Tracing activation efficiency of the bio-filtration bacteria in a recirculating aquaculture system using stable isotope analysis

Abstract

One challenge in starting a new recirculation aquaculture system (RAS) is to find the most efficient method to activate the nitrifying bacterial biofilm in the bio-filtration system. It has been suggested that chemical startup with ammonia and nitrite surpasses the “cold start” method where biofilm originates from the fish introduced to the un-activated system. However, there is no information on how the start-up method affects the actual nitrification activity of the bio- filtration system. The objectives of this study were to evaluate how different activation techniques affect the overall function and the nitrification activity of the bio-filtration units, and the interactions between heterotrophic and nitrifying bacteria in the RAS. The knowledge is poor on how different start up methods affects nitrification activity. All previous studies have focused on ammonium reduction rates and not on the actual nitrification activity process rates. This is the first study to do an in depth analysis of comparing activation techniques used for bio-filtration units in a Recirculating Aquaculture System. Also this is the first study to analyze how the activation technique affected the activity of the bio-filter and the proportion of heterotrophic bacteria vs. nitrifers that were contained in the biofilm by using carbon and nitrogen stable isotope analyses. To study these questions, I conducted an experiment at the Natural Resources Institute Finland, in Laukaa, Finland. We had eight experimental RAS units, four different start-up methods tested (cold start method with fish, ammonia addition, ammonia + nitrite addition, ammonium + nitrite + glucose addition) with two replicate units per treatment. The experiment was divided to two phases. The activation phase continued for five weeks (Feb 10 - Mar 16, 2016) after which rainbow trout (average size appr. 0.6 kg, biomass 20 kg per tank) where added to the units. Once the fish were added all of the additions in the treatments were stopped. Fish were fed 24 hours per day, at the rate of 1.6% per day. The addition of the fish in all the treatments after activation of the biofilter system was measured for a period of 25 days. In the activation period, the ammonia levels were highest in the ammonia treatment. After the fish were added, all treatments, besides the treatment with glucose, had an increase in the nitrate concentrations, signaling the nitrification activity. The highest nitrate 2 Foore, Nathan: Tracing activation efficiency of the bio-filtration bacteria in a Recirculating Aquaculture System using Stable Isotope Analysis Supervisors: Sanni Aalto & Jouni Vielma

concentration was in the cold start treatment followed closely by the ammonia and the ammonia and nitrite treatments. In the glucose treatment, the ammonia levels continued to rise, signaling low nitrification activity. During the activation period, the carbon content of the biofilm in the cold start and glucose treatments was already high, whereas in the ammonia and in the ammonia + nitrite treatments the biofilm carbon content rose rapidly after the addition of rainbow trout, implying the development of the biofilm biomass. The δ13C of the biofilm in all other treatments than the glucose treatment reflected the isotope value of the faeces and DIC, while in the glucose treatment the δ13C originated from the glucose used by the heterotrophs. The δ15N of the biofilm correlated positively with nitrate concentration, with highest δ15N values found from ammonia, ammonia + nitrite and cold start treatments, indicating their highest nitrification potential. In the glucose treatment, the δ15N of biofilm reflected mainly heterotrophic remineralization of organic matter. The cold start treatment has been demonstrated to be equally as effective as the chemical startup treatments. Evidence also suggests that the cold start treatment 1) amplifies the nitrification rate of the biofilm in the bio-filtration system, among the tested treatments, and 2) improves maturation time, just as effectively as the chemical startup treatments do. These results may come as a surprise to many who believed in the unassailable superiority of the chemical treatments. As for the glucose-subsidized method, it led to poor nitrification activity, indicating that adding glucose increases the amount of heterotrophic bacteria in the biofilm, which means the nitrifiers are less efficient at nitrification. Combining lab and field work with stable isotope analysis it is possible to come to accurate and detailed findings about the nitrification activity rates of bio-filtration treatments.