Feeds represent a major cost in aquaculture, particularly for carnivorous fish species, which still rely heavily on fish meal (FM) as the main protein source. However, depletion of wild fish stocks and increasing FM prices demand sustainable alternatives. Plant feedstuffs (PF) are commonly used but have important limitations, including anti-nutritional factors and direct competition with human food resources. Seaweeds (SW) are promising alternatives due to their availability, sustainable production, and nutritional and functional properties.
SW nutritional composition varies considerably, with protein ranging from 4–47% dry matter (DM) and lipids from 1–5% DM, mainly composed of n-3 and n-6 polyunsaturated fatty acids (PUFA). They are also rich in bioactive compounds, including antioxidants, phenolics, pigments, and polypeptides. However, polysaccharides account for 36–60% of SW biomass, with cell wall water-soluble polysaccharides (CWPS) representing 55–70% of total polysaccharides. Since monogastric animals cannot digest CWPS, their presence limits SW nutritional value and functional potential.
Current methods to improve SW nutrient bioavailability, including physical, chemical, and biological treatments, are often complex, time-consuming, and environmentally unsustainable. Commercial dietary enzymes are also inadequate, as they mainly target terrestrial plant polysaccharides rather than seaweed CWPS. Therefore, gut bacteria capable of producing CWPS-degrading enzymes (CWPSases) emerge as a promising alternative.
The isolation and administration of probiotic (PRO) bacteria may enable fish to utilize otherwise indigestible dietary compounds, improving feed efficiency and supporting cost-effective and sustainable diets. Additionally, probiotics may enhance fish health through antimicrobial and immunomodulatory effects. Fish gut microbiota strongly responds to dietary composition, and dietary modulation may enrich bacteria capable of metabolizing CWPS, as previously demonstrated with PF- and insect meal-based diets. Spore-forming bacteria, particularly Bacillus spp., are especially attractive for aquafeed applications because spores tolerate feed processing and gastrointestinal transit. Moreover, Bacillus species are known to produce enzymes able to hydrolyze the main SW CWPS.
PROALGAE aims to isolate autochthonous probiotic bacteria from the gastrointestinal tract of European sea bass (ESB; Dicentrarchus labrax) capable of producing CWPSases, improving the use of SW-rich diets while meeting EFSA safety requirements for probiotic eligibility. ESB was selected due to its high commercial relevance in European aquaculture, with expected applicability to other fish and livestock species.
The project is structured into eight tasks. In Task 1, bacteria will be isolated from the gastrointestinal tract of ESB fed diets containing Ulva rigida, Fucus vesiculosus, or Gracilaria spp., selected due to their common use in aquafeeds and distinct CWPS composition: ulvan, alginate/fucoidan, and agar/carrageenan, respectively. Gut microbiota diversity will be characterized through 16S rRNA sequencing to assess diet-induced microbial modulation.
Tasks 2, 3, and 5 involve in vitro screening of bacterial isolates. Task 2 will evaluate functional traits, including spore-forming ability and CWPS metabolism. Task 3 will assess probiotic suitability, focusing on EFSA biosafety criteria, gastrointestinal survival, and scalability for industrial production. In Task 4, selected isolates (approximately four) will be produced through spore production, purification, and lyophilization.
Task 5 will investigate cytotoxicity, pathogen interference, and immunomodulatory effects using gut-associated lymphoid tissue (GALT) lymphocytes from ESB and the RT-Gut epithelial cell line. Based on these results, two probiotic strains will be selected for in vivo evaluation.
In Task 6, digestibility and growth trials will be conducted in ESB fed SW-based diets supplemented individually with probiotic spores (1 × 10⁹ spores/kg feed) to assess their efficacy in improving SW utilization and CWPS digestion. Disease resistance will also be evaluated through Vibrio anguillarum challenge tests (Task 7), with immune responses analyzed in plasma and immune-related tissues. Finally, Task 8 will assess gut microbiota dynamics, histomorphology, and oxidative status to evaluate probiotic colonization and effects on gut health.
PROALGAE is expected to provide aquaculture with a multifunctional probiotic capable of improving seaweed utilization and disease resistance in carnivorous fish, addressing two major bottlenecks in sustainable aquaculture development. Notably, only one approved probiotic is currently available for aquaculture in the EU, and it neither forms spores nor targets seaweed digestibility.
