Influence of Nitrogen Bioavailability on the Anaerobic Co-Digestion of the Aegagropiles of the Seagrass Posidonia oceanica with Different Nitrogen-Rich Substrates: Process Performance and Kinetic Analysis

Academic Editors: Carmine Lubritto and Maria Rosa Di Cicco Received: 17 January 2025 Revised: 25 February 2025 Accepted: 4 March 2025 Published: 7 March 2025 Citation: de la Lama-Calvente, D.; Mancilla-Leytón, J.M.; GarridoMurillo, I.; Rojas-Carrillo, J.; Borja, R.; Fernández-Rodríguez, M.J. Influence of Nitrogen Bioavailability on the Anaerobic Co-Digestion of the Aegagropiles of the Seagrass Posidonia oceanica with Different Nitrogen-Rich Substrates: Process Performance and Kinetic Analysis. Appl. Sci. 2025,15, 2880. https:// doi.org/10.3390/app15062880 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article Influence of Nitrogen Bioavailability on the Anaerobic Co-Digestion of the Aegagropiles of the Seagrass Posidonia oceanica with Different Nitrogen-Rich Substrates: Process Performance and Kinetic Analysis David de la Lama-Calvente 1, Juan Manuel Mancilla-Leytón 2,* , Iván Garrido-Murillo 3, Javier Rojas-Carrillo 4, Rafael Borja 1and María José Fernández-Rodríguez 2 1Instituto de la Grasa (CSIC), Campus Universidad Pablo de Olavide, Edificio 46. Ctra. de Utrera, km. 1, 41013 Sevilla, Spain; [email protected] (D.d.l.L.-C.); [email protected] (R.B.) 2Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, 41080 Sevilla, Spain; [email protected] 3Estación de Ecología Acuática Alberto I de Mónaco, Empresa Metropolitana de Abastecimiento y Saneamiento de Aguas de Sevilla (EMASESA), 41003 Seville, Spain; [email protected] 4Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padua, 35122 Padua, Italy; javier[email protected] *Correspondence: [email protected] Abstract: The shedding of leaves by Posidonia oceanica (P. oceanica) in autumn results in the accumulation of shoreline debris, contributing to significant economic, social, and environmental problems. Due to the lack of alternative solutions, this waste biomass is disposed of in landfills, incurring an economic cost for the disposal process. In the context of the circular economy, anaerobic digestion (AD) can serve as a highly efficient biological alternative for treating and valorizing wastes with a high organic load. The aim of this research was to comparatively evaluate the performance and kinetics of the AD of ashore P. oceanica biomass and its anaerobic co-digestion (co-AD) with different nitrogen-rich co-substrates. To evaluate the effect of the nitrogen source in the co-AD system, peptone, casein, synthetic casein, urea, and the microalgae Raphidocelis subcapitata were used as co-substrates in biomethanization tests at a mesophilic temperature ( 35 ±2◦C ). The lowest methane yield was achieved for the sole AD of P. oceanica ( 79 ±3 NL CH4kg−1VS ), while the highest yields were found for the three co-ADs of P. oceanica with proteins (i.e., peptone, casein, and synthetic casein), showing no significant differences among them ( 380 ±30 −420 ±30 NL CH4kg−1VS ). Additionally, the first-order kinetics and the transference function model were proven and allowed for adequately fitting the experimental results of methane production with time. Keywords: marine angiosperm; biomethanization tests; methane; Raphidocelis subcapitata 1. Introduction Around 100 million years ago, terrestrial monocotyledonous plants, already reproducing through a floral system, began to colonize marine ecosystems, forming complex and well-structured habitats. Among these ecosystems, seagrasses have become one of the most productive and valuable areas on our planet, providing a wide range of essential services: vital habitat for thousands of fish and invertebrate species, food, nutrient cycling, carbon sink, etc. [1]. Posidonia oceanica (P. oceanica) is a marine angiosperm species that is endemic to the Mediterranean Sea. It forms extensive seagrass in shallow water, covering about 1.5% of Appl. Sci. 2025,15, 2880 https://doi.org/10.3390/app15062880 Appl. Sci. 2025,15, 2880 2 of 18 the Mediterranean basin and playing an important role in coastal ecosystem dynamics [ 2 , 3 ]. These meadows provide oxygen, food, shelter, and protection for many marine species. Additionally, these extensive meadows form a natural barrier that reduces wave energy, protecting against erosion and stabilizing the coastal profile [ 4 ]. Currently, P. oceanica beds are in danger of disappearance and have been listed as priority habitats by the European authorities [5]. P. oceanica presents a stem, long leaves grouped in bunches, and a root system that enables the individual specimens to cling to the substrate. Its leaves are removed from the rhizome in autumn after losing their photosynthetic capacity. These loosed leaves are released in large quantities along the shore and once they reach the sand, they start to decompose, causing serious environmental problems (methane release to the atmosphere, proliferation of mosquitoes, problems with navigation and fishing, etc.) [ 6 , 7 ]. Fresh deposits of P. oceanica debris usually appear at the coast during autumn and winter due to the weather and sea conditions, making this marine plant a renewable resource. Some leaves keep their fibrous structure for long periods after the easily biodegradable components are completely decomposed. These fibers are highly resistant to the mechanical and chemical action of the seawater. They are rolled up by the wave movement around a rhizomic nucleus, as a starting point for the formation of spherical agglomerates, from 3 to 12 cm in diameter (Figure 1). These agglomerates, called aegagropiles, are eventually left ashore where they remain and are sun-dried [8–10]. Appl. Sci. 2025, 15, x FOR PEER REVIEW 2 of 18 services: vital habitat for thousands of fish and invertebrate species, food, nutrient cycling, carbon sink, etc. [1]. Posidonia oceanica (P. oceanica) is a marine angiosperm species that is endemic to the Mediterranean Sea. It forms extensive seagrass in shallow water, covering about 1.5% of the Mediterranean basin and playing an important role in coastal ecosystem dynamics [2,3]. These meadows provide oxygen, food, shelter, and protection for many marine species. Additionally, these extensive meadows form a natural barrier that reduces wave energy, protecting against erosion and stabilizing the coastal profile [4]. Currently, P. oceanica beds are in danger of disappearance and have been listed as priority habitats by the European authorities [5]. P. oceanica presents a stem, long leaves grouped in bunches, and a root system that enables the individual specimens to cling to the substrate. Its leaves are removed from the rhizome in autumn after losing their photosynthetic capacity. These loosed leaves are released in large quantities along the shore and once they reach the sand, they start to decompose, causing serious environmental problems (methane release to the atmosphere, proliferation of mosquitoes, problems with navigation and fishing, etc.) [6,7]. Fresh deposits of P. oceanica debris usually appear at the coast during autumn and winter due to the weather and sea conditions, making this marine plant a renewable resource. Some leaves keep their fibrous structure for long periods after the easily biodegradable components are completely decomposed. These fibers are highly resistant to the mechanical and chemical action of the seawater. They are rolled up by the wave movement around a rhizomic nucleus, as a starting point for the formation of spherical agglomerates, from 3 to 12 cm in diameter (Figure 1). These agglomerates, called aegagropiles, are eventually left ashore where they remain and are sun-dried [8–10]. Although in protected natural areas, the collection of P. oceanica and its debris is forbidden due to its benefits on the conservation of the local habitat (Decree 132/2005, 2005), in certain locations, exceptionally, the responsible authorities allow and encourage the collection of this biomass. For instance, on beaches of Formentera (Spain), this waste is widely used as bedding for animals, crops, and as insulation in traditional houses. In Italy, the use as a co-substrate in composting is also allowed, provided that it does not exceed 20% of the final mixture in terms of wet matter [11]. The removal of tons of P. oceanica remains is a common practice in Mediterranean coasts to allow for recreational use in the summer months. Due to the lack of alternative solutions, this biomass is usually disposed of in landfills, which provokes an environmental problem as a significant amount of organic matter is removed from the ecosystem. Moreover, it carries an economic cost due to the disposal process [4,12]. Figure 1. Detail of the formation of the Posidonia oceanica aegagropiles. Figure 1. Detail of the formation of the Posidonia oceanica aegagropiles. Although in protected natural areas, the collection of P. oceanica and its debris is forbidden due to its benefits on the conservation of the local habitat (Decree 132/2005, 2005), in certain locations, exceptionally, the responsible authorities allow and encourage the collection of this biomass. For instance, on beaches of Formentera (Spain), this waste is widely used as bedding for animals, crops, and as insulation in traditional houses. In Italy, the use as a co-substrate in composting is also allowed, provided that it does not exceed 20% of the final mixture in terms of wet matter [ 11 ]. The removal of tons of P. oceanica remains is a common practice in Mediterranean coasts to allow for recreational use in the summer months. Due to the lack of alternative solutions, this biomass is usually disposed of in landfills, which provokes an environmental problem as a significant amount of organic Appl. Sci. 2025,15, 2880 3 of 18 matter is removed from the ecosystem. Moreover, it carries an economic cost due to the disposal process [4,12]. In the context of the circular economy, anaerobic digestion (AD) can be a highly efficient biological alternative for the treatment and valorization of wastes with a high organic load [ 13 ]. The interest in AD facilities has grown over the past few years because of the high methane content (around 50–60%) of the generated biogas, making it a renewable energy alternative to fossil fuels. Furthermore, the digestate can be used as an organic soil amender [ 13 , 14 ]. However, the viability of these processes is highly dependent on the type of substrate used. Therefore, before their implementation on an industrial or pilot scale, it is necessary to assess the methane potential of the biomass through laboratory-scale assays, such as the Biochemical Methane Potential (BMP) test, which allows the identification of substrate biodegradability, activity of the inoculum, and methane yield, providing some insights about the potential toxicity of the substrate to the AD process due to the presence of certain metabolites [14]. The use of P. oceanica for methane production by AD has been scarcely investigated in the literature. In an earlier study, the methane yield obtained was rather low ( 62 LCH4 kg−1 VS Pos ), most likely, due to the high lignin content (>30% VS) and the presence of minerals and salts [ 4 ]. Accordingly, the effect of thermal pre-treatment (132 ◦ C; 2.3 bar; 30 min) coupled with the removal of salts and the use of acid (HCl) during hydrolysis was also assessed by De Sanctis and Di Iaconi [ 4 ]. However, despite the applied pre-treatments, the methane production did not improve and even the yield was sometimes reduced by up to 73%. Another alternative could be the use of other substrates for an anaerobic co-digestion (co-AD) process. The co-AD process has important advantages, such as: (i) avoiding the use of chemicals or extra energy; (ii) the mixed feedstock could be prepared to present an optimal C/N ratio (25–30); (iii) mixing two co-substrates can dilute the presence of toxicants for the anaerobic process, such as salts or phenols; and (iv) it could enhance the valorization of other biomasses otherwise wasted [ 15 , 16 ]. Lignocellulosic biomasses, such as P. oceanica, are usually subjected to co-AD processes due to the lack of the necessary amount of nitrogen and, thus, the balanced C/N ratio to allow a stable performance. However, the bioavailability of the nitrogen source provided may influence the final methane production, although no sound conclusions can be drawn due to the significant lack of specific studies [17]. This study aimed to assess the influence of different nitrogen-rich substrates, which represent different nitrogen bioavailabilities, as co-substrates of the co-AD process of aegagropiles (ashore biomass) of the marine phanerogam P. oceanica, through BMP tests. This study represents a breakthrough in research into the valorization of P. oceanica waste, as it is the first time this biomass has been proposed to be used as a co-substrate in a co-AD process. In addition, it aims to further investigate the importance of the type of nitrogen source in anaerobic co-digestion systems. Another objective of this study was to assess the kinetics of anaerobic digestion of single P. oceanica biomass and mixtures of this biomass with different co-substrates rich in nitrogen, such as peptone, casein, synthetic casein (composed of a mixture of amino acids), urea, and the microalga Raphidocelis subcapitata. 2. Materials and Methods 2.1. Collection and Main Characterization of the Material Dried P. oceanica aegagropiles were collected during the winter season along a 150 m transect at a distance of 5–7 m from the coastline at ‘Playa de las Huertas’, Alicante (Spain). The samples (n= 72) were transported to the laboratory, where they were dried at room temperature (25 ◦ C) and low humidity for several days until a constant weight was reached. Subsequently, all samples were weighed and their volume estimated, assuming that all Appl. Sci. 2025,15, 2880 4 of 18 samples had an ellipsoidal shape. After biometric analyses, the samples were ground to obtain a homogeneous substrate with a particle size of up to 3.5 mm. The following methods were used to determine the specific physicochemical parameters of the P. oceanica biomass: APHA [ 18 ] standard methods (2540B and 2540E) were used to determine total solids (TS), volatile solids (VS), and mineral solids (MS). Raposo et al. [ 19 ] and APHA [ 18 ] standard method (5220D) were used to determine the total chemical oxygen demand (CODt) and soluble chemical oxygen demand (CODs), respectively. pH and total alkalinity (TA) were measured using a pH meter (model Crison 20 basic). The APHA [18] standard method (2320B) with titration to pH 4.3 was used to determine the TA. A LECO TruSpec ® Micro Elemental Analyzer from the Leco Corporation (St. Joseph, MI, USA) was used for elemental analysis. Metal elements were determined by following the U.S. EPA 3051A method [ 20 ]; briefly, samples were digested with a Mars Xtraction microwave (CEM, Matthews, NC, USA) and analyzed by inductively coupled plasma mass spectrometry (ICP-MS, AGILENT 7800, Madrid, Spain). 2.2. Biomethanization Tests BMP tests were performed in 250 mL reactors as described by De la Lama-Calvente et al. [ 21 ]. A mixture of anaerobic inocula from two industrial anaerobic digesters, one from a brewery (Heineken, Seville) and the other from a wastewater treatment plant ( EMASESA—EDAR El Copero, Seville, Spain), was used for BMP testing. Based on the volatile solids fraction of each inoculum, the mixing ratio was 1:1. The final inoculum mixture had values of 29.9 ± 0.1 (g kg −1 ) TS, 18.7 ± 0.5 (g kg −1 ) VS and 11.2 ±0.5 (g kg−1 ) MS. To achieve an appropriate C/N ratio (25) the following nitrogen-rich co-substrates were used (Table S1): (i) synthetic urea (Panreac); (ii) casein from bovine milk (SigmaAldrich); (iii) gelatin peptone of porcine origin (Cultimed); (iv) a mixture of amino acids prepared in the laboratory similar to commercial casein [ 22 ]; and (v) microalga Raphidocelis subcapitata , grown in Arnon-rich medium. The used acronyms along the text to identify the different substrates were: P.o.: P. oceanica; P.o.:Ur.: P. oceanica—Urea; P.o.:Cas.: P. oceanica —Casein; P.o.:Pep.: P. oceanica—Peptone; P.o.:Syn.Cas.: P. oceanica—Synthetic casein made of amino acids; P.o.:R.s.: P. oceanica—Raphidocelis subcapitata. The main physicochemical characteristics of the different substrates used are shown in Table 1. All the reactors were set up with an inoculum-to-substrate ratio (ISR) of 2 (based on VS), distilled water, and a micro-nutrient solution. The composition of the solution of trace elements was as follows: (in mg L −1 ) FeCl 2· 4H 2 O, 2000; CoCl 2· 6H 2 O, 2000; MnCl 2· 4H 2 O, 500; AlCl 3· 6H 2 O, 90; (NH 4 )6Mo 7 O 24· 4H 2 O, 50; H 3 BO 3 , 50; ZnCl 2 , 50; CuCl 2· 2H 2 O, 38; NiCl 2· 6H 2 O, 50; Na 2 SeO 3· 5H 2 O, 194; and EDTA, 1000. All assays were carried out in triplicate and were performed at a mesophilic temperature (35 ± 1 ◦ C). In addition, 3 reactors without added substrate were included as blanks to determine the endogenous methane production of the inoculum. Additionally, 3 reactors were fed with cellulose (Fluka Avicel PH101) and treated as the positive control to confirm the good activity of the inoculum. Before sealing, nitrogen gas was applied to all the reactors to ensure anaerobic conditions. The biogas produced in the reactors was passed through a NaOH 2N solution capable of trapping the CO 2 from the biogas mixture. The remained gas was measured volumetrically and assumed to be methane, as supported by several publications [ 23 , 24 ]. The experimental accumulative methane values are given in standard conditions of pressure and temperature (101.325 kPa and 273.15 K). Finally, the BMP tests were stopped when methane production was less than 1% of the accumulative value for 3 consecutive days. Appl. Sci. 2025,15, 2880 5 of 18 Table 1. Main physicochemical characteristics of the different studied co-substrates. Values are given by means ±standard deviations. Parameters Posidonia Oceanica Urea Casein Peptone Synthetic Casein Raphidocelis subcapitata TS (g kg−1)880 ±10 930 ±30 917 ±9 936 ±2 993.1 ±0.4 58 ±9 VS (g kg−1)780 ±10 921 ±60 899 ±3 878 ±3 993 ±1 55 ±9 MS (g kg−1)100 ±20 1.3 ±0.1 18 ±6 58 ±5 0.2 ±0.2 3.3 ±0.3 VS/TS 0.88 0.99 0.98 0.94 1.00 0.94 C (%) 42.9 ±0.2 20 * 47.8 ±0.5 41 ±3 48 ±1 51.3 ±0.7 N (%) 0.28 ±0.04 46.6 * 12.87 ±0.07 14 ±1 11.5 ±0.2 4.36 ±0.03 C/N 153 ±5 0.43 * 3.71 ±0.06 2.96 ±0.01 4.1 ±0.2 11.78 ±0.09 * Calculated stoichiometrically from the chemical formula. TS: Total solids; VS: Volatile solids; MS: Mineral solids. 2.3. Kinetic Study The first-order kinetic model and the transference function model (TFM) were applied to perform the kinetic study of methane production from the different co-digestion mixtures. Further information regarding the used equations can be found in the Appendix A. 2.4. Statistical Analysis Analysis of variance (ANOVA) and Tukey’s HSD test were used to compare the results of the different treatments and to determine whether there were significant differences. Before this, a normality test (Shapiro–Wilk test) was performed. A p-value ≤ 0.05 was considered statistically significant. For the calculation of biodegradability, the theoretical BMP test values were calculated using the formula described by Nielfa et al. [ 25 ] (Table S1). All the statistical analyses were carried out by using the SPSS Statistics software v.29 (IBM, Business Machines Corp, New York, NY, USA). 3. Results and Discussion 3.1. Posidonia Oceanica Characterization The collected samples showed a normal distribution in terms of biovolume (Table 2), ranging from 10 to 850 cm 3 , with an average value of 300 ± 200 cm 3 and a mode of 50 cm 3 . The aegagropiles were milled and the particle size distribution was measured. The results showed that due to the morphology of the material, all the fibers passed through the 3.5 mm mesh sieve. As suggested by the specific literature [ 26 ], the fraction related to a particle size between 1 mm and 3.5 mm was selected for further analysis and the BMP tests. The results showed a VS/TS ratio above 0.8, which is considered a suitable value for AD and is within the same range as other lignocellulosic biomasses [ 27 ]. Although the samples were kept at room temperature until a constant weight, the main humidity loss occurred in the collected site. Thus, it could be considered that the aegagropiles were sun-dried before further analysis and use. This low humidity (12%) was similar to other biomasses dried naturally. For example, different varieties of Sorghum bicolor presented humidity values within the range of 7–9% when sun-dried [ 27 ]. Similarly, the sun-dried biomass of the ashore macroalgae Rugulopteryx okamurae showed humidity values between 28 and 7% [28]. Appl. Sci. 2025,15, 2880 6 of 18 Table 2. Biometric and physicochemical characterization of the aegagropiles of Posidonia oceanica. Values are given by means ±standard deviations. Parameters Posidonia oceanica Physical Bio-Parameters Average weight (g) 9±8 Average width (cm) 4±2 Average length (cm) 5±2 Average height (cm) 3±1 Biovolume (cm3)300 ±200 Q1 (cm3)61 Q3 (cm3)450 Range (cm3)10–850 Mode (cm3)50 Elemental Analysis C (g kg−1)429 ±2 N (g kg−1)2.8 ±0.4 B (g kg−1)3.0 ±0.2 Na (g kg−1)17.9 ±0.8 Mg (g kg−1)4.3 ±0.2 Al (g kg−1)0.63 ±0.03 K (g kg−1)1.0 ±0.1 Ca (g kg−1)8±1 p(ppm) <LOD As (ppm) 10.4 ±0.4 Ti (ppm) 12 ±1 Cr (ppm) 3±1 Cu (ppm) 3±2 Co (ppm) <LOD Mn (ppm) <LOD Ni (ppm) 0.5 ±0.1 Fe (ppm) 1.6 ±0.1 Hg (ppm) 2±1 Pb (ppm) 3±2 Mo (ppb) 310 ±70 Zn (ppb) <LOD Sn (ppb) <LOD Sb (ppb) <LOD Q1: 1st quartile of biovolume normal distribution; Q3: 3rd quartile of biovolume normal distribution; LOD: Limit of detection. Regarding the elemental analysis, the C/N ratio (153 ± 5) indicated a low N content (Table 1), which may unbalance AD performance by affecting the bacterial growth and subsequently their hydrolytic activity [ 29 ]. This result supported the idea of carrying out a co-AD process using N-rich co-substrates. Seagrasses are also characterized by their potential to absorb heavy metals present in the seawater and act as bioindicators [ 30 ]. However, the heavy metal uptake capacity is strongly linked to the species and the location [ 30 , 31 ]. Regarding P. oceanica, strong relationships between the location, the plant part, the collection season, and the heavy metal composition have been previously reported [ 31 , 32 ]. The results obtained in this study are within the range of those previously reported, with very few exceptions [ 31 – 33 ]. For example, Zn is commonly detected in the 100–1000 ppm range, while in the sample used in the present study this element could not be detected. Similarly, Fe and Pb have been reported in the 100–3000 ppm and 10–100 ppm ranges, respectively, however, in the present study, Fe was found at a concentration of 1.6 ± 0.1 ppm and Pb at 3±2 ppm [31–33] . While the data reported in the literature is related to healthy and alive Appl. Sci. 2025,15, 2880 7 of 18 specimens of P. oceanica, in the present study, the P. oceanica samples were collected ashore, sun-dried, and after a natural decomposition process. These differences in the biomass origin could significantly alter the elemental profile as it has been recently reported [ 28 ]. Furthermore, heavy metals are strongly linked to a good performance of the AD process. While it is well known that the presence of trace elements is essential for efficient AD performance, at certain concentrations, these may disrupt the process by inhibiting the microbiome activity [ 34 ]. The most studied and common elements are Cu, Fe, Ni, Cd, Cr, Pb, and Zn. Although there is a significant amount of data supporting their impact on AD, there is still a lack of consensus regarding specific limits to identify the potential toxicity of the substrates. Nevertheless, the values reported in Table 2were not considered of major concern when compared with those reported in the literature [34–37]. 3.2. Co-Substrate Characterization The co-substrates used in the present study were commercial products of chemical degree, except for the microalga R. subcapitata, which was cultivated in an Arnon-rich medium. The solid fraction of these compounds represented more than 90% of the sample, while for the microalgae, the humidity represents 42% of the total weight (Table 1). In any case, the VS/TS ratio was above 0.94 across the board, suggesting that the organic fraction of each co-substrate could be considered quantitatively equal. Moreover, the elemental analysis revealed that the C/N ratio ranged from 11.78 (R. subcapitata) to 0.43 (urea), confirming the suitability of these co-substrates as feedstock for the co-AD with lignocellulosic biomasses, such as P. oceanica aegagropiles. 3.3. Digestate Main Characteristics The main physicochemical characteristics of the digestates resulting from the BMP tests carried out in this study are shown in Table 3. The pH ranged from 7.77 to 8.22 across the board, except for the P.o.:Ur. test which showed a higher value (9.2). pH values between 6.5 and 8.5 suggest a stable AD performance, although at pH under 6.3 or over 7.8 the methanogenesis stage may suffer failures and be partially hindered [ 38 ]. A higher pH may be related to the formation of ammonia during the hydrolysis stage, which is inhibitory to methanogens. Aqueous ammonia (NH 4+ ) is in equilibrium with free ammonia (NH 3 ); however, this equilibrium shifts to free ammonia formation as the pH increases. Concentrations of aqueous ammonia between 1.7 and 14 g NH 3 L −1 have been reported as inhibitory to methanogens, especially to acetoclastic species [ 38 ]. However, the total ammonium nitrogen (TAN) limits, from which inhibitory effects are observed, are highly related to other factors, such as substrate, inoculum, environmental conditions, and microbial acclimation [ 39 ]. In any case, it is generally considered that TAN values below 2500 mg N-NH 4+ L −1 do not indicate significant inhibition [ 26 ]. Another parameter directly linked to the stability of the AD process is the buffer capacity of the system, measured as the TA. It is generally accepted that TA, as a measure of carbonate buffer capacity, should be above 3000 mg CaCO 3 kg −1 [ 26 ]. However, there is very little information about a TA upper limit, which may indicate an inhibition process. In fact, in this study, the TA value of the digestate derived from the inhibited P.o.:Ur. test was as high as 15,100 mg CaCO 3 kg −1 . Appl. Sci. 2025,15, 2880 8 of 18 Table 3. Physicochemical parameters of the digestates derived from the biomethanization tests. Values are given as means ± standard deviations. Values with different letters in the same row mean significant differences. P.o. P.o.:Ur. P.o.:Cas. P.o.:Pep. P.o.:Syn.Cas. P.o.:R.s. TS (g kg−1)15 ±5a.b 16 ±2b.c 19 ±1b.c 12 ±5a19.0 ±0.6 c23.5 ±0.6 d VS (g kg−1)8±3a.b 11 ±1b.c 12 ±1c8±3a12.4 ±0.8 c15.6 ±0.3 d MS (g kg−1)7±2a.b 5±1a.c 6.5 ±0.6 a.b 4±2c6.6 ±0.4 a.b 7.9 ±0.6 b pH 7.82 ±0.07 a9.2 ±0.2 b8.2 ±0.1 c8.22 ±0.03 c8.16 ±0.05 c7.77 ±0.08 a TA (mg CaCO3kg−1)5590 ±70 a15,100 ±200 b7800 ±200 d8960 ±90 c6850 ±30 e4500 ±500 f CODs (mg O2kg−1)2100 ±500 a.d 7700 ±500 b1600 ±500 a.d.e 3100 ±300 c1400 ±400 e1500 ±200 a.d.e TAN (mg N-NH4+kg−1)990 ±40 a4000 ±200 b1830 ±50 c2030 ±60 c1600 ±200 d970 ±30 a BMPth (NL CH4kg−1VS) 526.2 83.0 455.8 399.0 498.1 537.9 BMPexp (NL CH4kg−1VS) 79 ±3a0b420 ±30 c389 ±7c380 ±30 c194 ±4d BD (%) 15 0 93 97 75 36 P.o.:Posidonia oceanica;Ur.: Urea; Cas: Casein; Pep.: Peptone; Syn.Cas.: Synthetic casein made of amino acids; R.s.:Raphidocelis subcapitata.TS: Total solids; VS: Volatile solids; MS: Mineral Solids; TA: Total alkalinity; CODs: Soluble chemical oxygen demand; TAN: Total Ammonium Nitrogen; BMP th : Theoretical Biochemical Methane Potential; BMPexp: Experimental Biochemical Methane Potential; BD: Biodegradability. Based on the physicochemical characteristics, the assayed digestates could be roughly separated into three main groups: (i) P.o. and P.o.:R.s.; (ii) P.o.:Cas., P.o.:Pep., and P.o.:Syn.Cas.; (iii) P.o.:Ur. The first group is characterized by showing the lowest pH, TA, and TAN values, although within acceptable values for the adequate performance of the AD process. The experimental methane yield was significantly lower than those achieved from the second group, despite the higher theoretical value (Table S2). This could be related to the lignocellulosic character of sun-dried P. oceanica. Moreover, the relatively low COD value suggested that the main limiting step was the hydrolytic stage, although the differences between both P.o. and P.o.:R.s. tests might also indicate that the methanogenesis was more limited in the P.o. test, based on the higher COD value and the lower methane yield. These differences might be related to the more balanced C/N ratio of the P.o.:R.s. test, which allowed for more consistent and stable bacterial and archaea growth. Similar results have been observed recently when other lignocellulosic biomasses, such as thermo-chemical pretreated rice husk, were co-digested with cow manure at different C/N ratios (13.8–75.7) [ 40 ]. The results showed that the highest methane yield was achieved at a C/N ratio of 24.7, meaning a yield improvement of 76% against the control [ 40 ]. In the present study, an improvement of 146% in methane yield of P.o.:R.s. compared to the mono-digestion of the sole P. oceanica was observed (Figure 2). The evaluation of P. oceanica as feedstock for an AD process is scarce in the literature. Only two studies reported specific methane yields using P. oceanica as a substrate. De Sanctis and Di Iaconi [ 4 ] reported values from 29 to 62 L CH 4 kg −1 VS, depending on the operating conditions. These results were slightly lower than that obtained in this study from the sole digestion of P. oceanica ( 79 ±3 NL CH4kg−1VS ). Additionally, De Sanctis and Di Iaconi [ 4 ] also evaluated the effect of some pretreatments, such as washing, thermal, or acidification with HCl, on methane production. The highest value (94 L CH 4 kg −1 VS) was observed for the unwashed biomass thermally pretreated at 132 ◦ C for 30 min after sample acidification with HCl. Again, in the present study, the co-AD of P. oceanica with a N-rich co-substrate, such as the microalga R. subcapitata, led to higher values (194 ±4 NL CH4kg−1VS, Figure 2). Appl. Sci. 2025,15, 2880 9 of 18 Appl. Sci. 2025, 15, x FOR PEER REVIEW 9 of 18 Figure 2. Experimental and accumulative methane of the different biomethanization (BMP) tests carried out versus time. P.o.: P. oceanica; Ur: urea; Cas.: casein; Pep.: gelatine peptone; Syn.Cas.: amino acids mixture simulating a synthetic casein; R.s.: Raphidocelis subcapitata. Values are given as means ± standard deviations. The second group was characterized by the highest methane yield (380 ± 30−420 ± 30 NL CH 4 kg −1 VS), with no significant differences between the three results. Moreover, biodegradability above 75% may indicate an almost complete degradation of the substrate [41]. COD values similar to those found in the first group (Table 3) also indicate that there was not an accumulation of solubilized organic matter, suggesting that both the hydrolytic and the methanogenetic stages operated properly. Only the P.o.:Pep. test showed a significantly higher COD value; however, it also showed the highest BD (97%). Peptone is a partially degraded protein, which makes it much more easily digestible and bioavailable for microorganisms, providing a quick source of nitrogen. This could suggest that the substrate was completely solubilized, unlike the other two tests where some parts of the substrates may have remained intact or not biodegraded. Due to its complex structure, casein is less soluble in water and takes longer to be digested by microorganisms. In biological systems, microorganisms need to break down casein into simpler components before they can metabolize it. The co-digestion of complex substrates, such as casein proteins and lignocellulose can create a more stable environment for anaerobic microorganisms, thereby optimizing biogas production. The hydrolysis of proteins, particularly complex ones, such as casein, is likely to be synchronized with the hydrolysis of complex lignocellulose, allowing for a more balanced release of nitrogen and carbon compounds. This synchronized release can maintain an optimal soluble C/N ratio, which is crucial for the proper development of anaerobic microorganisms. The parallel hydrolysis of nitrogenous and carbonaceous compounds promotes a more efficient digestion process, supporting the microbial community’s stability and increasing methane production. This is in line with the principles established in anaerobic digestion, where proper synchronization of substrate degradation is key to maximizing biogas yields. In any case and despite the apparent good performance, pH, TA, and TAN analyses of the digestates indicated that the systems were close to failure due to inhibition processes. In fact, when compared with the first group, the TAN values were around 2-fold higher, the pH was higher at least by 0.5 units, and the TA also increased up to 9000 mg CaCO 3 kg −1 (Table 3). Although these values were still Figure 2. Experimental and accumulative methane of the different biomethanization (BMP) tests carried out versus time. P.o.: P. oceanica; Ur: urea; Cas.: casein; Pep.: gelatine peptone; Syn.Cas.: amino acids mixture simulating a synthetic casein; R.s.: Raphidocelis subcapitata. Values are given as means ±standard deviations. Thesecondgroupwascharacterizedbythehighestmethaneyield( 380 ±30−420 ±30 NL CH 4 kg −1 VS), with no significant differences between the three results. Moreover, biodegradability above 75% may indicate an almost complete degradation of the substrate [ 41 ]. COD values similar to those found in the first group (Table 3) also indicate that there was not an accumulation of solubilized organic matter, suggesting that both the hydrolytic and the methanogenetic stages operated properly. Only the P.o.:Pep. test showed a significantly higher COD value; however, it also showed the highest BD (97%). Peptone is a partially degraded protein, which makes it much more easily digestible and bioavailable for microorganisms, providing a quick source of nitrogen. This could suggest that the substrate was completely solubilized, unlike the other two tests where some parts of the substrates may have remained intact or not biodegraded. Due to its complex structure, casein is less soluble in water and takes longer to be digested by microorganisms. In biological systems, microorganisms need to break down casein into simpler components before they can metabolize it. The co-digestion of complex substrates, such as casein proteins and lignocellulose can create a more stable environment for anaerobic microorganisms, thereby optimizing biogas production. The hydrolysis of proteins, particularly complex ones, such as casein, is likely to be synchronized with the hydrolysis of complex lignocellulose, allowing for a more balanced release of nitrogen and carbon compounds. This synchronized release can maintain an optimal soluble C/N ratio, which is crucial for the proper development of anaerobic microorganisms. The parallel hydrolysis of nitrogenous and carbonaceous compounds promotes a more efficient digestion process, supporting the microbial community’s stability and increasing methane production. This is in line with the principles established in anaerobic digestion, where proper synchronization of substrate degradation is key to maximizing biogas yields. In any case and despite the apparent good performance, pH, TA, and TAN analyses of the digestates indicated that the systems were close to failure due to inhibition processes. In fact, when compared with the first group, the TAN values were around 2-fold higher, the pH was higher at least by 0.5 units, and the TA also increased up to 9000 mg CaCO 3 kg −1 (Table 3). Although these values were still within the normal limits for good AD performance, they were closer to the upper limits which indicates inhibition due to the presence of ammonia. This may Appl. Sci. 2025,15, 2880 16 of 18 References 1. Murphy, G.E.P.; Dunic, J.C.; Adamczyk, E.M.; Bittick, S.J.; Côté, I.M.; Cristiani, J.; Wong, M.C. From coast to coast to coast: Ecology and management of seagrass ecosystems across Canada. Facets 2021,6, 139–179. [CrossRef] 2. 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