Efficiency of Salicornia neei in removing nitrogen and producing 1 biomass from a hypersaline and artificial wetland to treat 2 aquaculture effluent

11 Background: One of the main challenges for the sustainability of land-based marine 12 aquaculture systems is the treatment of saline effluent saturated with nitrogenous waste. 13 In this study, we evaluated the potential of Salicornia neei, a halophyte plant native to 14 South America, to remove nitrogen and produce biomass in sandy substrate with nitrogen 15 concentrations similar to marine aquaculture effluent. Plants were collected from the 16 natural environment and cultivated under three treatments: 1) seawater fertilized with 17 nitrate + ammonium (Nit+Amm); 2) seawater fertilized with nitrate (Nit); and 3) seawater 18 without fertilizer (Control). 19 Results: The nitrogen removal rate increased from 1.67 to 2.76 mg L d and from 1.95 20 to 2.96 mg L d in the Nit+Amm and Nit treatments, respectively. In the two treatments, 21 nitrogen removal efficiency varied between 87 ± 0.39 and 92 ± 0.40%. The salinity 22 increased from 40 to 52 g L of NaCl during the experiment, with no observed detrimental 23 effects on the nitrogen removal efficiency. At the end of the crop cycle, the biomass 24 production was not significantly different between the treatments of Nit+Amm and Nit 25 (mean Nit+Amm = 3,584 ± 249.3 g; mean Nit 3,004 ± 249.3 g) but was different with 26 respect to the control (mean Control = 1,527 ± 70.0 g). 27 Conclusions: Our results demonstrate that artificial wetlands of S. neei can be used for 28 wastewater treatment in marine aquaculture and for biomass production in South 29 America. 30 31

and relative humidity were sourced from climate records of the Chilean Meteorological First, the means of nitrogen removal and biomass formation were compared using a 1 4 5 one-way ANOVA (RStudio, Ver 3.6.0. probabilities of p<0.05 were considered significant. Additionally, to obtain a clearer view of the change in nitrogen concentration in the 1 4 7 measurements, the Pearson correlation coefficient was used, and the data that showed a 1 4 8 negative linear relationship were subsequently analyzed using the linear model (LM).

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Finally, the residuals were verified, determining their normality and the homogeneity of 1 5 0 the variance (homoscedasticity). The LM analysis provided the removal rate (slope) and 1 5 1 the initial concentration (intercept) for the proposed treatments and the control. During the 74 days of culture, the ambient temperature and relative humidity conditions 1 5 7 and the temperature, pH and salinity of the cultivation system showed different levels of 1 5 8 variability, and no rainfall was recorded during the experiment. The ambient temperature 1 5 9 had a mean of 16 ± 4 °C but was highly variable during the day with extreme values of 9 1 6 0 and 31 °C, while the relative humidity was 77.8 ± 8.7%, with extreme values of 60% and 1 6 1 95% (Fig. 2). The temperature in the culture systems was usually higher than the ambient 1 6 2 temperature, with a mean of 20.5 ± 1.24 °C and a range of 19.1 to 21.7 °C, with no 1 6 3 observed differences between treatments (Table 1). The pH remained relatively constant 1 6 4 and without differences between treatments, while the salinity had a noticeable increase 1 6 5 from a mean of 40 g L -1 of NaCl on day 1 to a mean of 51.5 ± 0.19 g L -1 of NaCl at the end 1 6 6 of the experiment (Table 1). No significant differences in salinity between treatments were 1 6 7 observed (p<0.05). Nitrate removal was high from the start of cultivation and had a clear tendency to increase 1 7 1 as biomass production increased (Fig. 3, Fig. 4). Specifically, at the beginning of the 1 7 2 culture, the nitrogen removal rate was between 1.67 and 1.95 mg L -1 d -1 , and at the end of 1 7 3 the culture, it increased to 2.76 and 2.96 mg L -1 d -1 in the Nit+Amm and Nit treatments, respectively, with no significant differences observed between treatments. Consequently, 1 7 5 the nitrogen removal efficiency was high throughout the crop and varied between 87% 1 7 6 and 92% (Table 2). Regarding biomass production, the treatments with Nit+Amm and Nit showed a significant 1 7 8 increase in fresh weight from 245 ± 35 g to 896 ± 123 g and from 253 ± 7 g to 751 ± 51 g, 1 7 9 respectively, while the control group did not show a significant increase in biomass ( Fig.   1 8 0 4). In this way, RAS cultivation systems reached a yield between 6.6 and 8.3 kg m -2 , with 1 8 1 no observed significant differences between treatments. This study determined that the Salicornia neei substrate interaction is an effective strategy 1 8 6 for the recovery of nitrogen compounds contained in saline effluent typical of marine 1 8 7 aquaculture. As shown in recent research, the integration of halophytes as a biofilter in 1 8 8 recirculating systems in marine aquaculture is an adequate alternative to decontaminating 1 8 9 waters with increased nitrogen compounds. In addition, this plant type offers 1 9 0 characteristics that are favourable in various markets (e.g.: for pharmaceuticals, biofuel  Physicochemical parameters of the effluent, such as temperature and pH, showed affect the determinant processes in the removal of nitrogen compounds. In this study, 1 9 8 temperature and pH were maintained within the optimal ranges (20-21 °C and 7.8-8.2) 1 9 9 and therefore did not affect the nutrient removal processes (Table 1). This finding is 2 0 0 consistent with Lee et al. [35], who reported that, for denitrification processes in wetland 2 0 1 systems, the optimal temperature ranges between 20 and 40 °C and the optimal pH is 2 0 2 approximately 8.0. Another important parameter evaluated in this study was the high 2 0 3 effluent salinity, which reached concentrations of up to 50 g L -1 of NaCl. This increase was 2 0 4 mainly due to the known environmental factor of evapotranspiration (Table 1) [37]. In this study, S. neei was selected to aid in nitrogen removal, mainly due to its natural eliminate of nitrogenous waste from aquaculture. Since, the daily removal rate recorded in 2 1 5 this study was up to 2.9 mg L d -1 (Table 2), values higher than those reported with other 2 1 6 halophyte species in high salinity [14]. Studies in related species have reported that they  that S. neei due to its natural growth in saline soils with scarce nitrification processes, but with the presence of more stable forms of N such as NH4 + also should be have these  Nitrogen bioaccumulation was not determined empirically in this study but can be derived  In response to this uncertainty, other researchers have studied and obtained low removal  [32] observed that of the 73% of nitrogen removed, only 11% had been fixed in plants.

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Notwithstanding the above, Webb et al. [25], observed significant differences between the 2 4 9 nitrogen removal capacity in beds planted with and without halophytes. In their study, they 2 5 0 demonstrated a higher removal yield in planted beds (62.0 ± 34.6 mmol N m −2 d −1 ) than in 2 5 1 unplanted beds (23.0 ± 26.8 mmol N m −2 d −1 ). Therefore, it can be inferred that the strong 2 5 2 root system formed by this class of plants supports the establishment of certain 2 5 3 microorganisms that, acting synergistically, improve the removal rate of nitrogen loads.

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Thus, it is not possible to determine whether plants or microorganisms have the more 2 5 5 important role in the performance of natural removal systems, but they should be 2 5 6 considered elements with significant functions to fulfil. The formation of S. neei biomass during the evaluation period reached a total net weight 2 6 0 of 13.4 kg and 14.9 kg m -2 over a period of six weeks in the treatment irrigated Nit+Amm.  This research was supported by a FIC BIP 30154272 grant from the "Gobierno regional de 3 0 6 Valparaíso" (Chile).    The authors declare that they have no competing interests.  Tables   3  1  7  3  1  8   Table 1 Temperature, pH, and salinity (mean ± SE) recorded at the effluent of the culture systems (lysimeter, n=15) 3 1 9 with Salicornia neei. Salinity is expressed as gram of natrium chloride per liter (g L -1 of NaCl). Each Input 3 2 0 corresponds to the treatments irrigated nitrate and ammonium (Nit + Amm) and nitrate (Nit). Control: irrigated with 3 2 1 sea water only.    (sand and gravel separated by a mesh), and irrigation micro-sprinklers.  Each Input corresponds to the treatments irrigated with nitrate and ammonium (Nit + Amm) and nitrate (Nit). Control

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Irrigated with sea water only. Input corresponds to the treatments irrigated with nitrate and ammonium (Nit + Amm) and nitrate (Nit). Control: treatment 3 4 9 with sea water only. Lower-case letters represents significant differences between treatments. ammonium. b irrigated with sea-water.