Introduction
Integrating aquaponics into recirculating aquaculture systems (RAS) leverages the interactions among fish, plants, and the microbiome, supporting optimized growth and a sustainable production model (Joyce et al., 2019). Within the AWARE project, the use of treated wastewater in aquaponic systems has been shown to produce safe food in accordance with European regulations (Cabo et al., 2025). Although microbial communities are recognized as key ecosystem stabilizers, their role within aquaponics systems remain insufficiently understood (Kushwaha et al., 2025). The aim of this study is to compare the temporal evolution of bacterial surface communities at three locations within RAS units operated with treated wastewater and with dechlorinated water. Through a metataxonomic approach, the study pretend to generate insights into the composition, diversity, and potential functional roles of these communities within the aquaponic system.
Methodology
Experimental system: Experiment was conducted in a small-scale, Recirculatory Aquaculture System (RAS) installed in Castellana Grotte coupled with a municipal wastewater plant. The experimental system comprised three different RAS modules, RAS1, 2 and 3. RAS1 operated using dechlorinated tap water (CONTROL), while RAS2 and RAS3 were supplied with reclaimed water. RAS were stocked with Nile tilapia (Oreochromis niloticus) and a 5 m2 of crop planting surface with capacity for 90 lettuce plants (Lactuca sativa Batavian Red).
Surface sampling: was conducted at the beginning of the experiment and subsequently after two months period in accordance with the procedures outlined in ISO 18593 for the assessment of both wet and dry surfaces on the vegetable growing tables (GTs), tanks (TKs) and sumps (Ss).
Microbiome characterization: bacteriome was characterised using next-generation sequencing (NGS) techniques. Following DNA extraction (PureLink��� Microbiome DNA Purification Kit, Invitrogen) and purification (OneStep��� PCR Inhibitor Removal Kit, Zymo Research), amplification and library preparation were performed using the 16S Barcoding Kit 24 V14, from Oxford Nanopore Technologies (ONT), following the standard protocol. Finally, 16S rRNA amplicon sequencing was carried out using the ONT��s MinION platform, which enables real-time, long-read sequencing..The sequences generated were analyzed in the cloud served EPI2ME, from ONT, with the 16s workflow. Sequences below 800 bp and longer than 2000 bp were discarded. Finally, filtered reads were classified with minimap2 using SILVA as reference database.
Results and Discussion
The evolution of the bacterial communities in the growing tables (GTs), sump (S) and tanks (TKs) of RAS1, RAS2 and RAS3 was characterized through metataxonomic analyses. The composition of the bacterial communities was analyzed though a non-metric multidimensional scaling (NMDS) analysis based on Bray-Curtis dissimilarity distance and subsequent clusterization.
Overall, the analysis of the microbiota present on RAS surfaces clearly revealed locationdependent differences in the temporal evolution of bacterial communities throughout the experiment. At the beginning of the trial (t0), the bacterial composition detected in the GTs clustered with that found on the Ss, both characterized by a high relative abundance of Rhizobiales Incertae Sedis, Rhodobacteraceae, Reyranellaceae, Hyphomicrobiaceae, Chitinophagaceae, Legionellaceae, and Burkholderiaceae. Notably, members of the order Rhizobiales are known to participate in nutrient cycling in aquatic systems (Jia et al., 2019).
By the end of the experiment (t2), however, the bacterial communities in the GTs were dominated by members of the family Aeromonadaceae across all three RAS units, together with Enterobacteriaceae, which reached its highest relative abundance in the GT of RAS1 (GT1). Importantly, the absence of Aeromonas hydrophila in the food produced within the RAS ensured product safety.
In the case of the Tks, the initial surface-associated communities formed a distinct cluster. Nevertheless, after two months (t2), the microbial communities in the RAS units operating with reclaimed water (RAS2 and RAS3) became similar to those of the Ss, showing a predominant relative abundance of Exiguobacteriaceae. The order Rhizobiales remained detectable, particularly in the Tk and S of RAS2. This pattern suggests that microbial composition at these locations is likely shaped by the extreme environmental conditions typical of the Castellana region during summer, as members of Exiguobacterium are known to withstand stressors such as high temperature, salinity, and heavy metal exposure.
By the end of the trial, the microbiota on Ss and Tks in systems using reclaimed water converged, being dominated by Exiguobacteriaceae. In contrast, the plant GTs of all three RAS units exhibited a distinct microbial trajectory, with Aeromonas becoming dominant (up to 91% relative abundance). This indicates that, while reclaimed water strongly influences the bacterial communities of Ss and Tks, it has a more limited impact on those of the GTs.
Conclusion: Bacteriome evolution on Ss and Tks surfaces is influenced by the use of reclaimed water, whereas its impact on GT surfaces appears to be more limited. Differences in microbial dynamics across RAS locations may arise from systemspecific factors such as water circulation configuration, as well as the influence of biofilter, plant, and fish microbiota. Finally, considering the spatial context of each surface within the RAS, it can be inferred that submerged S and Tk surfaces more closely reflect the systemwide microbiome, whereas the exposed GT surfaces likely mirror the microbiota associated with lettuce roots.
References
Cabo M., E. Guti��rrez, O. Boghenco, A. Garrido-Maestu, M. Bern��rdez, P. Niclas, M. Slater, A. Cobelo, M. Lois, J.L. Romalde, Le Goueff, Camelo, Silva, Nunes, Manaia, Di Sansebastiano, Ugolini. Food safety and animal welfare in a new reclaimed water-based aquaponic system. European Aquaculture Society Meeting. Aquaculture Europe 2025
Jia L., Jiang B., Huang F., Hu X. (2019). Nitrogen removal mechanism and microbial community changes of bioaugmentation subsurface wastewater infiltration system. Bioresource Technology, Vol (294).
Joyce, A., M. Timmons, S. Goddek, y T. Pentz. 2019. Bacterial Relationships in Aquaponics: New Research Directions. En: Aquaponics Food Production Systems. S. Goddek, A. Joyce , B. Kotzen y G.M. Burnell (Eds,): 145-161.
Kuswaha, J., Priyadarsini, M., Rani, J., Pati Pandey, K. & Dhoble, A. 2025. Aquaponic trends, configurations, operational parameters, and microbial dynamics: a concise review. Environment, Development and Sustainability . 27: 213-246.