| Summary: | <i>Pseudomonas fluorescens</i> and <i>Aeromonas hydrophila</i> bacteria are opportunistic pathogens that occur naturally in the aquatic environment and in the gut flora of healthy fish. Both species can pose a serious threat for fish that are highly sensitive to water pollution. The aim of this study was to determine the extent to which the amount of administered fish feed and fish biomass affect the distribution and abundance of <i>Ps. fluorescens</i> and <i>A. hydrophila</i> bacteria in a recirculating aquaculture system (RAS) during farming of European grayling (<i>Thymallus thymallus</i> L.) broodstock. A total of 68 water samples from the inflow, two rearing tanks and the outflow as well as 17 feed samples were collected and analyzed separately. Bacterial populations were analyzed by the culture-dependent method and a molecular method (fluorescence in situ hybridization, FISH) to detect culturable strains and viable but non-culturable strains, respectively. Fish biomass, feed and 16 water quality parameters (temperature, pH, concentration of dissolved oxygen, oxygen saturation, five-day biochemical oxygen demand (BOD<sub>5</sub>), total phosphorus, total organic phosphorus and nitrogen, orthophosphates, total nitrogen, nitrite and nitrate nitrogen, ammonia nitrogen, ammonium nitrogen, total suspended solids, and total organic carbon) were the explanatory factors. Statistically significant differences (RM-ANOVA, p ≤ 0.05) were stated in bacterial abundance in samples from the inflow, rearing tanks and the outflow. Water samples from the RAS were abundantly colonized by non-culturable <i>Ps. fluorescens</i> and <i>A. hydrophila</i> bacteria. Feed was not a source of bacteria, but a redundancy analysis (RDA) revealed that the amount of feed, fish biomass, BOD<sub>5</sub>, and total suspended solids and total organic carbon were positively correlated in both <i>Ps. fluorescens</i> and A. hydrophila. These parameters also influenced the distribution of both potentially pathogenic bacterial populations and contributed to the bacterial contamination of water in the RAS. Our results are particularly valuable for aquacultures that help to replenish wild stocks and rebuild populations of threatened species in natural aquatic environments.
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