Myxobacteria isolated from RAS: Ecology and significance as off-flavor producers

Despite advances in the operation of recirculating aquaculture systems (RAS), accumulation of earthy-muddy off-flavors in the fish remains a potential risk. Myxobacteria (Myxococcota) are reported to be among the most abundant geosmin synthase-harboring groups in RAS, but previous isolation attempts have been unsuccessful, limiting the knowledge of their role in off-flavor production. For the first time, we successfully isolated two geosmin-producing myxobacteria from RAS: Myxococcus virescens AT3 and Corallococcus exiguus AT4. Cell-specific geosmin production varied with the nutrient content in different media but was highest in a low-nutrient medium and when cultivated in water from RAS. Cultivation in RAS water also stimulated the production of other volatile organic compounds (VOCs). Newly identified potential off-flavor compounds included 4-methyl-2-heptanone ("forest" odor), 3-methyl-1-butanol ("medicinal" and "chemical"), and a presumptive sesquiterpenoid described as "musty," "earthy," and "flowery." The previously known off-flavor compound dimethyl sulfide was also detected. Myxobacteria have previously been proposed as keystone bacteria in the environment due to their predatory lifestyle. In predation assays using isolated bacteria from RAS, M. virescens AT3 and C. exiguus AT4 could successfully feed on 15 of 16 tested strains, suggesting a large influence on the biology of RAS microbiomes. The combination of predatory behavior and potent production of geosmin and other VOCs underscores the ecological and sensory impact of these bacteria in RAS. Understanding their behavior and metabolic outputs is critical to developing strategies for mitigating off-flavors in RAS. IMPORTANCEIssues with off-flavored fish in recirculating aquaculture systems (RAS) due to the presence of the earthy-musty smelling compounds geosmin and 2-MIB is considered one of the industrys most economically significant challenges. Knowledge of conditions that affect off-flavor production is essential information in the development of viable solutions for its mitigation. Little is known about the function of these microbially produced compounds or the conditions that trigger their production, especially in the underexplored myxobacteria. Investigation of natural isolates is crucial to determine the function of the genes involved and their differential expression in response to environmental cues. While myxobacteria in RAS have been previously shown to harbor the geosmin synthase gene through molecular studies, the present study is the first attempt to isolate these bacteria from RAS and quantify their geosmin production under different nutrient conditions. Through cultivation-based methods, we demonstrate their production of both known and novel compounds with earthy attributes.