Earthy and moldy off-flavors in aquaculture products reduce consumer preferences for farmed fish and consequently, the off-flavors also cause economic loss for RAS farmers (Abd El-Hack et al., 2022). Finding solutions to this reduce the presence of these compounds is essential, both for economic reasons as well as for the reputation of the entire aquaculture industry. Such off-flavors have historically been affiliated with the compounds geosmin and 2-MIB. These microbiologically produced compounds have mainly been attributed to Cyanobacteria and Streptomyces, but as gene sequence databases recently have expanded, molecular studies now indicate that the more obscure Myxobacteria may be the leading bacterial group responsible for earthy off-flavors in aquaculture (Lukassen et al., 2022, Lukassen et al., 2019). For the first time, we succeeded in isolating these bacteria from RAS, enabling their production of geosmin, 2-MIB and other off-flavors to be studied in details by GC-MS. After cultivation in growth media with a variable nutrient composition, production of geosmin and 2-MIB by selected Myxobacteria was characterized to estimate which compounds might control off-flavor production in RAS. These findings provide valuable information to the aquaculture sector to optimize their practice to combat off-flavor issues produced by these bacteria.
Material and Methods
Samples from different compartments of two different Recirculating Aquaculture Systems (RAS) in Denmark were used for the isolation of the bacteria. Isolated strains were identified through 16S rRNA gene amplicon sequencing and subsequently whole genome sequenced using Nanopore. The isolates were cultivated in growth media composed of varying levels of relevant nutrients (nitrogenous compounds, phosphorus, carbon), and volatile organic compounds (VOCs) were extracted using stir bar sorptive extraction (SBSE). VOCs were analyzed through Gas Chromatography-Mass Spectrometry (GC-MS). Production of VOCs in the different media were normalized to cellular production by enumeration of cells through fluorescence microscopy.
Results and Discussion
For the first time, isolation of Myxobacteria from RAS was successful. This bacterial group has the largest genomes among prokaryotes, ranging from 9-16 Mb, which might explain their notoriously slow growth. The slow growth, in combination with their slime-production and swarming colonies, makes isolation and purification of isolates especially hard. Utilizing baiting techniques for both predatory and saprophytic Myxobacteria, three different strains were isolated and purified. Results from 16S rRNA gene amplicon sequencing identified two of the isolates as belonging to the genera Myxococcus and Corallococcus, with high sequence identity similarity to species Myxococcus virescens and Corallococcus exiguus. The closest relative to the third isolate was found to be the genus Pseudenhygromyxa. Growth of the isolates in rich media, e.g., containing high levels of all considered nutrients, produced insignificant levels of 2-MIB. However, under the same conditions, geosmin was produced in levels >1200 ng/L. For reference, the human odor threshold for geosmin in water is about 5 ng/L (Srinivasan and Sorial, 2011). The high geosmin production supports that these bacteria may have a significant contribution to off-flavours in RAS-reared products. Results from other cultivation conditions are currently undergoing data analysis but will be presented at the conference.
Myxobacteria are prolific producers of the prominent off-flavor compound geosmin in RAS. Strategies to combat the production of off-flavor in these systems can only be developed through knowledge regarding the production of the specific bacterial producer in question. The present study provides further knowledge of how the nutritional characteristics of the rearing water will influence geosmin production in these bacteria.