## 4 Conclusions  
The impact of the nitrogen source during the anaerobic co-digestion (co-AD) of the ashore marine seagrass _Posidonia oceanica_ (_P. oceanica_) and N-rich substrates has been assessed in this study. There were no significant differences observed in the methane yields (380 \(\pm\) 30\(-\)420 \(\pm\) 30 NL CH\({}_{4}\) kg\({}^{-1}\) VS) derived from the co-AD of _P. oceanica_ and protein-like substrates (i.e., casein, peptone and synthetic casein), being these the highest yields achieved. However, the physicochemical characteristics of the resulting digestates suggested that long-term and continuous anaerobic processes may fail due to ammonia accumulation.  
The use of urea as a nitrogen source to reduce the C/N ratio of _P. oceanica_ from 153 \(\pm\) 5 to 25 has been proven to be inefficient and inhibitory to the co-AD process. TAN values of 4000 \(\pm\) 200 mg N-NH\({}^{4+}\) kg\({}^{-1}\) were obtained in the digestate and showed complete inhibition of the microbial community.  
The co-AD of _P. oceanica_ with _R. subcapitata_ enhanced the methane yield by 145% when compared with the sole AD of _P. oceanica_. Although the methane yield was lower than that obtained from the protein-like co-substrates (194 \(\pm\) 4 NL CH\({}_{4}\) kg\({}^{-1}\) VS), the resulting digestates showed higher stability, suggesting that nitrogen derived from complex organisms, such as microalgae, would be a better option in scaled-up co-AD systems.  
Data from AD experiments were well described by the first-order and transference function model (TFM). The highest specific rate constant (first-order model) obtained in the present research was found for the mixture _P. oceanica_--peptone (0.46 \(\pm\) 0.01 days\({}^{-1}\)). This value was 130% higher than that obtained for the sole _P. oceanica_ biomass. In addition, the highest maximum methane production rate (\(R_{max}\)) (TFM) was also found for the above-mentioned mixture, which was 10.9 times higher than the lowest value, which was found for the single _P. oceanica_ biomass tested (16.8 \(\pm\) 0.9 mL CH\({}_{4}\) (g VS-d)\({}^{-1}\)).  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app15062880/s1](https://www.mdpi.com/article/10.3390/app15062880/s1)]([https://www.mdpi.com/article/10.3390/app15062880/s1](https://www.mdpi.com/article/10.3390/app15062880/s1)), Table S1. C/N ratio, grams of volatile solids (VS) added in the BMP and Theoretical BMP value of each co-substrate; Table S2. Theoretical and experimental BMP of each co-substrate and calculated synergistic effect.  
D.d.l.L.-C.: Conceptualization, Investigation, Validation, Supervision, Writing--original draft, Writing--review & editing; J.M.M.-L.: Conceptualization, Validation, Supervision, Writing--original draft, Writing--review & editing; I.G.-M.: Investigation, Writing--originaldraft; J.R.-C.: Investigation, Writing--original draft; R.B.: Supervision, Resources, Funding acquisition, Writing--review & editing; M.J.F.-R.: Conceptualization, Investigation, Validation, Supervision, Writing--original draft, Writing--review & editing. All authors have read and agreed to the published version of the manuscript.  
The authors wish to express their gratitude to the Spanish Ministry of Science, Innovation and Universities for providing financial support through the project PID2023-151811 OB-I00. This research was co-funded by Spanish State Research Agency, grant number FJC2020-045654-I.  
Not applicable.  
The data presented in this study are available in the article.  
The authors also wish to thank [PERSON] and [PERSON] for their technical support and dedication.  
Author [PERSON] was employed by the company Empresa Metropolitana de Abastecimiento y Saneamiento de Aguas de Sevilla (EMASESA). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.