ALBERT

Description: Introduction Disinfection is a very important part of recirculation aquaculture systems (RAS). RAS allows for controllable environments in which main variables relevant to animal welfare and a successful production can be manipulated as required to improve efficiency and profitability. Common disinfection methods include chemical disinfectants, antibiotics, biocides, UV radiation and ozone. They can be used to treat disease outbreaks, or to reduce the bacterial load of the system which otherwise could lead to the overgrowth of potential pathogens or opportunistic bacterial groups competing with biofilter bacteria for space and resources. Ozone is the most used disinfection method requiring expensive technology and trained stuff. Hydrogen peroxide (H2O2) has been on focus as a “green” alternative. High H2O2 doses are associated with acute toxicity symptoms for some species. Low doses are harmless, offer additional system oxygenation and contribute to water quality improvement. After testing the use of low doses in a small research RAS, the present study aims to describe the first case study up-scaling a continuous hydrogen peroxide application to commercial fish production in RAS with focus on the determination of required concentrations, application monitoring and variations on microbiome composition. Material and Methods The present study was performed at RAS facilities of Danish Salmon A/S in Hirtshals, Denmark, one of the European pioneers in rearing salmon in land based aquaculture facilities and producing about 1.200 metric tons/year of Atlantic salmon (Salmo salar). Two identical Grow-out RAS with own water treatment elements and eight pre-grow tanks were used as treatment and control systems to compare the effects of continuous H2O2 application in combination with ozone to common operational practice. Oxygen Cones and additional aeration stones ensured the basic Oxygen supply in the tanks. Defined H2O2 quantities were applied with a dosing lance connected to a peristaltic pump and an International Bulk Container with PERSYNT® 50, (EVONIK Industries) into the distribution pipe feeding a collection tank from where the water was evenly distributed to all tanks. Based on previous experiments, a final dosing of about 20 l/h was projected. Water samples were collected at the start (REF) and after slowly enhancing the dosage over time (24h and 30d), for the determination of the total microbial count (certified chromogenic Compact Dry TC plates from R-Biopharm), microbial viability (BacLight Viability Kit) and bacterial community composition (FISH). Water parameters (Ammonia, Nitrite, Nitrate, Phosphate, COD, Turbidity and H2O2 concentration) and production related information (feeding rate, fish biomass, oxygen consumption, ozone production) were also regularly evaluated. Results and Discussion H2O2 application started on August 06, 2020 with 1 l/h (0.51mg/L) and was increased over time up to 14 l/h (7.09 mg/L) (Fig1). On September 30, 2020 the application was stopped due to detected changes on feeding behavior of the fish as well as incorrect redox measurements in the treatment system which could probably be attributed to accumulation of oxidative species not having enough organic material to react. The test and control systems had similar biomass during the experimental period (test: 34.9-52.5 tones and control 36.4-51.4 tones) and the feed intake was accordingly adjusted (mean feed intake test: 496 kg/day; control 450 kg/day). The oxygen demand registered on the treated system (82 l/min - 90 l/min) was lower than the control (104 l/min - 115 l/min). In general, there was a reduction of turbidity and decreased nitrogen species and phosphate in the treated system. System maintenance (biofilter cleaning and backwashing) might had influenced the COD and total microbial counts measurements. Total microbial counts reflected a steady increase in the number of CFU/ml in both systems (Fig.2) and evidenced microbial accommodation. There was an increased rate of microbial mortality according to the viability results with higher values in the treated system reflecting the longer exposure of this system to oxidative stress (Fig 3). The community composition varied according to the treatment (Fig.4) and changes in the abundance of the different bacterial groups analyzed could be attributed to variable vulnerability of members of these groups to the disinfection potential of H2O2 or its combination with ozone.

Description: Recirculation Aquaculture Systems (RAS) reduce water consumption but require costly filtering methods needed in order to maintain adequate levels of accumulating compounds, such as nitrogenous compounds and sludge. Sludge (faeces and waste diet) is mechanically removed by drum filters and settling systems and disposed of, while nitrogen compounds are reduced via nitrification-denitrification filters, the latter of which requires of supplementary commercial carbon sources. Sludge is a source of particulate carbon, potentially for use in denitrification, equivalent to ca 20% of feed input (Lekang 2013). The reutilization of sludge implies the correct accumulation and storage to avoid uncontrolled decomposition by partial exposure to aerobic and anaerobic conditions and the disintegration of complex molecules into easily biodegradable nutrients. Ozone exposure is a potential method to promote mineralization and allows formation of biodegradable intermediates. The present study investigated the potential of sludge as an alternative carbon source for denitrification and asks: i) How much time should the sludge be exposed to ozone in order to achieve the highest levels of carbon intermediates in solution? ii) What are the remaining constituents of the solid phase of the sludge post- ozonisation prone be used as nutrients by denitrifying bacteria? iii) Is there an influence of different fish diet composition on the fatty acid content of sludge? iv) Is denitrification possible when using ozone-treated sludge as a carbon source? As particulate matter represents a great amount of the sludge discharge, this study aimed to estimate the dissociative effect of ozonisation on the solid phase and the physicochemical changes that this process could bring to the water matrix in terms of carbon and nitrogen content present in solution and in particulate state. The preliminary results of a batch pilot study using ozone-activated sludge vs. a common commercial carbon source for denitrification are also provided.

Description: Introduction Water treatment plays a central role in the successful operation of recirculating aquaculture systems (RAS), because high stocking densities and feeding rates rapidly impair the quality of the limited water body. In RAS, nitrogen load is typically removed by multistep biological filtration whereby the first step is ammonium oxidization to nitrite. This biological degradation is vulnerable to disturbances e.g. changing water conditions and medications of the cultivated species what can lead to an accumulation of toxic nitrogen components in the system water. Ammonium can alternatively be removed from water by using zeolite. In emergencies zeolite can decrease fish mortality by reducing dangerous ammonium concentrations. In the present study a marine RAS, stocked with Sea bass (Dicentrarchus labrax), was operated with the bioreactor replaced by zeolite in the RAS water. Water parameters, microbial community and reared animals were determined to evaluate the impact of an alternative ammonium removal method on RAS performance. Material and methods Two identical 5m3 RAS each equipped with 3 rearing tanks, drum filte , ozone- injected protein skimmer and moving bed bioreactor were operated at constant water parameters (temperature: 17.0°C, conductivity: 50.4mS cm-1, pH: 7.9, O2: 97.8%). RAS were stocked in each experiment with 9kg m³-1 juvenile Sea bass (102.7 ± 25.3g) and fishes were fed 8 times a day (1.02% body weight per day) during a period of 21 days. These two RAS were controlled in 3 different scenarios: 1) RAS 1 and 2 with bioreactor and ozone, 2) RAS 1 and 2 with bioreactor without ozone and 3) with zeolite and ozone (RAS 1) or without ozone (RAS 2). Nitrogen components and abiotic water parameters in the water were measured daily. At the beginning and the end of each phase bacterial count and the composition of the bacterial community were determined. At the end of each phase the blood of 12 fishes per RAS was collected and analysed for relevant parameters (cortisol, glucose, lactate, ammonium, lysozyme, haematocrit). Results During phase 1 and 2 ammonium and nitrite concentration in the tank waters were similar in both RAS (Figure 1). Zeolite treatment (phase 3) led to a large increase in ammonium concentration and a decrease in nitrite concentration when ozone was applied (RAS 1), while without ozone ammonium increase was much lower but nitrite was strongly elevated (RAS 2). Nitrate level in the water increased during phase 3 in both RAS but with a higher slope in the system with ozone. During phase 1 and 2 ozone treatment had minimal effect on the bacterial count in both systems. Numbers of bacterial units increased strongly during zeolite treatment, especially when ozone was not applied (Table I). Zeolith treatment had no effect on stress related blood plasma parameters of the fishes. Discussion Zeolite had strong effects on water parameters in both RAS. Ammonium content in the water were elevated without bio- filtration, and probably caused bacterial growth in the RAS leading to high nitrite level in RAS 2 without ozone. An additional ozone treatment could prevent the accumulation of toxic nitrite, because of the spread of the bacterial mass was reduced. Blood parameter data indicate that the zeolite treatment could be suitable to continue the normal operation of a RAS when biofiltration is affected allowing emergency situations to be managed without the need for performance reducing emergency measures such as stopping feeding or reducing stock biomass.

Description: Recirculating Aquaculture Systems (RAS) reduce water consumption by efficient filtration to maintain appropriate levels of accumulating compounds and sludge. Sludge is mechanically separated by drum filters and disposed of to the detriment of overall system water budgets. Dissolved nitrogen compounds are reduced via nitrification–denitrification filters, requiring commercial external carbon sources. The reuse of sludge after ozone pre-treatment may represent the next step in RAS optimization. The present study analyzes the content of sludge from RAS and tests ozonation as a pre-treatment for recycling as carbon source. The dissociative effect of ozone and the physicochemical changes due to ozonation lead to a significant increase in soluble carbon availability. Predominantly long-chain fatty acid (FA) (saturated and unsaturated) with 16 and 18 carbon atoms independently of the treatment were found in the profiles. Saturated FA concentrations in solution increased after 20, 40, and 60 min ozonation. The solid content of the sludge was practically unaffected by ozonation in terms of FA profile: only saturated FA slightly increases after 40 min treatment. The implications of these findings for denitrifying bacteria are discussed.