Introduction
Denitrification is a biofiltration process which is commonly applied in RAS to control nitrate concentrations by converting nitrate-nitrogen (NO3-N) to nitrogen gas. This process requires high amounts of organic carbon, which is commonly supplied by external carbon sources that increase farm operational costs. Faecal waste can be used alternatively as an internal carbon source for denitrification but its insufficient carbon bioavailability often limits NO3-N removal. The quantity of bioavailable faecal carbon depends on diet digestibility and the respective faecal composition. Lately numerous alternative ingredients have been used in aquafeeds, frequenly resulting in increased indigestible carbohydrate fractions. As such, faeces capacity to act as an internal carbon source for denitrification may vary with diet composition (Meriac et al., 2014). In this respect, the present study aimed to explore the effect of dietary carbohydrate on the denitrification potential of a marine RAS using internal faecal carbon sources.
Materials and methods
In vitro trial: Settleable feaces originating from a preceding feeding trial (Syropoulou et al., 2022) testing six dietary ingredients (shrimp shell meal; SSM, feather meal, insect meal; IM, seaweed, single-cell meal, dried distillers grain with solubles; DDGS) on European seabass were collected. Faecal material was incubated in anoxic batch reactors under isocarbonic conditions for a 14-day period. Abiotic parameters including NO3-N were monitored daily and faecal samples were obtained every 24 h for the first three days (Day1, 2, 3) and every alternate day onwards. Samples were immediately analyzed for volatile fatty acids (VFAs) to determine the degree of fermentation (Letelier-Gordo et al., 2017). Data was normalized to the Day0 values and was expressed per unit of organic matter (OM) faeces and per unit of feed, respectively.
In vivo trial: Four identical RAS equipped with an up-flow sludge blanket denitrification reactor (DR), were stocked with juvenile European seabass (1.78 ± 0.02 kg/RAS). Fish were fed over a six-week period with two of the aforementioned six diets (IM, DDGS) in duplicate. Feeding level was gradually increased until Week2, when a fixed amount of 50 g feed/RAS/day was established. During the trial, presettled feacal waste produced per system was constantly fed to the respective DR. NO3-N levels in the outlet of the fish tanks were analyzed weekly, whereas in the inlet and outlet of the DR at the end of the trial over a 24-h scheme. Finally, after the completion of the experiment, both faeces and DR sludge were analyzed for their nutrient composition in order to create nutrient balances on a DR level.
Results & Discussion
Degree of fermentation, expressed as VFA production per kg OM feed, was influenced by OM digestibility, faecal removal efficiency via settling and carbon bioavailabity of feaces. Even though, fish fed with SSM excreted a low amount of feaces, feacal quality improved faecal removal efficiency and thus more material was available to be fermented per kg OM feed. Additionally, feacal carbon bioavailability was highest for this diet which explains the highest VFA production not only per kg OM feed (Figure A) but also per kg OM faeces. Among the tested diets, IM produced the second highest amount of VFAs whilst DDGS the second lowest. Additionally, the two diets yielded a highly different VFA profile, with propionate being produced only from IM faeces and lactate only from DDGS faeces. Due to the higher amount of VFAs produced in the in vitro trial along with the more favorable VFA profile, we hypothesized that IM would act as a more efficient internal carbon source for denitrification in RAS between the two. However, when denitrification efficiency was studied in the in vivo trial, no significant differences were found for NO3-N removal between the two dietary treatment groups (p>0.05; Figure B). This discrepancy between trials might be attributed to their different experimental duration, indicating that possibly long-term anaerobic digestion of feacal material may allow for better utilization of feacal carbon. Moreover, nutrient conditions differed upon case, since NO3-N was soon depleted in the in vitro trial, whereas NO3-N was constantly supplied to the DRs in the RAS setup. This likely resulted in the development of different microbial communities with different capacities to utilize faecal carbon. Additional microbial data will help identify the reason of this inconsistency. In conclusion, the present study showed that batch fermentation of faecal material is not a good indicator for the potential of faeces to act as an internal carbon source for denitrification.
References
Letelier-Gordo, C. O., Larsen, B. K., Dalsgaard, J., & Pedersen, P. B. (2017). The composition of readily available carbon sources produced by fermentation of fish faeces is affected by dietary protein:energy ratios. In Aquacultural Engineering (Vol. 77, pp. 27–32). Elsevier B.V. https://doi.org/10.1016/j.aquaeng.2017.01.006
Meriac, A., Eding, E. H., Kamstra, A., Busscher, J. P., Schrama, J. W., & Verreth, J. A. J. (2014). Denitrification on internal carbon sources in RAS is limited by fibers in fecal waste of rainbow trout. Aquaculture, 434, 264–271. https://doi.org/10.1016/j.aquaculture.2014.08.004
Syropoulou, E., Prakash, S., Smeenge, D., Prabhu, P. A. J., Sipkema, D., Schrama, J. W., & Kokou, F. (2022). Dietary carbohydrate sources for European seabass reared in recirculating systems: Impacts on digestibility and waste production. https://aquaeas.org/Program/PaperDetail/39834