Introduction
The biological removal of nitrogen compounds such as ammonium and nitrite in recirculating aquaculture systems (RAS) is primarily facilitated by nitrifying bacteria residing within the biofilter. This structure is often referred to as an "ecosystem within an ecosystem" due to the diverse microbial assemblages it supports—each with distinct metabolic and physiological traits—which collectively convert toxic ammonium excreted by fish into the less harmful nitrate through a two-step nitrification process. Monitoring studies have revealed that the biofilter represents a unique microbial niche, characterized by a notably low abundance of pathogen-related species, such as Moritella viscosa, Psychrobacter immobilis, and Aeromonas salmonicida. One plausible hypothesis is that the resident microbial communities exert inhibitory effects on the colonization of these pathogens, thereby maintaining a protective microbiological environment within the biofilter. However, the mechanisms governing pathogen establishment and the influence of various environmental and operational factors on this process remain largely unexplored. It is further hypothesized that the introduction of pathogens into the biofilter—potentially triggered by disease outbreaks or microbial imbalances in the RAS—could disrupt the native microbial community. Such disturbances may negatively impact both the diversity and functional performance of the biofilter, thereby impairing nitrification efficiency and leading to suboptimal water quality characterized by elevated levels of toxic ammonium and nitrite. Additionally, many mechanical components within RAS are not easily accessible, often leading to the generalized assumption that uniform cleaning and disinfection protocols are effective across the system. In reality, spatial heterogeneity can significantly influence the efficacy of these disinfection practices. A deeper understanding of pathogen establishment pathways is therefore essential for the development of targeted and effective disinfection strategies.
Material and methods
A 30-day biofilter experiment was conducted under controlled environmental conditions at Nofima Tromsø, with the system maintained at 12 °C and a salinity of 12 ppt within an environmental chamber. The study was designed to evaluate the effects of Yersinia ruckeri introduction on biofilter function, using three treatments as part of an infection challenge. The first treatment involved waterborne exposure to Y. ruckeri at a defined concentration (XY concentration). The second treatment consisted of biomedia inoculation, wherein biofilter media were submerged for 24 hours in a Y. ruckeri suspension, subsequently rinsed with phosphate-buffered saline (PBS), and transferred to the experimental culture bottles. A preliminary laboratory trial confirmed that Y. ruckeri successfully adheres to autoclaved biocarriers after 24 hours of exposure. A third group, serving as a control, remained unexposed to Y. ruckeri. Nine 1-liter Schott bottles were used for the experimental setup, each filled with a mix of 85% mature biomedia and 15% autoclaved biomedia. Continuous aeration was provided via air stones connected to membrane pumps. Every three days, total ammonia nitrogen (TAN) levels were elevated to 4 mg L⁻¹ to simulate nitrogenous waste input, and removal efficiency was assessed after six hours. During the first two weeks, biomedia samples were washed with PBS and marine broth, then plated on Y. ruckeri-selective agar to quantify colony-forming units (CFUs). Concurrently, microbial communities present in the water phase were analyzed by filtering the culture water and extracting microbial DNA for further analysis. At the conclusion of the 30-day experiment, a pH-shift disinfection protocol was applied to evaluate whether disinfected biomedia could act as a reservoir for Y. ruckeri, thereby ensuring that desinfection measures effectively eliminate any remaining pathogenic bacteria.
Results
Yersinia ruckeri was confirmed to attach to autoclaved biomedia within 24 hours of exposure. Among the experimental groups, the highest denitrification performance was observed in the treatment where the inoculated biomedia were washed with PBS buffer prior to their introduction into the system. Over the course of the experiment, a gradual decline in colony-forming units (CFUs) of Y. ruckeri was observed, indicating a reduction in the presence of the pathogen over time.
Discussion
The results indicate that Yersinia ruckeri does not actively proliferate within the biofilm of the biomedia but can still be detected more than one week after a single infection event. While the presence of Y. ruckeri had no measurable impact on the denitrification performance of the biofilter, the PBS-washing of biomedia prior to use significantly enhanced nitrogen removal efficiency. This improvement may be attributed to the removal of older or inactive biofilm layers, thereby promoting more effective colonization by active nitrifying communities. Results from the disinfection phase of the experiment are still under analysis at the time of writing.