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Nitrogen budget in recirculating aquaponic systems with different fish stocking density

As in any agroecosystem, also in aquaponics the nitrogen (N) balance represents an important tool to evaluate sustainability, and to identify factors that can improve N use efficiency (NUE) and reduce N losses. In this respect, fish stocking density has been little investigated, hence this research aimed to evaluate the N balance of a low technology aquaponic (AP) system managed at two fish densities in comparison with a hydroponic system (HP). In the fish tanks common carp at two initial stocking densities were reared (2.5 and 4.6 kg m–3 in low and high AP, hereafter named APL and APH, respectively) and the vegetated sector was cultivated with a leafy vegetable crop succession (Catalogna chicory, lettuce, Swiss chard). The N balance considered N input as fish feed or fertiliser, and N content in the initial water and the N output as N in the incremented fish biomass, in the harvested vegetables, in the sediments, and in the remaining water. Total N loss was estimated by difference. The total N input and the N loss through gas emission in the atmosphere were much higher in AP than in HP, particularly at high stocking density. The opposite trend was observed for the N input recovered in vegetable aboveground biomass. The N input recovered as fish biomass was slightly higher in APL compared to APH. The better results of APL than APH suggest that in low-tech AP system lower initial fish density should be adopted at the system start up to maximise both production and N recovery.


Introduction
Aquaponics (AP), the combination of hydroponics and recirculating aquaculture, is a promising atypical and complex food production technology (Somerville et al., 2014;König et al., 2016;Maucieri et al., 2018;Goddek et al., 2019). This integrated production system is meant to increase the sustainability of both fish and vegetable production. Indeed, as summarised by Yogev et al. (2016), the main advantages of AP are related to high water use efficiency, as the volume of water used to produce both fish and vegetables is lower compared to conventional agriculture; low fertiliser use, as vegetable nutrition, is mostly fulfilled by fish feed; use of organic practices, as chemicals are frequently toxic for the fish; low land use, as no fertile soil is required for vegetable production; high smallholder welfare, as it is able to give high vitamin and protein production per unit surface area. As in typical agroecosystems, also in AP nitrogen (N) balance represents a key point because the vegetable N requirements can compensate the low fish N use efficiency (NUE), with positive effect on both profitability and environmental impact. Indeed, as reviewed by Crab et al. (2007), purchase of commercial feed for fish farming comprises 50% or more in the production costs, mainly due to the cost of the protein component (the major source of N). In addition, in aquaculture systems, only 11% to 36% of the N input with feed is converted into harvestable products whereas about 75% is excreted in the water by fish (Hargreaves, 1998;Gross et al., 2000). This greatly affects environment compromising quality of discharged water and increasing NH3 volatilisation and N2O emissions in the atmosphere (Muralidha et al., 2017). In this context, AP technology can be a valuable solution to reduce the environmental impact of fish production reducing N2O emission and water use along with increasing the NUE through vegetable production (Paudel, 2020). Only few studies are available on N balance in AP systems to evaluate the contribution of vegetables on N recovery from fish effluent. Endut et al. (2014) reported a N recovery capability of the hydroponic section of 88% of the total ammonia N released in the AP system by fish. Fang et al. (2017) estimated that 24.9% of N input was recovered in fish biomass and a further 22.3% in the vegetable biomass, while Jaeger et al. (2019) found that only 19.3% of N in fish feed was recovered in common carp and lettuce biomasses. A cumulative fish-vegetable NUE of 48.9% has been reported in a small-scale AP system producing common carp and pakchoi (Brassica chinensis) with minimal NUE variation between summer (43.8%) and autumn (44.6%) (Zou et al., 2016a(Zou et al., , 2016b. A plant species effect on N recovery efficiency in AP has been found by Hu et al. (2015) who obtained a NUE of 41.3% and N o n -c o m m e r c i a l u s e o n l y 34.4% in tomato (Solanum lycopersicum) and pakchoi based AP systems, respectively. Wongkiew et al. (2017a) pointed out that NUE can be improved by reducing the feed rate, that halved N loss when the rate was reduced from 50 to 35 g d -1 , or lowering the feed-to-plant ratio, that decreased N loss by about 50% when the ratio decreased by 30%. Finally, Paudel (2020) compared different initial plant-fish biomass ratio, and found that increasing this ratio from 0.06 to 0.95, NUE increased from 29.3 to 39.1%. To our knowledge, no studies have compared the effect of different fish stocking density, maintaining constant biofilter surface and plant density, on N balance in aquaponics.
In view of this, the aim of this study was to evaluate the N balance of a recirculating AP system managed at two fish stocking densities in a vegetable succession, in comparison with a hydroponic system. Our specific objectives were: i) assess the influence of initial fish density on N balance and NUE; ii) evaluate the contribution of fish and plants on N recovery; iii) estimate the N gaseous losses in the different conditions.

Experiment set-up
The pilot-scale experiment has been carried out in a PE film single-span greenhouse covered with a 50% shade net, located at the experimental farm of Padova University, North-East Italy (45°20′ N; 11°57′ E; 6 m a.s.l.). The experimental treatments were: aquaponics with low fish density (APL), aquaponics with high fish density (APH) and hydroponics with no fish (HP), as control. The experiment was arranged as in a randomised block design with three replications for a total of 9 independent units. Each experimental unit ( Figure 1) consisted of: A) a tank (500 L) where in the AP units fish was farmed, whereas in the HP units only nutrient solution was present; B) two tanks, filled with 225 L of expanded clay as growing media (LECA Laterlite, Solignano, Italy), that received the same water flux from tank A and acted both as biofilter and cultivation tank for vegetables; C) a water storage tank (50 L volume) where the output from vegetable tanks was collected before relaunching in the tank A. In total, a surface of 0.63 m 2 of tank A was combined with 1.57 m 2 of vegetable production. The three parts of the system had water surfaces at different heights so that the water flow inside was guaranteed by overflow. The water accumulated in the storage tank was relaunched to the fish tank by a pump (Newa Jet 1700, NEWA Tecno Industria Srl, Loreggia, Italy) with water flow rate of 120 L h -1 allowing a complete water system recirculation every 5 h. The water oxygenation in the tanks A was guaranteed by porous stones connected to an aerator with flow rate of 102 L min -1 (Scubla D100, Scubla Srl, Remanzacco, Italy).
The experiment started on 19 th June 2017, when tanks were filled with a total of 600 L tap water, and ended on 7 th November 2017 with the harvest of fish and vegetables. On 27 th June, after water reached constant temperate, fish were put in APL and APH treatments (which through their wastes acted as N fertilisation), whereas in HP treatment 607 g per unit of Ca(NO3)2·4H2O (1011 mg L -1 ) were added. Except N, all 9 units contained the same nutrient solution (220 mg L -1 of KH2PO4, 330 mg L -1 of K2SO4, 456 mg L -1 of MgSO4 · 7H2O, 31 mg L -1 of Fe-EDTA, and 13 mg L -1 of micronutrients). In the AP units, tanks A were stocked with full-scaled common carp (Cyprinus carpio L.) obtained from a commercial farm with an initial live weight of 169±56 g at initial stocking densities of 2.5 kg m -3 and 4.6 kg m -3 for APL and APH treatments (i.e. 7 and 14 fishes per unit), respectively. The follow-ing crops succession was cultivated in the vegetable tanks: Catalogna chicory (Cichorium intybus L. Catalogna group -from June 27 th to July 25 th , 9 plants m -2 ), lettuce (Lactuca sativa L.from July 26 th to August 29 th , 12 plants m -2 ) and Swiss chard (Beta vulgaris L. subsp. vulgaris, Cicla Group -from August 29 th to November 7 th , 10 plants m -2 ).

Operation and monitoring of the system
Fish health status and mortality were monitored daily. The fish were manually fed once a day with a commercial extruded sinking pelleted feed (Classic K, Skretting, Verona, Italy; 41.1% crude protein). In the units, water was never changed throughout the trial. Water lost by evapotranspiration of each unit was manually refilled daily.

Sampling and analytical methods
Two times per week water was monitored for: i) in situ temperature, dissolved oxygen (DO), pH, redox potential (ORP), and electrical conductivity (EC) using a portable multi-parameter meter (HQ40d Portable Multi-Parameter Meter, Hach Lange GmbH, Germany); ii) in laboratory, water NO2 − , NO3 − , and NH4 + concentration were determined by ion chromatography (ICS-900 system, Dionex Corp., Milan, Italy). Six times during the experimental period, the water used to refill evapotranspiration losses was analysed for NO2 − , NO3 − , and NH4 + concentrations. In the result section, data are expressed as NO2−N, NO3−N, and NH4−N. All fishes from each unit were weighed individually every month (five times) during the experimental period and the feed quantity to be administered daily was calculated for each AP unit on the base of biomass present at the moment of each weighing at rate ranging from 0.5% to 2% (Maucieri et al., 2019). Total feed N content was determined by Kjeldahl method. Total N content of carp biomass (2.7%) was estimated considering an average of literature data (Schwarz et al., 1998;Skibniewska et al., 2013).
At harvest time plants were gathered, divided into above and belowground biomass, and dried in a thermo-ventilated oven at 65°C until a constant weight was reached to determine dry weight and dry matter content. Total N content in the biomass was determined by Kjeldahl method.
At the end of the experiment the expanded clay content in the vegetable tanks was removed and washed with fresh water to collect tanks sediment. The sediment was dried in a thermo-ventilated oven at 65°C until a constant weight was reached. After that, total N content in the dry sediment was determined by CNS Macrovario combustion analyser (Elementar Analysensysteme GmbH, Germany) and values have been expressed as percentage on dry matter.

Estimation of nitrogen species and mass balance
The N mass balance was calculated considering the different N species measured in the experiment. In particular, N input was originated by the application of feed (in APL and APH), fertiliser (HP) and by the N water content (in initial and additional water). The measured N species were the total N contained in the fish, in the vegetables, in the sediments and in the residual water at the end of experiment. The difference between total input and output was accounted to gaseous losses. Hence, N balance was calculated according to the following equation: Nfeed/fertiliser in + Nwater in = Nfish out + Nplants out + Nsediment out + Nwater out + Ngas out Where: Nfeed/fertiliser in (g) = Feed supplied (g) × Total Kjeldahl Nitrogen (TKN) (%) or Fertiliser supplied (g) × N (%) Nwater in (g) = (Initial water volume + Evapotranspiration refill) (L) × (NO3-N + NO2-N + NH4-N)(g L -1 ) Nfish out (g) = (final fish weight -initial fish weight) (g) ×2.7% Nplants out (g) = Vegetables biomass (g) × TKN (%) Nsediment out (g) = Sediment dry weight (g) × N (%) Nwater out (g) = Final water volume (L) × (NO3-N + NO2-N + NH4-N) (g L -1 ) Ngas out (g) = the amount of N that was not recovered through water, sediments, plants and fish and represents the N lost through ammonia volatilisation and nitrification/denitrification (Wongkiew et al., 2017b). As this is the only unknown variable, it was calculated by difference.
Water dissolved organic N content was not considered in our budget for two reasons: i) it was absent in the water used to fill and refill systems as municipal drinking water was used; ii) it can be considered negligible in the residual water (Hargreaves, 1998). Fish solid wastes and uneaten feed, suspended in water, were trapped in the biofilter and their total N content was considered in the budget as N-sediment out whereas the residual dissolved organic N in the water was mineralised by proteolytic and heterotrophic bacteria to dissolved inorganic substances (especially ammonia) (Hargreaves, 1998).

Statistical analysis
Data were checked for homogeneity of variance across treatments by using Kolmogorov-Smirnov test. Normal distributed data (systems ET and N budget components) were analysed using analysis of variance (ANOVA) and when significant (P<0.05), means were separated by Tukey's HSD test.
For data not normally distributed (water temperature, dissolved oxygen, electrical conductivity and pH), Kruskal-Wallis nonparametric test (accepted at the level of P<0.05) was used to check the significance of differences.

Water management
Daily water consumption in the experimental units due to evapotranspiration was not significantly different among treatments with an average value of 3.7 L m -2 day -1 , equal to a daily evapotranspiration of 1.37% compared to the total water content of the system. A significant seasonal effect was found according to the crop cycle with the highest average consumptions monitored during summer cycles (Catalogna 5.1 L m -2 day -1 and lettuce 5.3 L m -2 day -1 ) and the lowest during summer-autumn Swiss chard cycles (2.1 L m -2 day -1 ) ( Figure 2). The lower water consumption during Swiss chard cycle is likely due to the low temperature that both reduced evapotranspiration and plant growth rate (Maucieri et al., 2019). Daily water evapotranspiration of our experimental AP gave typical performance of AP commercial systems (daily evapotranspiration from 0.05% to 5% of system total water content) (Maucieri et al., 2018). The cumulative AP system water consumption per cultivated unit surface was 143 L m -2 , 175 L m -2 , and 147 L m -2 for Catalogna chicory, lettuce, and Swiss chard cycles, respectively. Values are in line with water consumptions that these species have in traditional cultivation systems (120-200 L m -2 , 150-200 L m -2 , and 150-250 L m -2 for Catalogna chicory, lettuce, and Swiss chard, respectively). Considering that the AP system, in addition of vegetables also provided fish biomass, the water use efficiency was actually higher, confirming that AP is a water saving technology. At the beginning of the experiment each aquaponic unit was filled with 600 L of water whereas in the whole experimental period 1096 L, 1123 L, and 1069 L were supplied to refill evapotranspired water in the HP, APL, and APH treatments, respectively, with an average concentration of 4.04 mg NO3-N L -1 and 0.19 mg NH4-N L -1 . Cumulatively, 7.18±0.33 g N, 7.29±0.20 g N, 7.06±0.03 g N were added with water (Nwater in) in HP, APL, and APH treatments, respectively.
The highest NH4-N concentration was measured at the beginning of the experiment. In about one week NH4-N concentration dramatically decreased with, in the same time, a dramatic increase of NO2concentration. Unlike NH4-N and NO2-N, NO3-N concentration followed a treatment specific trend: i) in HP treatment values decreased progressively from the beginning to the end of the experiment; ii) in the APL treatment the NO3concentration increased from the third week to the end of September, then tendentially decreased; iii) in the APH treatment the NO3-N concentration remained stable near zero for all the experimental period ( Figure 3). Evolution over time of N forms in AP treatments showed similar trends of which observed by Hu et al. (2015) and Zou et al. (2016a). The highest NH4 + and NO2concentrations, observed during the first three experimental weeks, were due to the slow growth of nitrifying bacteria that determined an accumulation of intermediate N forms (NH4 + and NO2 -) (Hu et al., 2015). Particularly, in AP systems N was daily supplied in organic form (feed protein) that was metabolised by fish and released in form of ammonia. Subsequently, ammonia can be oxidized to NO2by ammonia oxidizing bacteria and then in NO3by nitrite oxidizing bacteria (Wongkiew et al., 2017b). However, the growth rate of the bacteria involved in the nitrification process is unpaired determining intermediate accumulation ( faster growth rate than nitrite oxidizing bacteria at temperature above 25°C) (Yamamoto et al., 2008). The lack of NO3concentration increase in the APH treatment during all experimental period was due to the higher fish density that determined, in the hydroponic section, a higher accumulation of organic matter and a lower oxygen content than APL. These conditions have probably determined a favourable environment for a greater denitrifying bacteria growth which determined NO3reduction (Endut et al., 2010). At the end of the experiment the N concentrations of residual water in the system were significantly different among treatments with the highest and lowest residual N found in HP and APH treatments, respectively (Table 1).

Environmental conditions
Water temperature was not different among treatments whereas significantly lower values were observed during Swiss chard cycle (summer-autumn crop) than Catalogna and lettuce cycles (summer crops) (Figure 4). Water temperature (range 14-31 °C) was always within the indicative absolute cold and heat stress temperatures of 5° and 35 °C for common carp (Koehn, 2004), and during the majority of the experiment close to the optimum temperature (i.e. 15-31 °C) indicated by Jaxion-Harm and Ladich (2014), and able to guarantee optimal performance at both nitrification (Kinyage and Pedersen, 2016) and denitrification (Akratos and Tsihrintzis, 2007;Xu et al., 2016) processes.
Dissolved oxygen was significantly different among treatments and crops with higher values observed in the HP treatment compared to AP treatments and in Catalogna and Swiss chard crops compared to lettuce ( Figure 5). This water parameter is one of the factors that influence nitrification with a consumption approximately ranging from 4.18 mg to 4.57 mg of oxygen per mg of NH4-N oxidized to NO3-N (Chen et al., 2006;Vymazal, 2007). Thus, the lower dissolved oxygen values in the AP treatments, in addition to fish metabolism and respiration, was due to nitrification process. As far as crop effect is concerned, lettuce had much higher growth rate compared to Catalogna (Maucieri et al., 2019), hence consuming more oxygen for aerobic respiration (Ehret et al., 2010;Lara et al., 2011); then, Swiss chard had lower growth rate, but water temperature was lower, increasing oxygen solubility (Trejo-Téllez and Gómez-Merino, 2012;Al-Rawahy et al., 2019).
A different trend among treatments was observed considering water EC with a constant decrease in the HP treatment, and steady pattern in the other two treatments in Catalogna chicory and lettuce crops; a decrease in values was observed in Swiss chard as well, but beginning from the second half of the cycle (Figure 6). In the HP treatment, all nutrients were added with fertiliser to the water only at the beginning of the experiment, and then nutrient concentrations decreased because of the plant up-take. Differently, in AP treatments N was not provided with fertiliser, and thus was lower. In the Catalogna and lettuce crop cycles EC remained constant because nutrients released through fish feces were taken up by plants at the same rate. In the last crop cycle, the initial growth rate of Swiss chard plants was very low, leading to an increase of ions   that were not promptly taken up, but later plants nutritional needs augmented and thus EC decreased. Water pH ranged between 7.0 and 9.0 without differences among treatments; higher water pH values were measured during the first crop cycle whereas similar values were detected during lettuce and Swiss chard cycles (Figure 7). This parameter is one of the key factors in AP systems because it influences fish, plants and microbes at the same time but optimal values are different for each of the three organisms (Tyson et al., 2011;Goddek et al., 2015). In this experiment pH ranged from 7.0 to 9.0, within the tolerated pH for aquaponics, even if it has to be taken into account that NUE decreases as pH value increases (Zou et al., 2016c).

Article
In our study, during the first crop period the average pH was near 8.5, values that speed up nitrifying bacteria development (Tyson et al., 2004) while reducing denitrifying bacteria activity (Zou et al., 2016c). However, in order to keep pH values closer to vegetables needs, in the other two cycles pH values were maintained (adding phosphoric acid) near to 8.0, in accordance with optimum pH for aquaponics as stated by Goddek et al. (2015).

Fish performance
Fish health was very good during the whole trial and only two fish died (1 from APL and 1 from APH treatments) and, at the end of the trial, fishes weighed 446±147 g, on average. About feed, 3443.6±79.0 g system -1 (APL) and 6387.5±187.4 g system -1 (APH) were added during experimental period which represent the 96.9% and 98.3% of total N input in the APL and APH treatments, respectively. In other terms, while APL showed a significant lower N input with fish feed of 46.1% than APH, the N output with fish biomass was only 35.4% lower in the APL than APH treatment (Table 2). In the HP treatment, where mineral N was added, the N input was 72.0 g system -1 (Nfertiliser in).

Vegetables performance
Vegetables yield was significantly different among treatments during all crop cycles (Maucieri et al., 2019;Maucieri et al., 2020). Vegetables biomass N uptake was significantly different among treatments with better performance for APL and HP treatments whereas for all cropping cycle the APH showed the worse performance (Table 3)     three crop cycles the best performance was obtained for APL treatment with a higher uptake of 12.0% and 44.3% than HP and APH, respectively. The N uptake for vegetated unit surface was 13.7 g N m -2 and 9.5 g N m -2 equal to a daily average uptake of 97.2 mg N m -2 d -1 and 67.4 mg N m -2 d -1 for APL and APH, respectively. Considering the daily average Nfeed in (1.0 and 1.9 g N m -2 d -1 for APL and APH, respectively), the daily vegetables uptake represents only the 9.7% and 3.5% of N supplied with feed in APL and APH treatments, respectively.

Sediment
Sediment production was different among treatments with the highest value in APH treatment and the lowest value in HP. This was expected, as sediments derive from deposition of fish feed and feces (Grant et al., 2019) which of course increased with increasing stoking density. No statistical differences among treatments were obtained considering sediment N concentration and content (Table 4).

Nitrogen mass balance
During the experimental period the total N input (Nfeed/fertiliser in + Nwater in) was 79.2±0.3 g, 234.0±5.2 g, and 427.6±12.4 g per unit for HP, APL, and APH treatments, respectively. At the end of the experimental period the N recovered as fish, plants, sediment and water was 91.4%, 53.9%, and 32.3% of the total N input for HP, APL, and APH, respectively ( Figure 8). The useful N recovery (plant + fish) was 16.1, 45.0 and 54.1 g m -2 for HP, APL, and APH treatments, equal to 24.2%, 34.8%, and 25.2% of total N input, respectively. The fraction to reach the 100% (Ngas out) was represented by N that was released in the atmosphere as gas compounds (e.g. N2, N2O) during N nitrification and denitrification and N released in the atmosphere as NH3 (Wongkiew et al., 2017b).
The N supplied with feed and recovered with fish biomass was 26.5% and 22.1% in APL and APH, respectively, in line with literature data (11%-36%) (Hargreaves, 1998;Gross et al., 2000;Hu et al., 2015). These data confirm the low aquaculture NUE and highlight the great potential of AP technology in increasing of aquaculture NUE, reducing at the same time, the environmental impact related to the treatment and disposal of aquaculture wastewater.
Denitrification is an anoxic process, in which NO3− is reduced to NO2− and subsequently to N2(gas) by heterotrophic denitrifying bacteria, directly and/or indirectly affected by several factors as carbon source, nitrate loading rate, oxygen availability, temperature and pH (Li et al., 2007;Vymazal, 2007;Hang et al., 2016). This is also supported by N balance results, which showed that 46.1% and 67.7% of supplied N in the APL and APH systems, respectively, were not recovered at the end of the experiment. Generally, denitrification is a significant pathway in AP systems contributing to N loss from 25% to 60% (Hu et al., 2015;Zou et al., 2016c), to which must be added the losses by ammonia volatilisation which is not negligible when aeration is provided and with a pH above 8.0 (Wongkiew et al., 2017b), as in the conditions of the present study. Considering that in AP systems the N can be converted into resources as long as the N is not transformed into high microbial biomass or nitrogen gas via denitrification (Wongkiew et al., 2017b), our results suggest that initial fish stocking density is a key aspect that have a relevant and prolonged effect on AP system performance. Indeed, as reported in Maucieri et al. (2018b), at the end of the experiment APL treatment reach the same fish density of which observed in APH one at the beginning of the trial but oxygen concentration and NO3availability were not negatively influenced. Plants N uptake is the main pathway of fish residual N recycling in AP and so an efficient system should show high crops yield and low amount of N loss in atmosphere (Wongkiew et al., 2017b). In our study the higher fish stocking density decreased the plants N uptake as a consequence of low NO3availability, which likely occurred because of the low DO and the relatively high pH that also increased the N loss, thus reducing the system efficiency.

Conclusions
N balance showed that initial fish stocking density has a relevant and prolonged effect on AP system production performance with lower NUE and higher N loss through gas emissions with initial fish stocking density of 4.6 kg m -3 respect to 2.5 kg m -3 . Therefore, our findings suggest that in a low-tech AP system a low initial fish density should be adopted at the system start up to maximise production and reduce N losses. Daily water evapotranspiration of proposed AP system gave typical performance of AP systems and was comparable with horticulture typical conditions showing that AP technology have higher water use efficiency than aquaculture and hydroponic section considered alone. Further researches are needed to confirm our results and to evaluate strategies to improve both nitrogen recovery in fish and vegetables and reduce N loss trough gas emissions.

Highlights
-The higher initial fish density had higher N input, but resulted in a lower N recovered in fish and vegetable biomass, and much higher N loss as gas emissions. -The lower initial fish density combined a lower N input to a higher recovery in fish and vegetable biomass, and a lower N loss as gas emissions. -The lower initial fish density allowed to maximise both production and environmental preservation. Table 4. Sediment production, nitrogen concentration and content.