Recirculating aquaculture system (RAS) biofilters: focusing on bacterial communities complexity and activity

This research has been carried out in order to better understand the relationships between bacterial communities, established within the biofilter of a recirculating aquaculture system, and the influence exerted by forcing factors on the system itself. The work was divided into three main objectives that were aimed at studying: - the composition of the metabolically active bacterial fraction through the construction of cDNA clone libraries and the application of fingerprinting techniques (e.g., the T-RFLP); - the effect of particulate organic carbon on both the nitrification process and the microbial communities in different types of biological filters; - the effect of a moderate increase in the oxidation-reduction potential (ORP), through the injection of ozone, towards the activity and structure of the bacterial communities. The cDNA clone libraries allowed subdividing the active community in 48 phylotypes, each corresponding to a species. The Gammaproteobacteria (59.7%) were predominant, followed by Alphaproteobacteria (11.5%) and Bacteroidetes (7.9%). Most clones, especially among the Gammaproteobacteria, belonged to species that are potentially pathogenic to fish, even these latter were in an excellent health state during the experimentation period. This might suggest the existence of a shelter effect by the autochthonous bacterial flora against opportunistic pathogens, which are always present in such systems. Results showed a negative relationship between the nitrification efficiency and C/N ratio (defined as the ratio of particulate organic carbon and dissolved inorganic nitrogen), with a significant decrease in nitrification when the C/N ratio increased from 0 to 4. The increase of such ratio led to a dramatic increase in bacterial abundance (viable and total counts) on both the packing media and the water outlet. This suggests that the increase in organic carbon could allow to the predominance of heterotrophic bacteria on those autotrophic, which are responsible for the nitrification process, with the consequent drastic decrease in the filtration efficiency. Data regarding the effects caused by the ozonation process on water and packing media samples showed that the oxidation of organic macromolecules, by ozone, has - 4 - consequences on the whole microbial compartment. In fact, based on results from the filtration efficiency (chemical analysis on nutrients) and metabolic activity (exoenzymatic activities) determinations, the use of various methods (both molecular and chemical) confirmed that both the composition and structure of the bacterial community (as it was determined by the application of flow cytometry and clone libraries) in addition to bacterial activity, were different in untreated and ozonated biological filters. In conclusion, results provide an important contribution to the current knowledge on the dynamics and relationships between the different compartments in complex systems such as recirculated aquaculture systems, mainly giving indications about the proper management of the filters in relation to the parameters that characterize the system itself.