Use of anaerobic digestate as biofertilizer: Another step forward in the valorisation of the invasive brown macroalgae Rugulopteryx okamurae

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in: Scientia Horticulturae on February 2024, available at: https://doi.org/10.1016/j.scienta.2023.112638”. Use of anaerobic digestate as biofertilizer: Another step forward in the valorisation 1 of the invasive brown macroalgae Rugulopteryx okamurae 2 David De la Lama-Calventea, Juan Manuel Mancilla-Leytónb 1 , Rafael Borjaa, María José 3 Fernández-Rodríguezb 4 a Spanish Scientific Research Council (CSIC) - Instituto de la Grasa (IG), Department of 5 Food Biotechnology, Campus Universidad Pablo de Olavide, Edificio 46. Ctra. de Utrera, 6 km 1, 41013 Seville, Spain. 7 b Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de 8 Sevilla, 41080 Seville, Spain. 9 Abstract 10 The rapid and aggressive colonization of the invasive macroalgae Rugulopteryx 11 okamurae on the southern coast of Spain has caused an unprecedented environmental, 12 health and economic impact. At present, the scientific community has focused on the 13 valorisation of R. okamurae waste, as tonnes of collected biomass from this species end 14 up in landfills. In this sense, the aim of this study was to assess, for the first time, the 15 viability of the R. okamurae digestate as biofertilizer to growth the Mediterranean forage 16 species Avena strigosa (black oat). Two anaerobic digestates, derived from the anaerobic 17 digestion of the thermally pre-treated (120 ºC; 45 min) and the zeolite-assisted 18 mechanically pre-treated invasive macroalgae, were used in this study. Both digestates 19 were compared with a commercial inorganic nutritive solution. The effect of different 20 fertilizers and water availability on forage height, aerial, root and seed biomass, as well 21 as photosynthetic activity and macro- and micronutrient forage content were studied. The 22 control treatment showed the lowest height at the end of the experiment (0.38 m) and the 23 1 Corresponding author: [email protected] digestate from the thermally pre-treated macroalgae treatment had the highest one (0.59 24 m). Final fresh and dry aerial biomass was separately significantly dependent on both 25 fertilizer type and water stress. Final root and seed biomass were not significantly 26 different among the treatments studied. The lowest photosynthetic activity was obtained 27 from the control treatment (3.61 µmol m⁻² s⁻¹), while the highest was reached by the 28 digestate where R. okamurae was thermally pre-treated (4.96 µmol m⁻² s⁻¹). The results 29 of this study show that both digestates tested can be used effectively as organic fertilizer 30 in the cultivation of black oats, obtaining production efficiency values similar (digestate 31 from mechanically pre-treated macroalgae) or higher (digestate from thermally pre- 32 treated macroalgae) than inorganic fertilizer. 33 Keywords: Circular economy model; Anaerobic digestate; Zeolite; Rugulopteryx 34 okamurae; Avena strigosa; Photosynthetic activity. 35 36 1. Introduction 37 An invasive alien species is that which threatens or adversely impact the biodiversity and 38 the related ecosystem of an area where it was recently introduced (EU, 2014). Among 39 these species, seaweeds had been pointed as the main reason of the biodiversity loss in 40 the Mediterranean Sea (Coll et al., 2010), where more than 60 different macroalgae have 41 been introduced (Piazzi and Balata, 2009), of which only one specie is currently included 42 in the list of invasive alien species of Union concern: the brown macroalgae Rugulopteryx 43 okamurae (R.o.) (EU, 2022). 44 The rapid and aggressive colonization of this macroalgae on the southern coast of Spain, 45 more specifically in the Strait of Gibraltar, has cost around 1.2 million euros per season 46 since its first appearance took place in 2015 (MITECO, 2022; Sempere-Valverde et al., 47 2021). The displacement of local biota, the negative impact on sea-dependant 48 anthropogenic activities, such as fishery, and the accumulation of the decomposing settled 49 algae in the beach, fermenting uncontrolled, has caused an unprecedented environmental, 50 health and economic impact (García-Gómez et al., 2021; MITECO, 2022). 51 Several options have been pointed out as possible ways to diminish the proliferation of 52 the invasive species (e.g. manual removal, use of chemicals, etc.) although none have 53 been so far successfully implemented (MITECO, 2022). Among the scientific 54 community, efforts have been focused on the valorisation of this biomass. In this sense, 55 the macroalgae R.o. have been proposed as a feedstock for the recovery of diterpene 56 compounds with pharmaceutical applications; the production of bioplastics; compost; or 57 the generation of biogas (Casal-Porras et al., 2021; Cuevas et al., 2021; De la Lama- 58 Calvente et al., 2021; Patón et al., 2022; Santana et al., 2022). 59 Thus, the anaerobic digestion (AD) of macroalgae has been pointed out as a feasible 60 solution to curb the above-mentioned environmental issue. Several authors have reported 61 that the theoretical biomethane production of macroalgae is within the range of 500 – 62 1000 NLCH4 kgVS-1, with actual reported values reaching up to 90% of its theoretical yield, 63 which confirms the potential of these biomasses as feedstock for an AD process (Darko 64 et al., 2020; Pardilhó et al., 2022). Additionally, the AD process, besides the biogas, 65 generates a digestate enriched in minerals and other macro and micronutrients which has 66 the potential of being used as a biofertilizer (Bergstrand 2022; Thompson et al., 2020). In 67 order to reduce the consumption of mineral resources and fossil fuels, there is a trend 68 toward the use of organic fertilizers as opposed to mineral fertilizers. It is known that the 69 use of digestate as a biofertilizer has many benefits on the soils where it is applied, helping 70 to improve environmental, social and productive problems (Liu and Lal, 2015). 71 Fernández-Rodríguez et al. (2021) reported that the digestate from the AD of olive mill 72 solid waste improved the growth, the photosynthetic rate, and the nutritional content of 73 the grass Lolium rigidum. Furthermore, Bustamante et al. (2019) indicated that the 74 composition of Rosmarinus officinalis L. was dependent on the fertilizer used when 75 applying the composted digestate derived from the AD of cattle and pig slurry. Thus, the 76 origin of the digestate needs to be assessed in order to determine its potential suitability 77 as a soil amender or biofertilizer. Specifically, the C/N ratio, the heavy metal and nutrient 78 content, and the presence of pathogens or phytotoxic compounds, such as polyphenols, 79 are the most important parameters that could impact the plant quality (Bustamante et al., 80 2019; Fernández-Rodríguez et al., 2021; Golovko et al., 2022; Simioni et al., 2022). As 81 most of the potentially toxic and recalcitrant compounds are present in the solid fraction 82 of the digestate, some authors have evaluated the use of both liquid and solid fractions 83 separately and in combination, being the most used the liquid part at different 84 concentrations depending on soil requirements (Fernández-Rodríguez et al., 2021; Hadidi 85 et al., 2021; Simioni et al., 2022). 86 Nevertheless, the literature shows few studies where anaerobic digestates have been used 87 as soil amendments or biofertilizers. It is known that digestate fertilizer characteristics 88 are dependent on the substrate used in the anaerobic process. Recent and current literature 89 on the use of digestate as a fertilizer or soil amendments mainly uses as substrate cattle 90 or pig slurry, microalgae, or food waste as substrate (Bustamante et al., 2019; Fernández- 91 Rodríguez et al., 2022; Golovko et al., 2022). Although some studies have analyzed the 92 digestates in order to compare them against the applicable regulations for their use as 93 fertilizers (De la Lama-Calvente et al., 2023; Thompson et al., 2020), to the best of our 94 knowledge, no actual experiments have been carried out up to now in order to assess the 95 viability of anaerobic digestate derived from the AD of marine macroalgae. 96 The aims of this study was to assess, for the first time, the viability of the anaerobic 97 digestates, derived from the treatment of the invasive R. okamurae, as biofertilizer during 98 the growth of the Mediterranean forage species Avena strigosa (black oat). The specific 99 objectives were: i) to study the effects of the type of fertilizer and the conditions of water 100 availability on the growth, seedling establishment, productivity of black oat and 101 performance of cultivated plants, specifically regarding their influence on gas exchange 102 characteristics; and ii) to investigate potential correlations between the effects of different 103 fertilizers and nutrient concentrations in plant tissues. 104 105 2. Materials and Methods 106 2.1. Anaerobic digestate 107 De la Lama-Calvente et al. (2023) recently evaluated the viability of R.o. waste biomass 108 as the feedstock of an AD process in order to produce renewable energy. In their study, 109 the highest methane yields were obtained when the algal biomass was pretreated with a 110 new low-cost mechanical process assisted by zeolite and when the biomass was thermally 111 pretreated at 120 °C for 45 min. These treatments enhanced the methane production by a 112 35% over the untreated macroalgae. The anaerobic digestates derived from the AD 113 process of the two pretreatments were filtered and the liquid fractions selected for this 114 research to provide the organic nutrient solutions (ONS): i) from thermally pretreated R.o. 115 at 120 ºC during 45 min (ONS-T); ii) from mechanically pretreated R.o. assisted by 116 zeolite (ONS-Z). 117 The anaerobic digestates used in this experiment were derived from biochemical methane 118 potential (BMP) tests carried out at mesophilic temperature (35 ± 2 ºC) using R.o. as 119 substrate. The initial anaerobic inoculum was collected from a nearby brewery industry 120 which used an up-flow sludge blanket reactor as a system to treat the generated 121 wastewater. Once the BMP assays finished, the selected digestates were centrifuged and 122 the liquid phase was stored at -20 ºC until further use. 123 In order to obtain a suitable amount of digestate, the selected assays were repeated in 2 L 124 reactors following the same conditions as previously reported. The accumulative methane 125 yields of the repeated tests and the AD performances, regarded to the actual digestates 126 used in this work are shown in the supplementary material (Figure S1). 127 128 2.1.1. Digestate characterisation 129 The liquid fractions of the digestates used in the present work were analyzed in 130 accordance with international standard methods (De la Lama-Calvente et al., 2023). A 131 portable pH meter model Hach sensION MM150 was used to determine pH and 132 conductivity. Total alkalinity (TA) was determined by using a pH meter model Crison by 133 titration to pH 4.3 as shown in the 2320B standard method (APHA, 2017). The solid 134 content (total solids (TS) and volatile solids (VS)) was analyzed following the standard 135 method 2540B & 2540E (APHA, 2017). C and N analyses of the lyophilized liquid 136 fractions were performed by using a LECO CN828 Elemental Analyzer (Leco 137 Corporation, USA). The U.S. EPA 3051A method (USEPA, 2007) was followed in order 138 to determine the metal and micronutrients content by using a Mars Xtraction microwave 139 (CEM, USA) and analyzed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS, 140 AGILENT 7800, Spain). 141 142 2.2. Crop species selection 143 The selected species for this study was A. strigosa Schreb (also known as black oat), a 144 high-yielding annual grass with rapid establishment in the field and high forage 145 production. It is very interesting for the climatic and soil conditions of the Iberian 146 Peninsula due to its excellent adaptation (it grows adequately in moderate climatic and 147 dry environments; it tolerates dryness, low fertility levels and acidic soils (pH 4.5 – 7.3)) 148 (Dial, 2014). This crop, although nearly to extinction at the end of the twentieth century 149 in the Iberian Peninsula, presents a high nutritional value and is easily digested by cattle 150 (Ugrenović et al., 2021). A. strigosa seeds were supplied by the company Fertiprado 151 España. 152 153 2.3. Experimental set-up 154 The fertigation treatments were performed in 400 cm3 pots filled with sterile sand and 155 perlite in the base with a density of 9 plants per pot. Besides the two different digestates 156 studied (ONS-T and ONS-Z), a third assay in which the plant was fertigated with a 157 commercial inorganic nutritive solution (INS) was also set up as a positive control and in 158 order to assess the viability of the proposed biofertilizers against a commercial inorganic 159 solution. The selected INS was the 50% Hoagland nutrient solution (Hoagland and 160 Arnon, 1950) as it has been widely used in these types of experiments (Fernández- 161 Rodríguez et al., 2021; Liu and Lal, 2015). 162 Prior to the experiment, 20 pots for each treatment were split into two sets of 10 and 163 placed in plastic trays. Each treatment was subjected to two different situations of water 164 availability: i) water stress (simulated precipitation of 350 mm year -1, S) and ii) optimal 165 water availability (simulated precipitation of 650 mm year -1, R). These situations 166 correspond to a reduction or an increase of 30% of the average values of precipitation for 167 the cultivation areas of the oat in the South of Europe (Rodríguez-Berbel et al., 2022). 168 This situation was simulated by watering the pots weekly with a solution containing a 169 nutrient-poor medium (5% Hoagland nutrient solution). 170 Finally, two sets (10 pots each) were also prepared as control. The same system as 171 described before was used but no nutrient solution was added during the whole 172 experiment (C-S: water stressed control treatment; C-R: optimal water availability 173 control treatment). 174 In fertilized treatments, each pot was fertigated with 14 mL of nutrient solution (either 175 ONS or INS) adjusted to provide the required nitrogen level (210 kg N ha-1) in accordance 176 with the limits established in the Spanish regulation for vulnerable lands (RD 47/2022). 177 The fertigation was provided manually 15 days after the plant’s first emergence (stem ≥ 178 1 cm). 179 All the pots were kept in a greenhouse (General Research Services (CITIUS), University 180 of Seville) with 40 – 60 % relative humidity, a minimum to a maximum temperature of 181 24 – 26 ºC, and natural daylight. A brief resume of the different treatments carried out is 182 shown in Table S1 provided in the supplementary material. 183 184 2.3.1. Crop characterization 185 At the end of the experiment, the net photosynthesis rate (A) was measured on fully 186 developed leaves (n=10) for each study treatment at a CO2 concentration of 400 μmol 187 mol−1 and at 1000 μmol photons m−2 s−1 with a LICOR 6800 (Inc., Neb., USA). 188 Once the experiment was concluded (60 days from the emergency), the plants were 189 collected and each steam was divided into three fractions for further analysis: i) aerial 190 parts; ii) seeds; iii) roots. C, N, and heavy metal content were determined as described 191 above. Dry biomass was obtained after placing the samples in an oven at 80 ºC over a 192 period of 48 h. 193 2.3.2. Statistical analysis 194 The usual tests for normality and homoscedasticity (Kolmogorov-Smirnov test and 195 Levene's test, respectively) were used to assume the hypotheses necessary for parametric 196 tests. Data were analyzed by two-way analysis of variance (ANOVA). Tukey's test was 197 used to identify differences, two-to-two comparisons, when there were significant results 198 other treatments. Mn is also required for photosynthesis. Values below 90 mg kg-1 are 354 considered deficient. By contrast, values higher than 200 mg kg-1 are considered to be 355 toxic for plants (Tripathi et al., 2015). In controls and INS treatments, Mn values were 356 lower than 90 mg kg-1, while organic treatments showed concentrations higher than 90 357 mg kg-1. The last micronutrient is Zn, which is also essential for early growth and plant 358 development. All treatments presented Zn values established as optimal for plants (10- 359 120 mg kg-1) (Tripathi et al., 2015). No significant differences were found between the 360 different treatments studied. 361 362 4. Conclusions 363 The introduction of a circular economy model in areas affected by the invasive 364 macroalgae R. okamurae can favour and achieve energy self-sufficiency and improve the 365 quality of life of its inhabitants. The results of the present study indicated that: i) the two 366 R. okamurae anaerobic digestates tested digestates showed chemical and nutritional 367 characteristics (macro and micro nutrients) suitable for the cultivation of black oats, 368 achieving higher or similar efficiencies to inorganic fertilizers; ii) the water deficit 369 reduced the production values of the black oat crop (6-19%) compared to the treatments 370 with optimum water availability; iii) the ONS-T treatments, as a consequence of an 371 improvement in nutrient content and photosynthetic activity, resulted in significantly 372 higher production values than those obtained in the INS and ONS-Z treatments. 373 374 Acknowledgements 375 The authors wish to express their gratitude to the Spanish Ministry of Science and 376 Innovation for providing financial support through the project PID2020-114975RB- 377 100/AEI/10.13039/5011000/11033. M.J. 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Agriculture (Switzerland), 11(1), 1-14. 568 doi:10.3390/agriculture11010055 569 570 Table 1. Elemental analysis and main physicochemical characteristics of the soluble fractions of the different fertilizers studied. Results are based on 571 fresh or dry matter. Values are given mean (standard deviation). 572 INS: Inorganic nutritive solution; ONS-T: Organic nutritive solution digestate from Rugulopteryx okamurae thermally pretreatment; ONS-Z: Organic nutritive solution digestate from R. okamurae with zeolite pre-treatment. 573 1dry bases 574 2units in mg/L 575 Parameters ONS-Z ONS-T INS Parameters ONS-Z ONS-T pH 8.33 (0.02) 8.35 (0.01) 6.9 (0.1)2 Al (g/kg)1 0.131 (0.007) 0.117 (0.007) Conductivity (mS cm-1) 14.12 (0.01) 15.48 (0.03) 0.9 (0.1)2 Ca (g/kg)1 2.0 (0.07) 1.7 (0.07) Alkalinity (mg CaCO3 kg-1) 6730 (30) 6800 (100) - Cr (ppm)1 1.0 (0.2) 0.9 (0.2) VS (g/kg) 9.8 (0.2) 3.8 (0.3) - Mn (ppm)1 3 (4) 3 (4) TS (g/kg) 10.8 (0.2) 4.1 (0.3) - Fe (g/kg)1 2.5 (0.2) 2.4 (0.2) H (g/kg)1 42 (2) 47.3 (0.8) - Co (ppm)1 0.2 (0.5) 0.2 (0.5) O (g/kg)1 230 (2) 231 (7) - Ni (ppm)1 2.6 (0.3) 2.3 (0.3) C (g/kg)1 330 (20) 350 (6) - Cu (ppm)1 0.93 (0.05) 0.84 (0.05) N (g/kg)1 25 (3) 23.9 (0.7) 107 (2)2 Zn (ppm)1 7 (1) 7 (1) P (g/kg)1 0.21 (0.07) 0.19 (0.07) 21 (5)2 As (ppm)1 2.6 (0.6) 2.1 (0.6) K (g/kg)1 3.0 (0.2) 3.0 (0.2) 117 (9)2 Mo (ppb)1 40 (20) 30 (20) N:P:K ratio 210:2.3:25 210:2.2:26 210:40:229 Cd (ppb)1 50 (20) 40 (20) C/N ratio 13 (1) 14.6 (0.2) - Sn (ppb)1 70 (50) 70 (50) B (ppm)1 21 (22) 17 (22) - Ba (ppm)1 2.1 (0.2) 1.8 (0.2) Na (g/kg)1 5.1 (0.6) 4.3 (0.6) - Hg (ppm)1 0.006 (0.007) 0.005 (0.007) Mg (g/kg)1 0.8 (0.4) 0.7 (0.4) 17 (3)2 Pb (ppm)1 0.5 (0.3) 0.5 (0.3)