Harnessing Microalgal Microbiomes for Pathogen Control in Aquaculture by Probiotic Consortia

The global expansion of aquaculture, essential for sustainable food production, is significantly challenged by bacterial disease outbreaks, often leading to substantial economic losses and increased reliance on antibiotics, which in turn exacerbates the problem of antimicrobial resistance (AMR). Probiotics offer a promising sustainable alternative for disease control. However, traditional probiotic strategies, often relying on single bacterial strains, have shown limitations in efficacy and persistence. Meanwhile, the complex ecological interactions within natural microbial communities, such as those associated with live-feed microalgae, remain a largely untapped resource for novel biocontrol solutions. The purpose of this PhD project was to investigate the probiotic potential of microalgal microbiomes, particularly from Isochrysis galbana and Tetraselmis suecica, against the significant bacterial fish pathogen Vibrio anguillarum, to characterize the inhibitory microbial consortia, and to elucidate the underlying molecular mechanisms of pathogen inhibition.

This work first established a novel GFP-based fluorescence assay for efficiently screening the anti-pathogenic activity of complex microbial communities against a fluorescently labelled target pathogen, overcoming limitations of traditional agar-based methods. Using this assay, the native microbiomes of the microalgae Isochrysis galbana and Tetraselmis suecica were assessed, revealing that I. galbana microbiomes consistently demonstrated potent inhibition against both low and highly virulent strains of V. anguillarum. Metataxonomic analysis of these inhibitory communities highlighted the frequent dominance of bacterial families such as Alteromonadaceae, Rhodobacteraceae and Halomonadaceae. While some individual isolates, notably Phaeobacter spp., exhibited direct antagonism, a key finding was the discovery of synergistic inhibition by co-cultures of isolates, such as Vreelandella alkaliphila strain D2 and Sulfitobacter pontiacus strain D3, which showed enhanced or emergent inhibitory activity not observed in monocultures. This highlighted the importance of microbial interactions in pathogen suppression.

To unravel the mechanisms driving these synergistic effects, the V. alkaliphila D2 and S. pontiacus D3 co-culture was investigated in detail. The increased pathogen inhibition was found to be dependent on close physical proximity of the strains and was particularly pronounced under iron-limited conditions. Integrated multi-omics approaches, including metagenomics, metatranscriptomics, and metabolomics (LC-MS and MSI), identified desferrioxamines, a class of siderophores, produced by V. alkaliphila as key mediators of this inhibition. The proposed mechanism involves iron sequestration, starving V. anguillarum, which was found to lack the specific uptake receptor for these xenosiderophores. Biosynthetic gene clusters (BGCs) responsible for desferrioxamine production were identified and their expression confirmed in the inhibitory consortia. Further investigation into the ecological relevance of these interactions, through re introduction of the co-culture to an axenic I. galbana system, revealed that the culture context significantly influences microbial dynamics and inhibitory outcomes, with S. pontiacus D3 becoming dominant and exhibiting strong pathogen inhibition, in the algal system, even in monoculture.

This thesis concludes that microalgal microbiomes, particularly from the common live feed
microalga Isochrysis galbana, represent a rich source for the development of effective, multi-
strain probiotic consortia for aquaculture. The research highlights that synergistic interactions
within these communities are essential for robust pathogen inhibition, found to be mediated
by mechanisms such as siderophore-based iron competition. The findings underscore the
necessity of evaluating probiotic candidates within ecologically relevant contexts, as
microbial dynamics and efficacy can differ significantly from simplified laboratory
conditions. This work contributes novel insights into the chemical ecology of bacterial
interactions in microalgal microbiomes and provides a foundation for engineering beneficial
microbiomes for sustainable disease management in aquaculture, thereby reducing the
reliance on antibiotics.