Introduction
Off-flavours are of great concerns in various systems such as aquaculture, drinking water and food production. Geosmin (GSM) and 2-methylisoborneol (MIB) are the most widespread off-flavour compounds in recirculating aquaculture systems (RAS), known for their earthy/muddy smell and extremely low detection threshold for humans . GSM and MIB are secondary metabolites of microbial origin and the main off-flavour producers are Actinobacteria , Myxococcota , and Cyanobacteria (Lukassen et al., 2017) . Typically, two distinct routes of GSM and MIB biosynthesis occur in prokaryotes: the 2-C-Methyl-D-erythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway (Lange et al., 2000). The latter pathway also has an auxiliary leucine-dependent isoprenoid pathway . In this study, we investigated microbial communities of 50 samples from five RAS in three Swedish fish farms by metagenomic analysis.
Materials and methods
Two of the sampled RAS were aquaponic systems combining production of rainbow trout with tomatoes or vegetable cultivation, two produced eel, and one produced sturgeon. Three RAS used moving biofilm carriers for nitrification, one used a stationary trickling filter, and one relied on nitrification in gravel bed. Four RAS had drum filters for particles separation and one used sedimentation tanks. Samples of microbial communities were collected from various locations within the system including biofilters, sedimentation tanks, gravel bed, and wall growth. Following DNA extraction, the samples were analysed using shotgun metagenomic sequencing. The raw data were filtered and trimmed by using Fastp (v0.20.0). Then seven batches of metagenomics data were co- assembled into contigs using MEGAHIT (v1.1.3), and the scaffolds longer than 2000 bp were selected for binning with MetaBAT (v2.12.1) to obtain metagenome-assembled genomes (MAGs) . CheckM (v1.2.1) was used to evaluate the completeness and redundancy of MAGs. The coverage of MAGs (%) in each sample was estimated using CoverM (v0.6.1). Taxonomic classifications of MAGs were conducted using GTDB-Tk (v2.1.0), and phylogenetic analysis was performed by using PhyloPhlAn(v3.0.3).
Results and discussion
A total of 76 potential off-flavour producers, affiliated with 10 bacterial phyla (Acidobacteriota , Actinobacteriota , Myxococcota , Proteobacteria , Nitrospirota , Planctomycetota , Bacteroidota , Chloroflexota , Gemmatimonadota , Verrucomicrobiota) and 1 archaeal phylum (Iainarchaeota), were identified from 1378 MAGs based on functional genes annotation. After conducting a blast (basic local alignment search tool) search for functional genes responsible for the MEP and MVA pathways, we identified 38 MAGs with complete MEP pathway, 33 MAGs with complete MVA pathway, and 5 MAGs with both complete MEP and MVA pathways (Fig. 1). The GSM and MIB synthase genes (geoA and MIBS) were found in all the 76 MAGs, indicating a high diversity of previously unknown off-flavor producers in RAS. In addition, a large number of bacteria (203 MAGs, affiliated with 16 bacterial phyla) with almost complete MEP or MVA pathways but lacking the final genes of geoA and MIBS were also identified (Fig. 2) , suggesting that these bacteria may interact with potential off-flavor producers in RAS by providing precursors or intermediates.
References
Lange, B.M., Rujan, T., Martin, W., Croteau, R. 2000. Isoprenoid biosynthesis: The evolution of two ancient and distinct pathways across genomes. Proceedings of the National Academy of Sciences, 97(24), 13172-13177.
Lukassen, M.B., Saunders, A.M., Sindilariu, P.-D., Nielsen, J.L. 2017. Quantification of novel geosmin-producing bacteria in aquaculture systems. Aquaculture, 479, 304-310.