Introduction
Disease outbreaks continue to represent a major challenge for aquaculture production, compromising animal welfare and causing economic losses, which highlights the need for alternatives to antibiotics capable of enhancing fish health and resilience to pathogens. Sustainable functional feed additives have gained increasing attention, with brown seaweed-derived polysaccharides emerging as promising candidates due to their immunostimulatory and potential prebiotic properties (Thépot et al., 2021). In this regard, seaweed polysaccharides are not digestible by the fish, suggesting that their beneficial effects may be mediated by the gut microbiota, which ferments these compounds into short-chain fatty acids (SCFAs). In turn, SCFAs may act as mediators of gut epithelial integrity, immune regulation, and host-microbe signalling (Petit et al., 2022; Liu et al., 2023). Despite the proposed microbiota-mediated mechanism, comparative evidence of their direct prebiotic effects across freshwater and marine fish microbiomes remains scarce. Therefore, this study aimed to evaluate the prebiotic potential of laminarin, fucoidan, and alginate using in vitro fermentation systems inoculated with microbiota from freshwater Atlantic salmon (Salmo salar) and marine European seabass (Dicentrarchus labrax).
Materials and Methods
In vitro fermentation bioreactors were set up using faecal material collected by settling from fish reared in recirculating aquaculture systems at Wageningen University (Syropoulou et al., 2024). Four treatments were established in triplicate by supplementing the bioreactors with either no additive (control), laminarin, fucoidan, or alginate from brown seaweeds at a concentration of 5 mg/L. Separate fermentation trials were conducted under the specific rearing conditions for Atlantic salmon (14°C) and European seabass (23°C) and under anoxic conditions for 14 days. Samples were collected periodically for SCFA quantification by gas chromatography, dissolved carbon and nitrogen species quantification, organic matter analyses, and microbial community profiling through V1-V9 16S rRNA gene sequencing with the MinION Mk1C platform (Oxford Nanopore Technologies).
Results and Discussion
The production of SCFAs increased over time in both species, confirming microbial fermentation, while organic matter decreased, likely reflecting substrate consumption for bacterial growth. In Atlantic salmon, the treatment significantly affected the concentrations of propionic, butyric, and caproic acids. Specifically, after 7 days, alginate induced higher butyrate levels than laminarin and the control groups, which further increased by day 14. This pattern may be linked to the higher relative abundance of Clostridium, especially Clostridium beijerinckii, which ferments complex carbohydrates into butyric acid (Olorunsogbon et al., 2022), whose abundance also increased over time. On the other hand, the most predominant species of the control group at the end of the trial was Lactococcus lactis, involved in fermentation of mono- and disaccharides into lactic acid. Ammonium nitrogen (N-NH4+) increased over time, indicating microbial mineralization; however, final concentrations were lower in the alginate treatment than in the rest of the experimental groups. Together with the lower total dissolved nitrogen, the decrease in organic carbon, and the concomitant increase in inorganic carbon observed in this treatment, these results suggest that alginate fostered enhanced microbial assimilation, and/or heterotrophic nitrification and subsequent denitrification under oxygen-limited conditions. In European seabass, the treatment effects were more moderate, mainly affecting the levels of valeric, isocaproic, and caproic acids, which were higher in the alginate treatment. Acetic acid concentrations also showed a numerical increase when supplementing the media with alginate, which might be consistent with the slight increase in Cetobacterium somerae at day 14 in the media containing alginate compared with the control group, as this species conducts carbohydrate fermentation into acetic acid (Finegold et al., 2003). In addition, the in vitro trial of European seabass did show no differences among treatments in dissolved carbon and nitrogen species concentrations, while organic carbon levels increased over time, potentially reflecting bacterial growth and/or SCFA production. Taken together, these findings suggest that alginate promoted the highest fermentative response across both models, supporting its potential as the most effective prebiotic candidate among the tested brown seaweed polysaccharides.
Acknowledgment
The AquaUP project is co-funded by the European Union and the Dutch Research Council (NWO; reference: EP.1511.23.001), as part of the Sustainable Blue Economy Partnership programme (2024-2027).
References
Liu, X. F., Shao, J. H., Liao, Y. T., Wang, L. N., Jia, Y., Dong, P. J., Liu, X. Z., He, D. D., Li, C., & Zhang, X. (2023). Regulation of short-chain fatty acids in the immune system. Frontiers in immunology, 14, 1186892.
Olorunsogbon, T., Adesanya, Y., Atiyeh, H. K., Okonkwo, C. C., Ujor, V. C., & Ezeji, T. C. (2022). Effects of Clostridium beijerinckii and medium modifications on acetone-butanol-ethanol production from switchgrass. Frontiers in Bioengineering and Biotechnology, 10, 942701.
Petit, J., de Bruijn, I., Goldman, M. R., van den Brink, E., Pellikaan, W. F., Forlenza, M., & Wiegertjes, G. F. (2022). β-Glucan-Induced immuno-modulation: A role for the intestinal microbiota and short-chain fatty acids in common carp. Frontiers in Immunology, 12, 761820.
Syropoulou, E., Sipkema, D., Smit, S. E., Schrama, J. W., & Kokou, F. (2024). Aquaculture faecal waste generates different products during anaerobic digestion depending on nutrient composition. Journal of Environmental Management, 370, 122826.
Thépot, V., Campbell, A. H., Rimmer, M. A., & Paul, N. A. (2021). Meta-analysis of the use of seaweeds and their extracts as immunostimulants for fish: a systematic review. Reviews in Aquaculture, 13(2), 907-933.
Finegold, S. M., Vaisanen, M. L., Molitoris, D. R., Tomzynski, T. J., Song, Y., Liu, C., Collins, M. D., & Lawson, P. A. (2003). Cetobacterium somerae sp. nov. from human feces and emended description of the genus Cetobacterium. Systematic and applied microbiology, 26(2), 177-181.