Introduction
Microalgae are increasingly recognized as a sustainable solution to environmental challenges, particularly in the field of wastewater treatment. Certainly, these remarkable microorganisms are able to extract excess nutrients from wastewater and transform them into high-value biomass - eco-friendly resource - that can be turned into nutritious aquaculture feed. Among various microalgae, Nannochloropsis stands out due to its robust growth, high protein and lipid content, and significant levels of polyunsaturated fatty (Han et al., 2019). However, the in-depth potential of Nannochloropsis for the valorisation of waste products (e.g., brewery waste) into aquafeeds remains largely unexplored. The aim of this study was to investigate some of the physiological consequences in fish of the use of such microalgae-based production systems with focus at the systemic, tissular and environmental level from a holobiont perspective (i.e., the interconnected system of the host and its associated microorganisms), using gilthead sea bream as a model of Mediterranean farmed fish.
Materials and methods
The potential of Nannochloropsis diets to revalorize brewery waste was analysed in a 96-days gilthead sea bream trial from April to July under natural temperature and daily photoperiod at the IATS latitude (40◦ 5′ N; 0◦ 10′ E). The experimental design with triplicate tanks per dietary treatment (210 fish per diet, 11 g initial BW) included four diets: a commercial-based diet as a negative control (D1), and three fishmeal-free diets with varying inclusion levels of Nannochloropsis oceanica (D2, 0%; D3, 2%; D4, 14%). For the most extreme diet formulation (D4), fish oil was replaced by a DHA-enriched microalgae oil, resulting in a diet devoid of fishmeal and fish oil and with an EPA+DHA content (1.80%) above the established nutrient requirements for juveniles of gilthead sea bream. All four diets were formulated by LSAqua (Hooglede; Belgium) to be isoproteic (48% dry matter) and isolipidic (18% dry matter) feeds with different inclusion levels of fishmeal, fish oil, plant proteins and other alternative ingredients (soya oil, poultry meal, processed animal proteins, single cell proteins, and microalgae biomass) to facilitate aquaculture eco-intensification through increased circularity and resource utilization. At the end of trial, 12 fish per dietary treatment were sampled for analyses of total plasma antioxidant activity, host gene expression, and bacteria composition of the adherent mucus layer and water environment. Antioxidant capacity was measured as Trolox activity. Gene expression was made using customized tissue-specific arrays with 89 selected markers of growth, lipid metabolism, antioxidant defence, and immunological status. Microbial characterization was performed amplifying full-length 16S rRNA using Oxford Nanopore sequencing. Bioinformatic analysis comprised demultiplexing, quality filtering and taxonomic assignment. Statistically significant differences (p < 0.05) on growth performance, and gene expression were assessed by one-way ANOVA/Kruskal-Wallis test followed by a Holm-Sidak post-hoc test.
Results and discussion
All fish groups grew efficiently and only slight differences on final weight (< 5%) were found between D1-fed fish and the other three experimental groups fed fishmeal-free diets (D2-D4), which remained almost undistinguishable in terms of growth and feed conversion. However, fish fed the D4 diet showed an up-regulated hepatic expression of two key endocrine regulatory factors, igf1 and ghr1, suggesting an improved growth potential at the transcriptional level in fish fed with the highest microalgae inclusion level. No signs of histopathological damage were found in the liver and intestines of any experimental group, but D4 fish displayed a reduced lipid vacuolization of enterocytes accompanied by a depletion of hepatic lipid droplets. Certainly, PLS-DA highlighted a consistent down-regulation of genes involved in fatty acid synthesis, such as desaturases (scd1a, fads2), elongases (elovl1, elovl5, elovl6), and fatty acid transporters (hfabp), alongside the up-regulation of hepatic lipase (hl). This shift is indicative of a metabolic adaptation that favours tissue-fatty acid uptake over de novo fatty acid synthesis. Likely this metabolic feature was primarily due to the highest PUFA content (DHA-rich algae oil) of D4, while the increased plasma antioxidant capacity of D3 and D4 fish would be the result of a combination of enzymatic and non-enzymatic antioxidant mechanisms that become activated with the increased dietary supply of PUFA and microalgal biomass. The microalgae-based diets also modulated the expression of several intestinal genes, including interleukins, galectin binding receptors, brush border enzymes, and markers of tight junction integrity (il1b, il34, tnfα, lgals, alpi, tjp1, csflr1, cxadr), encompassing both pro- and anti-inflammatory roles. Such finding denotes a delicate balance in the host intestinal transcriptome of D3-D4 fish, which helps to avoid excessive inflammatory responses while keeping the immune system alert to prevent the entry of invading pathogens. Such adaptive gene expression pattern would be favoured by the reshape of gut microbiota with an overrepresentation of 17 bacterial genera with potential detoxifying, antioxidant and anti-inflammatory properties. Among them, up to seven bacteria genera were markedly overrepresented in the intestine of D4 fish (Vibrio, Hydrogenispora, Marinobacter, Palleronia-Pseudomaribius, Ruegeria, Atopostipes, Thaumasiovibrio). The microbiota of the water environment was also altered by the dietary treatment, denoting the presence of active feeding fish, changes in diet composition and, more specifically, the degree of Nannochloropsis supplementation.
Concluding Remarks
Nannochloropsis supplementation largely reshaped the microbiota inhabiting the intestine and the water environment, promoting in turns a healthy intestinal core microbiota. Microalgae biomass, together with DHA-rich algae oil, would drive the expression pattern of key enzymes of lipid metabolism, antioxidant defence and immunological surveillance, while changes in lipid metabolism could be primarily attributed to a dietary surplus of PUFA rather than to an increased supply of microalgal biomass.
Funding
This work was supported by the ERA-NET SUSFOOD2 FOSC AlgaeBrew European project (Project ID: 2021SUSFOODFOSCEN101).
References
Han et al., 2019. Appl. Sci. 9, 2377.