Delgado and Márquez-Reyes, 2018; Hossain et al., 2020). However, our results were in agreement with previous studies conducted on rainbow trout (Perera et al., 1995; Zamani et al., 2020), Atlantic salmon (Marit Berge et al., 2005) and Pacific white shrimp (Hamidoghli et al., 2019), in which inclusion of dietary bacterial SCP did not affect or enhance feed intake, but lead to a poorer growth performance. This suggests that in our case the issue with SCP from C. glutamicum was not due to its palatability, but rather to the utilisation potential of the ingredient.

Across a range of SCP sources, bacteria are generally recognised as relatively digestible and their inclusion has been successfully demonstrated in several major finfish species of commercial interest without affecting growth or fish health (Glencross et al., 2020; Jones et al., 2020). In particular, the same SCP ingredient was tested by Marchi et al. (2023) on gilthead seabream, reporting no negative effects on the growth performance and an interesting probiotic effect on the gut microbiota, even at a high substitution rate. However, still some inconsistency appeared in the data being perceived as a limiting factor for their broader application (Glencross et al., 2023). Some works suggest that the thickness of the cell-wall may be a constraint for the digestibility of a number of SCP sources (Ugalde and Castrillo, 2002; Yamada and Sgarbieri, 2005; Ritala et al., 2017; Tibbetts et al., 2017; Teuling et al., 2019). C. glutamicum is gram-positive bacteria with a thick cell wall made out of a complex structure of peptidoglycan and arabinogalactan (Eggeling and Sahm, 2001; Cheng et al., 2021). In general, finfish can cope well with cell-wall lysis by producing acidic secretion of HCl and thus decreasing the stomach pH to values below 3 (Lobel, 1981). By observing the digestive system of grey mullet, the species presented a stomach with an alkaline pH (approx. 8.5), the same as the intestine (Payne, 1978). In the absence of a specific acid secretion of HCl in the stomach, the species has evolved relying on a mechanical trituration of feed particles carried out in the pyloric stomach, called the gizzard (Payne, 1978; Lobel, 1981; Crosetti and Blaber, 2015). A gizzard-like pyloric stomach is frequent in those species that eat detritus and flocculant benthic biofilm (e.g. algae, bacteria and non-living organic matter) such as P. lineatus (Bowen, 2022), H. niloticus (Agbugui et al., 2021), S. stomachicus (Wilson and Castro, 2010) and D. cepedianum (Watson et al., 2003). The wall of the gizzard is made of rings of smooth, non-striated muscle fibres and often contains sand or small debris to assist in grinding plant and fibrous material (Crosetti and Blaber, 2015). A work conducted by Sánchez et al. (1993) hypothesised that the poor growth that occurred in grey mullet fed a microalgae-based diet may have been due to the lack of access to sand in captive conditions, therefore preventing the development of a functional gizzard. To the best of our knowledge, no other scientific publications dealt with the possible effects of a non-functional gizzard on diet digestion in finfish species. On the other hand, this phenomena is well described in poultry nutrition in which it is known that the access to structural components such as whole cereals or coarse fibres, improve growth and nutrient absorption in chickens by enhancing gizzard functionality (Svihus, 2014; Idan et al., 2023). Flathead grey mullet used in this experiment were raised in a captive condition in fiberglass tanks, therefore having no access to sand or coarse material and possibly impairing the proper development of a functional gizzard. Thus, the lower growth performance observed in fish fed SCP may be related to poor nutrient digestion caused by an incomplete lysis of the C. glutamicum cell-wall, as described previously (Sánchez et al., 1993). The hypothesis of a deficiency in nutrient digestion appears to be reasonably acceptable after a look to the digestive enzyme activity. SCP inclusions lead to a reduction in the specific activity of the pancreatic and intestinal proteases (e.g. trypsin, chymotrypsin and leucine aminopeptidase), which is well correlated with an impaired protein absorption and low growth performance (Silva et al., 2010; Gisbert et al., 2016). A similar reduction in the activity of digestive proteases has been observed in carnivorous fish when fed too high inclusion of plant ingredients, and associated with poor growth and histopathological changes (e.g. enteritis) (Silva et al., 2010; Rodiles et al., 2012; Chikwati et al., 2013; Willora et al., 2022). How was suggested in a work conducted by Pujante et al. (2016) on thick-lipped grey mullet (Chelon labrosus) the enzymatic digestion of proteins mainly takes place in the anterior intestine by the action of alkaline proteases. A functional evidence for this is evinced from the lack of acid proteases activity (e.g. pepsin) in flathead grey mullet (Gisbert et al., 2016). Alkaline proteases are most active at a pH range from 8 to 10 (Nasri et al., 2011; Liew et al., 2013; Sanatan et al., 2013). Among them, trypsin activates other enzymes initially generated as inactive zymogens, such as chymotrypsin and therefore is considered a key enzyme in protein digestion (Asgeirsson et al., 1989). Trypsin and chymotrypsin catalyse the hydrolysis of the bonds of peptides into smaller dipeptides and oligopeptides which are split by the action of leucine aminopeptidase into free amino acids that can be absorbed into the enterocytes (Willora et al., 2022). In marine finfish with a functional gastric stomach, the acidity of the chyme entering from the stomach into anterior intestine is buffered by the secretion of HCO₃ from the pancreas, which create the optimal pH condition for the activation of the alkaline proteases (Wilson et al., 2002; Márquez et al., 2012; Parma et al., 2019). It is interesting to note that in our trial, we have observed a decreased pH and buffer capacity in the diets containing the SCP from C. glutamicum. These findings are in line with a previous study conducted by Prodanovic and Sredanovic (2005) reporting that the buffer capacity of yeast SCP is significantly lower than that of SBM (9.15 and 21.15, respectively), as well as the pH (5.84 and 6.58, respectively) probably due to the higher content of nucleic acids, as suggested in a work conducted by Ritala et al. (2017). We do not know if this could have contributed to an inhibitory effect on the activity of alkaline proteases; however, it is likely that in our experiment the intestinal pH of grey mullet decreased as a function of bacterial SCP inclusion. The activity of the alkaline proteases is mediated by a complex cascade process involving pH, temperature and the concentration of ions. In fish fed SCP diets, we have also found significantly lower concentrations of ions of calcium (Ca²⁺) and potassium (K⁺) in the blood. Ca²⁺ is recognised as a trypsin activator, since its presence strengthens the interactions inside protein molecules and enhances thermal stabilisation of this pancreatic enzyme (Bezerra et al., 2005), while K⁺ has an effect on the activation of leucine aminopeptidase (Liew et al., 2013). No differences were observed in both α-amylase and bile salt-activated lipase activity between fish fed the different diets. If compared to carnivorous species, the higher α-amylase activity found in flathead grey mullet, broadly hints that carbohydrates may be preferred as a protein sparing substrate as it has been previously postulated by several authors (Pujante et al., 2016; Calixto da Silva et al., 2020; Solovyev and Gisbert, 2022).

If we consider the other performance indicators, we observed that fish fed the diet with the highest inclusion of SCP also showed a significantly lower Fulton’s condition factor (K). This length-weight relationship can be correlated with the wellness conditions of the fish, and its fluctuations with the interaction between feeding condition and energy reserves (Robinson et al., 2008; Datta et al., 2013; Yulianto et al., 2020). Because of the issue with nutrient digestion in fish fed SCP diets, flathead grey mullet belonging to these groups appeared underfed with lower energy reserves and therefore with a lower flesh/skeleton ratio, which also explain the increased content of ash in the body of this group of fish. In terms of nutritional indices, which are used for comparing the nutritional values of different ingredient sources, they appeared to be significantly decreased by SCP inclusion, which is reflected by the feed conversion rate and the growth. On the other hand, gross protein and lipid efficiency (32.3 ± 1.7 and 75.4 ± 9.6%, respectively) of fish fed the SBM diet were encouraging in order to further design compound feeds for this low trophic species. With a focus on the protein utilisation, grey mullet fed a diet with only 3% fishmeal, were able to convert almost 1/3 of the dietary protein into edible fillet. This ratio appears similar to that found for gilthead seabream (Busti et al., 2020a) and rainbow trout (Pelusio et al., 2020) and it is somewhat higher to previous findings with European seabass (Bonvini et al., 2018; Parma et al., 2020; Pelusio et al., 2022). These results may indicate that is possible to further decrease the level of fishmeal in aquafeeds for this species.

As mentioned previously, the SCP additive contains a high level of nucleic acids, mainly RNAs (Ritala et al., 2017). In our trial, we have seen an increasing concentration of urea in the blood of the fish fed SCP diets. This phenomena has been described in previous works and is ascribed to the catabolism of pyrimidines, which are presents in nucleic acid molecules (Agboola et al., 2021; Lin et al., 2022; Marchi et al., 2023). High concentrations of ammonia metabolites in the blood may also interfere with protein, fat and carbohydrate metabolism (Rumsey et al., 1992), which may also explain the lower performance of flathead grey mullet fed bacterial SCP diets in comparison to the control group. In our trial, the blood glucose concentration found in the fish appears higher than values found in literature for the same species (Wanshu, 1992; Calixto da Silva et al., 2020). Despite this, the significantly lower concentration of blood glucose in fish fed SCP diets is likely to support the hypothesis of there being a problem with the digestion of this ingredient rather than a pathological condition. Starvation or underfeeding conditions are generally associated with a decreased blood glucose level in many fish species (Polakof et al., 2012). The enzyme activity of the marker enzymes in the blood associated to liver damage (aspartate transaminase and alanine transaminase) and tissue damage (creatine kinase and lactate dehydrogenase) did not show any difference among the fish groups in our trial. Interestingly, the activity of the enzyme alkaline phosphatase showed a trend and appeared to be higher in fish fed the diet with no inclusion of SCP. This difference may be related to the higher nutrient absorption in this group of fish, since the enzyme is involved in the absorption and transport of lipid and carbohydrates from the intestine, and its activity is positively correlated with food ingestion and growth rates (Lemieux et al., 1999; Lallès, 2020).

The gut microbiota of fish plays an important role in regulating nutrient digestion, immune responses, disease resistance, and the colonisation of potential pathogens (Parma et al., 2016; Gu et al., 2017; Busti et al., 2020b; Pelusio et al., 2020; Xie et al., 2021). The composition of bacterial communities is strongly determined by the environment conditions both internal and external such as salinity (Kivistik et al., 2020) and temperature (Thompson et al., 2017; Sepulveda and Moeller, 2020), the immune/health status (Kashinskaya et al., 2019; Parshukov et al., 2019), the host species considered and their dietary components (Kashinskaya et al., 2018). In particular, the composition of flathead grey mullet microbiota in response to different dietary ingredients is a relatively unknown topic. While there is a published study conducted by Le and Wang (2020) aimed at the characterisation of the microbiota from wild flathead grey mullet, information is lacking for fish reared in captive conditions fed formulated diets. Our finding displayed Fusobacteria and Proteobacteria as the most abundant phyla, accounting for more than 70% of the total variability in all groups. Inclusion of C. glutamicum in the diet of flathead grey mullet promoted a shifting between the two phyla, increasing the amount of Proteobacteria over the Fusobacteria (with presence of Actinobacteria expected, particularly in the SCP10 and SCP20 groups). Using as a reference the gut microbiota observed in wild fish, with Proteobacteria and Firmicutes as the most abundant phyla (Le and Wang, 2020), it is interesting to note that this appears more similar to fish fed the SCP diets even though these groups displayed severely reduced growth performances. Proteobacteria are found to be dominant in the gut microbiota of several fish species fed marine-derived ingredients (Gu et al., 2017; Kim et al., 2021). The phylum Firmicutes accounts for only a minor part of the variability of the gut microbiota in all groups, even though it was more abundant in fish fed SCP supplemented diets. This phylum, and also especially the lactic acid bacteria, often dominate the composition of the microbiome of fish fed plant-based diets (Busti et al., 2020b; Pelusio et al., 2020), and it is normally associated with improved nutrient digestion (Parma et al., 2016; Parma et al., 2020; Marchi et al., 2023). In our trial, fish fed the diet without inclusion of SCP displayed an increased dominance of the phylum Fusobacteria, which is totally represented by the genus Cetobacterium. This genus has been found to be dominant in the gut of many freshwater species and its presence is associated with a positive impact on the digestion and metabolic efficiency of the host (Xie et al., 2021). At the genus level, we have seen an increased abundance of Streptococcus in fish fed SCP diets. Some species of streptococci, as potential spore forming species, may represent carry-over of bacteria from the SCP feed additive (Karlsen et al., 2022), as some feed components are now shown to bias the interpretation of microbiome analyses by acting as the vectors for introduction of species to the intestinal microbiome of the host. Moreover, several bacterial species within the genus Streptococcus spp. have been reported as important pathogens of fish species (Ringø et al., 2018; Halimi et al., 2020). Although without a significant difference, in fish fed SCP diets we have also seen an increasing trend for the family Vibrionaceae, a family associated with important pathogens of fish (Zhang et al., 2020; Sepúlveda et al., 2022). On the other hand, fish fed diets with inclusion of SCP showed in their gut microbiota a decreasing trend in the family Aeromonadaceae, while there was a trend toward increasing in the relative abundances of Rhodobacter, Corynebacterium and Lactobacillus. Different species of Aeromonas are described as important fish pathogens (Zmysłowska et al., 2009; Soto-Dávila et al., 2022). Rhodobacter and Lactobacillus are considered among the most promising probiotics used in aquaculture (Ringø et al., 2018; El-Saadony et al., 2021; Cao et al., 2022). Corynebacterium, as expected, was found significantly higher in fish fed SCP diets and the presence of some species has been associated with a probiotic effect (Horn et al., 2022; Marchi et al., 2023). Along these same lines of investigation of probiotics are studies that evince the quorum sensing interactions that bacterial food components can have (Davares et al., 2022). Quorum sensing utilises small molecules like acyl-homoserine lactones and oligopeptides for intercellular signalling. An unrefined product like SCP may include bioactive molecules with potential for interspecies bacterial communication. When using quorum sensing bacteria not only communicate with members of the same species but may also “eavesdrop” on the “conversation” of other species and modulate their behaviour in response to signal molecules they do not synthesise (Federle and Bassler, 2003). This can have unexpected consequences on diverse assemblages of bacteria from unique hosts, perhaps even some degree of dysbiosis and poor fitness of the microbiota, which could lead to some of the poor performance indicators found in this study. Overall the resulting microbiota data will contribute to improved characterisation of the taxonomic diversity of the gut bacterial community from grey mullet, which is essential for understanding the dynamics of the diet-host-gut-microbiota axis.

These results indicated that flathead grey mullet reared under captive conditions demonstrate incomplete utilisation of a protein source derived from C. glutamicum SCP. Probably due to the particular organisation of the digestive system of flathead grey mullet there was an incomplete protein digestion of SCP due to poor lysis of cell-wall components (this being due to lack of an acidic stomach digestion) and also associated with the captive conditions (failure in the development of a functional gizzard). Based on our assessment, the diet had significant negative effects on individual variables in analysis of both digestive enzymes and gut microbiota.

The datasets presented in this study can be found in online repositories. The name of the repository and accession number can be found below: NCBI; PRJNA954609.

The animal study was reviewed and approved by Ethical-Scientific Committee for Animal Experimentation of the University of Bologna.

ABe, PG, ABo and LP conceived and designed the experiment. ABe wrote the first draft of the manuscript. ABe, SN, EG, KA, DC, ABo and LP reviewed and edited the manuscript. ABe, SN, DC and LP carried out fish maintenance and sample collection. SN, EG and KA carried out digestive enzyme activity analysis and data processing. ABe, AD and VI carried out the microbiota analysis and data processing. FD carried out plasma biochemistry analysis. All authors reviewed, improved the writing, and approved the final manuscript. All authors contributed to the article and approved the submitted version.