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

Depósi o de in es igación de la Uni e sidad de Se illa h ps://idus.us.es/ “This is an Accep ed Manusc ip o an a icle published by Else ie in: Scien ia Ho icul u ae on Feb ua y 2024, a ailable a : h ps://doi.o g/10.1016/j.scien a.2023.112638”. Use o anae obic diges a e as bio e ilize : Ano he s ep o wa d in he alo isa ion 1 o he in asi e b own mac oalgae Rugulop e yx okamu ae 2 Da id De la Lama-Cal en ea, Juan Manuel Mancilla-Ley ónb 1 , Ra ael Bo jaa, Ma ía José 3 Fe nández-Rod íguezb 4 a Spanish Scien i ic Resea ch Council (CSIC) - Ins i u o de la G asa (IG), Depa men o 5 Food Bio echnology, Campus Uni e sidad Pablo de Ola ide, Edi icio 46. C a. de U e a, 6 km 1, 41013 Se ille, Spain. 7 b Depa amen o de Biología Vege al y Ecología, Facul ad de Biología, Uni e sidad de 8 Se illa, 41080 Se ille, Spain. 9 Abs ac 10 The apid and agg essi e coloniza ion o he in asi e mac oalgae Rugulop e yx 11 okamu ae on he sou he n coas o Spain has caused an unp eceden ed en i onmen al, 12 heal h and economic impac . A p esen , he scien i ic communi y has ocused on he 13 alo isa ion o R. okamu ae was e, as onnes o collec ed biomass om his species end 14 up in land ills. In his sense, he aim o his s udy was o assess, o he i s ime, he 15 iabili y o he R. okamu ae diges a e as bio e ilize o g ow h he Medi e anean o age 16 species A ena s igosa (black oa ). Two anae obic diges a es, de i ed om he anae obic 17 diges ion o he he mally p e- ea ed (120 ºC; 45 min) and he zeoli e-assis ed 18 mechanically p e- ea ed in asi e mac oalgae, we e used in his s udy. Bo h diges a es 19 we e compa ed wi h a comme cial ino ganic nu i i e solu ion. The e ec o di e en 20 e ilize s and wa e a ailabili y on o age heigh , ae ial, oo and seed biomass, as well 21 as pho osyn he ic ac i i y and mac o- and mic onu ien o age con en we e s udied. The 22 con ol ea men showed he lowes heigh a he end o he expe imen (0.38 m) and he 23 1 Co esponding au ho : [email protected] diges a e om he he mally p e- ea ed mac oalgae ea men had he highes one (0.59 24 m). Final esh and d y ae ial biomass was sepa a ely signi ican ly dependen on bo h 25 e ilize ype and wa e s ess. Final oo and seed biomass we e no signi ican ly 26 di e en among he ea men s s udied. The lowes pho osyn he ic ac i i y was ob ained 27 om he con ol ea men (3.61 µmol m⁻² s⁻¹), while he highes was eached by he 28 diges a e whe e R. okamu ae was he mally p e- ea ed (4.96 µmol m⁻² s⁻¹). The esul s 29 o his s udy show ha bo h diges a es es ed can be used e ec i ely as o ganic e ilize 30 in he cul i a ion o black oa s, ob aining p oduc ion e iciency alues simila (diges a e 31 om mechanically p e- ea ed mac oalgae) o highe (diges a e om he mally p e- 32 ea ed mac oalgae) han ino ganic e ilize . 33 Keywo ds: Ci cula economy model; Anae obic diges a e; Zeoli e; Rugulop e yx 34 okamu ae; A ena s igosa; Pho osyn he ic ac i i y. 35 36 1. In oduc ion 37 An in asi e alien species is ha which h ea ens o ad e sely impac he biodi e si y and 38 he ela ed ecosys em o an a ea whe e i was ecen ly in oduced (EU, 2014). Among 39 hese species, seaweeds had been poin ed as he main eason o he biodi e si y loss in 40 he Medi e anean Sea (Coll e al., 2010), whe e mo e han 60 di e en mac oalgae ha e 41 been in oduced (Piazzi and Bala a, 2009), o which only one specie is cu en ly included 42 in he lis o in asi e alien species o Union conce n: he b own mac oalgae Rugulop e yx 43 okamu ae (R.o.) (EU, 2022). 44 The apid and agg essi e coloniza ion o his mac oalgae on he sou he n coas o Spain, 45 mo e speci ically in he S ai o Gib al a , has cos a ound 1.2 million eu os pe season 46 since i s i s appea ance ook place in 2015 (MITECO, 2022; Sempe e-Val e de e al., 47 2021). The displacemen o local bio a, he nega i e impac on sea-dependan 48 an h opogenic ac i i ies, such as ishe y, and he accumula ion o he decomposing se led 49 algae in he beach, e men ing uncon olled, has caused an unp eceden ed en i onmen al, 50 heal h and economic impac (Ga cía-Gómez e al., 2021; MITECO, 2022). 51 Se e al op ions ha e been poin ed ou as possible ways o diminish he p oli e a ion o 52 he in asi e species (e.g. manual emo al, use o chemicals, e c.) al hough none ha e 53 been so a success ully implemen ed (MITECO, 2022). Among he scien i ic 54 communi y, e o s ha e been ocused on he alo isa ion o his biomass. In his sense, 55 he mac oalgae R.o. ha e been p oposed as a eeds ock o he eco e y o di e pene 56 compounds wi h pha maceu ical applica ions; he p oduc ion o bioplas ics; compos ; o 57 he gene a ion o biogas (Casal-Po as e al., 2021; Cue as e al., 2021; De la Lama- 58 Cal en e e al., 2021; Pa ón e al., 2022; San ana e al., 2022). 59 Thus, he anae obic diges ion (AD) o mac oalgae has been poin ed ou as a easible 60 solu ion o cu b he abo e-men ioned en i onmen al issue. Se e al au ho s ha e epo ed 61 ha he heo e ical biome hane p oduc ion o mac oalgae is wi hin he ange o 500 – 62 1000 NLCH4 kgVS-1, wi h ac ual epo ed alues eaching up o 90% o i s heo e ical yield, 63 which con i ms he po en ial o hese biomasses as eeds ock o an AD p ocess (Da ko 64 e al., 2020; Pa dilhó e al., 2022). Addi ionally, he AD p ocess, besides he biogas, 65 gene a es a diges a e en iched in mine als and o he mac o and mic onu ien s which has 66 he po en ial o being used as a bio e ilize (Be gs and 2022; Thompson e al., 2020). In 67 o de o educe he consump ion o mine al esou ces and ossil uels, he e is a end 68 owa d he use o o ganic e ilize s as opposed o mine al e ilize s. I is known ha he 69 use o diges a e as a bio e ilize has many bene i s on he soils whe e i is applied, helping 70 o imp o e en i onmen al, social and p oduc i e p oblems (Liu and Lal, 2015). 71 Fe nández-Rod íguez e al. (2021) epo ed ha he diges a e om he AD o oli e mill 72 solid was e imp o ed he g ow h, he pho osyn he ic a e, and he nu i ional con en o 73 he g ass Lolium igidum. Fu he mo e, Bus aman e e al. (2019) indica ed ha he 74 composi ion o Rosma inus o icinalis L. was dependen on he e ilize used when 75 applying he compos ed diges a e de i ed om he AD o ca le and pig slu y. Thus, he 76 o igin o he diges a e needs o be assessed in o de o de e mine i s po en ial sui abili y 77 as a soil amende o bio e ilize . Speci ically, he C/N a io, he hea y me al and nu ien 78 con en , and he p esence o pa hogens o phy o oxic compounds, such as polyphenols, 79 a e he mos impo an pa ame e s ha could impac he plan quali y (Bus aman e e al., 80 2019; Fe nández-Rod íguez e al., 2021; Golo ko e al., 2022; Simioni e al., 2022). As 81 mos o he po en ially oxic and ecalci an compounds a e p esen in he solid ac ion 82 o he diges a e, some au ho s ha e e alua ed he use o bo h liquid and solid ac ions 83 sepa a ely and in combina ion, being he mos used he liquid pa a di e en 84 concen a ions depending on soil equi emen s (Fe nández-Rod íguez e al., 2021; Hadidi 85 e al., 2021; Simioni e al., 2022). 86 Ne e heless, he li e a u e shows ew s udies whe e anae obic diges a es ha e been used 87 as soil amendmen s o bio e ilize s. I is known ha diges a e e ilize cha ac e is ics 88 a e dependen on he subs a e used in he anae obic p ocess. Recen and cu en li e a u e 89 on he use o diges a e as a e ilize o soil amendmen s mainly uses as subs a e ca le 90 o pig slu y, mic oalgae, o ood was e as subs a e (Bus aman e e al., 2019; Fe nández- 91 Rod íguez e al., 2022; Golo ko e al., 2022). Al hough some s udies ha e analyzed he 92 diges a es in o de o compa e hem agains he applicable egula ions o hei use as 93 e ilize s (De la Lama-Cal en e e al., 2023; Thompson e al., 2020), o he bes o ou 94 knowledge, no ac ual expe imen s ha e been ca ied ou up o now in o de o assess he 95 iabili y o anae obic diges a e de i ed om he AD o ma ine mac oalgae. 96 The aims o his s udy was o assess, o he i s ime, he iabili y o he anae obic 97 diges a es, de i ed om he ea men o he in asi e R. okamu ae, as bio e ilize du ing 98 he g ow h o he Medi e anean o age species A ena s igosa (black oa ). The speci ic 99 objec i es we e: i) o s udy he e ec s o he ype o e ilize and he condi ions o wa e 100 a ailabili y on he g ow h, seedling es ablishmen , p oduc i i y o black oa and 101 pe o mance o cul i a ed plan s, speci ically ega ding hei in luence on gas exchange 102 cha ac e is ics; and ii) o in es iga e po en ial co ela ions be ween he e ec s o di e en 103 e ilize s and nu ien concen a ions in plan issues. 104 105 2. Ma e ials and Me hods 106 2.1. Anae obic diges a e 107 De la Lama-Cal en e e al. (2023) ecen ly e alua ed he iabili y o R.o. was e biomass 108 as he eeds ock o an AD p ocess in o de o p oduce enewable ene gy. In hei s udy, 109 he highes me hane yields we e ob ained when he algal biomass was p e ea ed wi h a 110 new low-cos mechanical p ocess assis ed by zeoli e and when he biomass was he mally 111 p e ea ed a 120 °C o 45 min. These ea men s enhanced he me hane p oduc ion by a 112 35% o e he un ea ed mac oalgae. The anae obic diges a es de i ed om he AD 113 p ocess o he wo p e ea men s we e il e ed and he liquid ac ions selec ed o his 114 esea ch o p o ide he o ganic nu ien solu ions (ONS): i) om he mally p e ea ed R.o. 115 a 120 ºC du ing 45 min (ONS-T); ii) om mechanically p e ea ed R.o. assis ed by 116 zeoli e (ONS-Z). 117 The anae obic diges a es used in his expe imen we e de i ed om biochemical me hane 118 po en ial (BMP) es s ca ied ou a mesophilic empe a u e (35 ± 2 ºC) using R.o. as 119 subs a e. The ini ial anae obic inoculum was collec ed om a nea by b ewe y indus y 120 which used an up- low sludge blanke eac o as a sys em o ea he gene a ed 121 was ewa e . Once he BMP assays inished, he selec ed diges a es we e cen i uged and 122 he liquid phase was s o ed a -20 ºC un il u he use. 123 In o de o ob ain a sui able amoun o diges a e, he selec ed assays we e epea ed in 2 L 124 eac o s ollowing he same condi ions as p e iously epo ed. The accumula i e me hane 125 yields o he epea ed es s and he AD pe o mances, ega ded o he ac ual diges a es 126 used in his wo k a e shown in he supplemen a y ma e ial (Figu e S1). 127 128 2.1.1. Diges a e cha ac e isa ion 129 The liquid ac ions o he diges a es used in he p esen wo k we e analyzed in 130 acco dance wi h in e na ional s anda d me hods (De la Lama-Cal en e e al., 2023). A 131 po able pH me e model Hach sensION MM150 was used o de e mine pH and 132 conduc i i y. To al alkalini y (TA) was de e mined by using a pH me e model C ison by 133 i a ion o pH 4.3 as shown in he 2320B s anda d me hod (APHA, 2017). The solid 134 con en ( o al solids (TS) and ola ile solids (VS)) was analyzed ollowing he s anda d 135 me hod 2540B & 2540E (APHA, 2017). C and N analyses o he lyophilized liquid 136 ac ions we e pe o med by using a LECO CN828 Elemen al Analyze (Leco 137 Co po a ion, USA). The U.S. EPA 3051A me hod (USEPA, 2007) was ollowed in o de 138 o de e mine he me al and mic onu ien s con en by using a Ma s X ac ion mic owa e 139 (CEM, USA) and analyzed by Induc i ely Coupled Plasma Mass Spec ome y (ICP-MS, 140 AGILENT 7800, Spain). 141 142 2.2. C op species selec ion 143 The selec ed species o his s udy was A. s igosa Sch eb (also known as black oa ), a 144 high-yielding annual g ass wi h apid es ablishmen in he ield and high o age 145 p oduc ion. I is e y in e es ing o he clima ic and soil condi ions o he Ibe ian 146 Peninsula due o i s excellen adap a ion (i g ows adequa ely in mode a e clima ic and 147 d y en i onmen s; i ole a es d yness, low e ili y le els and acidic soils (pH 4.5 – 7.3)) 148 (Dial, 2014). This c op, al hough nea ly o ex inc ion a he end o he wen ie h cen u y 149 in he Ibe ian Peninsula, p esen s a high nu i ional alue and is easily diges ed by ca le 150 (Ug eno ić e al., 2021). A. s igosa seeds we e supplied by he company Fe ip ado 151 España. 152 153 2.3. Expe imen al se -up 154 The e iga ion ea men s we e pe o med in 400 cm3 po s illed wi h s e ile sand and 155 pe li e in he base wi h a densi y o 9 plan s pe po . Besides he wo di e en diges a es 156 s udied (ONS-T and ONS-Z), a hi d assay in which he plan was e iga ed wi h a 157 comme cial ino ganic nu i i e solu ion (INS) was also se up as a posi i e con ol and in 158 o de o assess he iabili y o he p oposed bio e ilize s agains a comme cial ino ganic 159 solu ion. The selec ed INS was he 50% Hoagland nu ien solu ion (Hoagland and 160 A non, 1950) as i has been widely used in hese ypes o expe imen s (Fe nández- 161 Rod íguez e al., 2021; Liu and Lal, 2015). 162 P io o he expe imen , 20 po s o each ea men we e spli in o wo se s o 10 and 163 placed in plas ic ays. Each ea men was subjec ed o wo di e en si ua ions o wa e 164 a ailabili y: i) wa e s ess (simula ed p ecipi a ion o 350 mm yea -1, S) and ii) op imal 165 wa e a ailabili y (simula ed p ecipi a ion o 650 mm yea -1, R). These si ua ions 166 co espond o a educ ion o an inc ease o 30% o he a e age alues o p ecipi a ion o 167 he cul i a ion a eas o he oa in he Sou h o Eu ope (Rod íguez-Be bel e al., 2022). 168 This si ua ion was simula ed by wa e ing he po s weekly wi h a solu ion con aining a 169 nu ien -poo medium (5% Hoagland nu ien solu ion). 170 Finally, wo se s (10 po s each) we e also p epa ed as con ol. The same sys em as 171 desc ibed be o e was used bu no nu ien solu ion was added du ing he whole 172 expe imen (C-S: wa e s essed con ol ea men ; C-R: op imal wa e a ailabili y 173 con ol ea men ). 174 In e ilized ea men s, each po was e iga ed wi h 14 mL o nu ien solu ion (ei he 175 ONS o INS) adjus ed o p o ide he equi ed ni ogen le el (210 kg N ha-1) in acco dance 176 wi h he limi s es ablished in he Spanish egula ion o ulne able lands (RD 47/2022). 177 The e iga ion was p o ided manually 15 days a e he plan ’s i s eme gence (s em ≥ 178 1 cm). 179 All he po s we e kep in a g eenhouse (Gene al Resea ch Se ices (CITIUS), Uni e si y 180 o Se ille) wi h 40 – 60 % ela i e humidi y, a minimum o a maximum empe a u e o 181 24 – 26 ºC, and na u al dayligh . A b ie esume o he di e en ea men s ca ied ou is 182 shown in Table S1 p o ided in he supplemen a y ma e ial. 183 184 2.3.1. C op cha ac e iza ion 185 A he end o he expe imen , he ne pho osyn hesis a e (A) was measu ed on ully 186 de eloped lea es (n=10) o each s udy ea men a a CO2 concen a ion o 400 μmol 187 mol−1 and a 1000 μmol pho ons m−2 s−1 wi h a LICOR 6800 (Inc., Neb., USA). 188 Once he expe imen was concluded (60 days om he eme gency), he plan s we e 189 collec ed and each s eam was di ided in o h ee ac ions o u he analysis: i) ae ial 190 pa s; ii) seeds; iii) oo s. C, N, and hea y me al con en we e de e mined as desc ibed 191 abo e. D y biomass was ob ained a e placing he samples in an o en a 80 ºC o e a 192 pe iod o 48 h. 193 2.3.2. S a is ical analysis 194 The usual es s o no mali y and homoscedas ici y (Kolmogo o -Smi no es and 195 Le ene's es , espec i ely) we e used o assume he hypo heses necessa y o pa ame ic 196 es s. Da a we e analyzed by wo-way analysis o a iance (ANOVA). Tukey's es was 197 used o iden i y di e ences, wo- o- wo compa isons, when he e we e signi ican esul s 198 o he ea men s. Mn is also equi ed o pho osyn hesis. Values below 90 mg kg-1 a e 354 conside ed de icien . By con as , alues highe han 200 mg kg-1 a e conside ed o be 355 oxic o plan s (T ipa hi e al., 2015). In con ols and INS ea men s, Mn alues we e 356 lowe han 90 mg kg-1, while o ganic ea men s showed concen a ions highe han 90 357 mg kg-1. The las mic onu ien is Zn, which is also essen ial o ea ly g ow h and plan 358 de elopmen . All ea men s p esen ed Zn alues es ablished as op imal o plan s (10- 359 120 mg kg-1) (T ipa hi e al., 2015). No signi ican di e ences we e ound be ween he 360 di e en ea men s s udied. 361 362 4. Conclusions 363 The in oduc ion o a ci cula economy model in a eas a ec ed by he in asi e 364 mac oalgae R. okamu ae can a ou and achie e ene gy sel -su iciency and imp o e he 365 quali y o li e o i s inhabi an s. The esul s o he p esen s udy indica ed ha : i) he wo 366 R. okamu ae anae obic diges a es es ed diges a es showed chemical and nu i ional 367 cha ac e is ics (mac o and mic o nu ien s) sui able o he cul i a ion o black oa s, 368 achie ing highe o simila e iciencies o ino ganic e ilize s; ii) he wa e de ici 369 educed he p oduc ion alues o he black oa c op (6-19%) compa ed o he ea men s 370 wi h op imum wa e a ailabili y; iii) he ONS-T ea men s, as a consequence o an 371 imp o emen in nu ien con en and pho osyn he ic ac i i y, esul ed in signi ican ly 372 highe p oduc ion alues han hose ob ained in he INS and ONS-Z ea men s. 373 374 Acknowledgemen s 375 The au ho s wish o exp ess hei g a i ude o he Spanish Minis y o Science and 376 Inno a ion o p o iding inancial suppo h ough he p ojec PID2020-114975RB- 377 100/AEI/10.13039/5011000/11033. M.J. Fe nández-Rod íguez is ecipien o a “Juan de 378 la Cie a Fo mación” g an om he Spanish S a e Resea ch Agency (FJC2020-045654- 379 I). The au ho s also wish specially o hank F. O iz (Fe ip ado España) o his ad ice 380 and M.J. Ba aú Pá aga o he help in he g eenhouse. 381 382 Re e ences 383 384 Be gs and, K. J. 2022. O ganic e ilize s in g eenhouse p oduc ion sys ems–a e iew. 385 Sci. Ho ic., 295, 110855. h ps://doi.o g/10.1016/j.scien a.2021.110855 386 B aun, J.C.A., Colla, L.M., 2022. Use o mic oalgae o he de elopmen o bio e ilize s 387 and bios imulan s. BioEne g. Res. h ps://doi.o g/10.1007/s12155-022-10456-8. 388 Bus aman e, M.A., Nogués, I., Jones, S., Allison, G.G., 2019. The e ec o anae obic 389 diges a e de i ed compos s on he me aboli e composi ion and he mal beha iou o 390 osema y. Sci. Rep. 9, 6489. h ps://doi.o g/10.1038/s41598-019-42725-6. 391 Casal-Po as, I., Zubía, E., B un, F.G., 2021. Dilkamu al: A no el chemical weapon 392 in ol ed in he in asi e capaci y o he alga Rugulop e yx okamu ae in he S ai o 393 Gib al a . Es ua . Coas . Shel Sci. 257, 107398. 394 h ps://doi.o g/10.1016/j.ecss.2021.107398. 395 CEC, 2003. P oposal o a Di ec i e o he Eu opean Pa liamen and he Council on 396 Sp eading o Sludge on Land (B ussels, 30 Ap il). 397 h ps://en i onmen .ec.eu opa.eu/ opics/was e-and- ecycling/sewage-sludge_en 398 Coll, M., Pi oddi, C., S eenbeek, J., Kaschne , K., Las am, F.B.R., Aguzzi, J., Balles e os, 399 E., Bianchi, C.N., Co be a, J., Dailianis, T., Dano a o, R., Es ada, M., F oglia, C., 400 Galil, B.S., Gasol, J.M., Ge wage, R., Gil, J., Guilhaumon, F., Kesne -Reyes, K., 401 Ki sos, M.S., Koukou as, A., Lampada iou, N., Laxamana, E., de la Cuad a, C.M.L.F., 402 Lo ze, H.K., Ma in, D., Mouillo , D., O o, D., Raice ich, S., Rius-Ba ile, J., Saiz- 403 Salinas, J.I., Vicen e, C.S., Somo , S., Templado, J., Tu on, X., Va idis, D., 404 Villanue a, R., Voul siadou, E., 2010. The biodi e si y o he medi e anean sea: 405 es ima es, pa e ns, and h ea s. PLoS ONE. 5 (8), e11842. 406 h ps://doi.o g/10.1371/jou nal.pone.0011842. 407 Cue as, B., A oba, A.I., de los Reyes, C., Gómez-Ja amillo, L., González-Mon elongo, 408 M.C., Zubía, E., 2021. Di e penoids om he b own alga Rugulop e yx okamu ae and 409 hei an i-in lamma o y ac i i y. Ma ine d ugs 19, 677. 410 h ps://doi.o g/10.3390/md19120677. 411 Da ko, C.N.S., Agyei-Tu ou , B., Faloye, D.F., Goosen, N.J., Nyankson, E., Dodoo- 412 A hin, D., 2022. Biome hane p oduc ion om esidual algae biomass (Ecklonia 413 maxima): E ec s o inoculum acclima iza ion on yield. Was e Biomass Valo i. 13, 414 497-509. h ps://doi.o g/10.1007/s12649-021-01497-9. 415 De la Lama-Cal en e, D., Cube o, J., Fe nández-Rod íguez, M.J., Jiménez-Rod íguez, 416 A., Bo ja, R., 2022. Anae obic co-diges ion o mic oalgae and indus ial was es: A 417 c i ical and bibliome ic e iew. in L. Q. Zepka, E. Jacob-Lopes, M. C. Dep á (eds.), 418 P og ess in Mic oalgae Resea ch - A Pa h o Shaping Sus ainable Fu u es, 419 In echOpen, London. h ps://doi.o g/10.5772/in echopen.104378. 420 De la Lama-Cal en e, D., Fe nández-Rod íguez, M.J., Balles e os, M., Ruiz-Sal ado , Á.R., 421 Raposo, F., Ga cía-Gómez, J.C., Bo ja, R., 2023. Tu ning an in asi e alien species in o a 422 aluable biomass: Anae obic diges ion o Rugulop e yx okamu ae a e he mal and new 423 de eloped low-cos mechanical p e ea men s. Sci. To al En i on. 856, 158914. 424 h p://dx.doi.o g/10.1016/j.sci o en .2022.158914. 425 Dial, H.L., 2014. Plan guide o black oa (A ena s igosa Sch eb.) USDA-Na u al Resou ces 426 Conse a ion Se ice; Tucson Plan Ma e ials Cen e : Tucson, AZ, USA, 85705. 427 De la Lama-Cal en e, D., Fe nández-Rod íguez, M.J., Llanos, J., Mancilla-Ley ón, J.M., 428 Bo ja, R., 2021. Enhancing me hane p oduc ion om he in asi e mac oalga Rugulop e yx 429 okamu ae h ough anae obic co-diges ion wi h oli e mill solid was e: p ocess pe o mance 430 and kine ic analysis. J. Appl. Phycol. 33, 4113-4124. h ps://doi.o g/10.1007/s10811-021- 431 02548-3. 432 Ehmann, A., Bach, I. M., Bilbao, J., Lewandowski, I., Mülle , T. 2019. Phospha es 433 ecycled om semi-liquid manu e and diges a e a e sui able al e na i e e ilize s o 434 o namen als. Scien ia Ho icul u ae, 243, 440-450. 435 h ps://doi.o g/10.1016/j.scien a.2018.08.052 436 EU, 2014. Regula ion (EU) No 1143/2014 o he Eu opean Pa liamen o he Council o 437 22 Oc obe 2014 on he p e en ion and managemen o he in oduc ion and sp ead o 438 in asi e alien species. h ps://eu -lex.eu opa.eu/legal- 439 con en /EN/TXT/?u i=celex%3A32014R1143. [accessed 22nd Sep embe 2022]. 440 EU, 2022. Commission implemen ing egula ion (EU) 2016/1141 o 13 July 2016 441 adop ing a lis o in asi e alien species o Union conce n pu suan o Regula ion (EU) 442 No 1143/2014 o he Eu opean Pa liamen and o he Council and i s amendmen s. 443 h ps://eu -lex.eu opa.eu/legal-con en /EN/TXT/?u i=CELEX%3A02016R1141- 444 20220802. [accessed 27 h Sep embe 2022]. 445 Fe nández-Rod íguez, M.J., J.M. Mancilla-Ley ón, Jiménez-Rod íguez, A., Bo ja, R., 446 Rincón, B., 2021. Reuse o he diges a e ob ained om he biome haniza ion o oli e 447 mill solid was e (OMSW) as soil amendmen o e ilize o he cul i a ion o o age 448 g ass (Lolium igidum a . Wimme a). Sci. To al En i on. 792, 148465. 449 h ps://doi.o g/10.1016/j.sci o en .2021.148465. 450 Fe nández-Rod íguez, M.J., Palenzuela, M.V., Balles e os, M., J.M. Mancilla-Ley ón, 451 Bo ja, R., 2022. E ec o di e en diges a es de i ed om anae obic co-diges ion o 452 oli e mill solid was e (omsw) and a ious mic oalgae as e ilize s o he cul i a ion 453 o yeg ass. Plan Soil 475, 331-342. h ps://doi.o g/10.1007/s11104-022-05370-z. 454 Fon aneli, R. S. ; San os, H. P. dos ; Fon aneli, R. S. ; Oli ei a, J. T. ; Lehmen, R. I. ; 455 D eon, G., 2012. Win e o age g asses. In: Fon aneli, R. S.; San os, H. P. dos ; 456 Fon aneli, R. S (Eds), Fo agei as pa a in eg ação la ou a-pecuá ia- lo es a na egião 457 sul-b asilei a, EMBRAPA, B asilia 458 Ga cía-Gómez, J.C., Flo ido, M., Olaya-Ponzone, L., Sempe e-Val e de, J., Megina, C., 459 2021. The in asi e mac oalga Rugulop e yx okamu ae: Subs a a plas ici y and spa ial 460 coloniza ion p essu e on esiden mac oalgae. F on . Ecol. E ol. 9, 631754. 461 h ps://doi.o g/10.3389/ e o.2021.631754. 462 Golo ko, O., Ah ens, L., Schelin, J., Sö engå d, M., Be gs and, K.J., Asp, H., Hul be g, 463 M., Wibe g, K., 2022. O ganic mic opollulan s, hea y me als and pa hogens in 464 anae obic diges a e based on ood was e. J. En i on. Manage. 313, 114997. 465 h ps://doi.o g/10.1016/j.jen man.2022.114997. 466 Hadidi, M., Bahlaouan, B., Asbai, Z., Benjelloun, G.R., El An i, S., Bou aleb, N., 2021. 467 Op imizing biogas and bio e ilize p oduc ion om abundan Mo occan indus ial 468 o ganic was es by he o mula ion and he use o ungal inoculum. Ad . En i on. 469 Technol. 4, 275-287. h ps://doi.o g/10.22104/AET.2022.5357.1450. 470 Heuzé V., T an G., Hassoun P., Lebas F., 2015. Black oa (A ena s igosa). Feedipedia, 471 a p og amme by INRAE, CIRAD, AFZ and FAO. 472 h ps://www. eedipedia.o g/node/581 Las upda ed on Oc obe 26, 2015, 17:48 473 Hoagland, D.R., A non, D., 1950. The wa e cul u e me hod o g owing plan s wi hou 474 soil. Cali . Ag ic. Exp. S a . Ci c. 2nd Edi ion. 347, 1–32. 475 Hollige , C., Al es, M., And ade, D., Angelidaki, I., As als, S., Baie , U., Boug ie , C., 476 Bu iè e, P., Ca balla, M., de Wilde, V., Ebe sede , F., Fe nández, B., Fica a, E., 477 Fo idis, I., F igon, J.C., de Laclos, H.F., Ghasimi, D.S.M., Hack, G., Ha el, M., 478 Hee enklage, J., Ho a h, I.S., Jenicek, P., Koch, K., K au wald, J., Lizasoain, J., Liu, 479 J., Mosbe ge , L., Nis o , M., Oechsne , H., Oli ei a, J.V., Pa e son, M., Pauss, A., 480 Pommie , S., Po queddu, I., Raposo, F., Ribei o, T., P und, F.R., S ömbe g, S., 481 To ijos, M., an Eeke , M., an Lie , J., Wedwi schka, H., Wie inck, I., 2016. 482 Towa ds a s anda diza ion o biome hane po en ial es s. Wa e Sci. Technol. 74, 483 2515–2522. h ps://doi.o g/10.2166/ws .2016.336. 484 Jeong, H., Ra, K., 2022. Seag ass and g een mac oalgae halimeda as biomoni o ing ools o 485 me al con amina ion in Chuuk, mic onesia: Pollu ion assessmen and 486 bioaccumula ion. Ma . Pollu . Bull. 178, 113625. 487 h ps://10.1016/j.ma polbul.2022.113625. 488 Liu, R., Lal, R., 2015. Po en ials o enginee ed nanopa icles as e ilize s o inc easing 489 ag onomic p oduc ions. Sci. To al En i on. 514, 131-139. 490 h ps://doi.o g/10.1016/j.sci o en .2015.01.104. 491 McKennedy, J., She lock, O., 2015. Anae obic diges ion o ma ine mac oalgae: A 492 e iew. Renew. Sus . Ene g. Re . 52, 1781-1790. 493 h p://dx.doi.o g/10.1016/j. se .2015.07.101. 494 MITECO, 2022. Es a egia de con ol del alga Rugulop e yx okamu ae en España. 495 h ps://www.mi eco.gob.es/es/biodi e sidad/publicaciones/pbl_ auna_ lo a_es a egi 496 a_R_okamu ae.aspx [accessed 28 h Sep embe 2022]. 497 O’B ien, B.J., Milligan, E., Ca e , J., Roy, E.D., 2019. In eg a ing anae obic co- 498 diges ion o dai y manu e and ood was e wi h cul i a ion o edible mush ooms o 499 nu ien eco e y. Bio esou . Technol. 285, 121312. 500 h ps://doi.o g/10.1016/j.bio ech.2019.121312. 501 Pa dilhó, S., Boa en u a, R., Almeida, M., Dias, J.M., 2022. Ma ine mac oalgae was e: 502 A po en ial eeds ock o biogas p oduc ion. J. En i on. Manage. 304, 114309. 503 h ps://doi.o g/10.1016/j.jen man.2021.114309. 504 Pa ón, D., Ga cía-Gómez, J.C., Lo ing, J., To es, A., 2022. Compos ing he in asi e 505 oxic seaweed Rugulop e yx okamu ae using i e in e eb a e species, and a mini- 506 e iew con compos ing mac oalgae. Was e Biomass Valo i. 507 h ps://doi.o g/10.1007/s12649-022-01849-z. 508 Piazzi, L., Bala a, D., 2009. In asion o alien mac oalgae in di e en Medi e anean 509 habi a s. Biol. In asions, 11, 193–204. h ps://doi.o g/10.1007/s10530-008-9224-3. 510 Raposo, F., Fe nández-Ceg í, V., De la Rubia, M.A., Bo ja, R., Béline, F., Ca ina o, C., 511 Demi e , G., Fe nández, B., Fe nández-Polanco, M., F igon, J.C., Ganesh, R., 512 Kapa aju, P., Koubo a, J., Méndez, R., Menin, G., Peene, A., Sche e , P., To ijos, 513 M., Uellendahl, H., Wie inck, I., de Wilde, V., 2011. Biochemical me hane po en ial 514 (BMP) o solid o ganic subs a es: e alua ion o anae obic biodeg adabili y using da a 515 om an in e na ional in e labo a o y s udy. J. Chem. Technol. Bio echnol. 86, 1088- 516 1098. h ps://www.doi.o g/10.1002/jc b.2622. 517 RD 1051/2022, de 27 de diciemb e, po el que se es ablecen no mas pa a la nu ición 518 sos enible en los suelos ag a ios. BOE 312, de 29 de diciemb e de 2022. BOE-A-2022- 519 23052. h ps://www.boe.es/busca /ac .php?id=BOE-A-2022-23052. 520 RD 47/2022, de 18 de ene o, sob e p o ección de las aguas con a la con aminación di usa 521 p oducida po los ni a os p oceden es de uen es ag a ias. BOE 17, de 20 de ene o de 522 2022, 5664-5684. BOE-A-2022-860. 523 h ps://www.boe.es/eli/es/ d/2022/01/18/47. 524 Rizzioli, F., Be asini, D., Bolzonella, D., F ison, N., Ba is a, F., 2023. A c i ical e iew 525 on he echno-economic easibili y o nu ien s eco e y om anae obic diges a e in 526 he ag icul u al sec o . Sep. Pu i . Technol. 306, 122690. 527 h ps://doi.o g/10.1016/j.seppu .2022.122690. 528 Rod íguez-Be bel, N., So ia, R., O ega, R., Lucas-Bo ja, M.E., Mi alles, I. (2022). 529 Ag icul u al Land Deg ada ion in Spain. In: The Handbook o En i onmen al 530 Chemis y. Sp inge , Be lin, Heidelbe g. h ps://doi.o g/10.1007/698_2022_924 531 San ana, I., Félix, M., Gue e o, A., Bengoechea, C., 2022. P ocessing and cha ac e iza ion o 532 bioplas ics om he in asi e seaweed Rugulop e yx okamu ae. Polym. 14, 355. 533 h ps://doi.o g/10.3390/polym14020355. 534 Sa eyn H, Ede P. End-o -was e c i e ia o biodeg adable was e subjec ed o biological 535 ea men (compos & diges a e): Technical Repo by he Join Resea ch Cen e o he 536 Eu opean Commission, h ps://op.eu opa.eu/en/publica ion-de ail/-/publica ion/66031c1b- 537 b894-4da3-b091-4e094e83 885/language-en; 2014 [accessed 09 Janua y 2023]. 538 Sempe e-Val e de, J., Os alé-Val ibe as, E., Maes e, M., González A anda, R., Bazai i, 539 H., Espinosa, F., 2021. Impac s o he non-indigenous seaweed Rugulop e yx 540 okamu ae on a Medi e anean co alligenous communi y (S ai o Gib al a ): The ole 541 o long- e m moni o ing. Ecol. Indic. 121, 107135. 542 h ps://doi.o g/10.1016/j.ecolind.2020.107135. 543 Simioni, T., Agus ini, C.B., De me , A., Gu e es, M., 2022. Use o anne y was e 544 anae obic diges a e ag icul u al e ilize : an analysis o oa g ow h and soil e ili y. 545 Was e Biomass Valo i. h ps://doi.o g/10.1007/s12649-022-01927-2. 546 Sobczak, M. F.; Oli o, C. J.; Gabbi, A. M.; Cha ao, P. S.; Heime dinge , A.; Sil a, J. H. 547 S. da; Pe ei a, L. E. T.; Ziech, M. F.; Rossa olla, G., 2005. E alua ion o an elephan 548 g ass pas u e mixed wi h black oa s managed unde ag o ecological p inciples in he 549 win e pe iod. Li es . Res. Ru al De ., 17 (6) a icle # 71 550 S eine , F., Zu o, A.M., Zoz, T., Zoz, A., Zoz, J., 2017. D ough ole ance o whea and 551 black oa c ops a ea ly s ages o seedling g ow h. Re is a de Ciências Ag á ias. 40(3): 552 576-586. h ps://doi.o g/10.19084/RCA16118h 553 Thompson, T.M., Young, B.R., Ba ou ian, S., 2020. Pelagic Sa gassum o ene gy and 554 e ilise p oduc ion in he Ca ibbean: a case s udy on Ba bados. Renew. Sus . Ene g. 555 Re . 118, 109564. h ps://doi.o g/10.1016/j. se .2019.109564. 556 Tieche , T. L., Tieche , T., Ce e a, C. A., Fe ei a, P. A. A., Nicoloso, F. T., So iani, H. 557 H., Tassina i, A., Pa anhos, J. T., De Con i, L., B une o, G. (2016). Physiological and 558 nu i ional s a us o black oa (a ena s igosa sch eb.) g own in soil wi h in e ac ion o 559 high doses o coppe and zinc. Plan Physiol. and Biochem., 106, 253-263. 560 doi:10.1016/j.plaphy.2016.05.015 561 T ipa hi, D.K., Singh, S., Singh, S. e al. Mic onu ien s and hei di e se ole in 562 ag icul u al c ops: ad ances and u u e p ospec i e. Ac a Physiol Plan 37, 139 (2015). 563 h ps://doi.o g/10.1007/s11738-015-1870-3 564 Ug eno ić, V., Popo ić, V., Ug ino ić, M., Filipo ić, V., Mačkić, K., Ljubičić, N., 565 Popo ić, S., Lakić, Ž. (2021). Black oa (a ena s igosa sch eb.) on ogenesis and 566 ag onomic pe o mance in o ganic c opping sys em and pannonian 567 en i onmen s. Ag icul u e (Swi ze land), 11(1), 1-14. 568 doi:10.3390/ag icul u e11010055 569 570 Table 1. Elemen al analysis and main physicochemical cha ac e is ics o he soluble ac ions o he di e en e ilize s s udied. Resul s a e based on 571 esh o d y ma e . Values a e gi en mean (s anda d de ia ion). 572 INS: Ino ganic nu i i e solu ion; ONS-T: O ganic nu i i e solu ion diges a e om Rugulop e yx okamu ae he mally p e ea men ; ONS-Z: O ganic nu i i e solu ion diges a e om R. okamu ae wi h zeoli e p e- ea men . 573 1d y bases 574 2uni s in mg/L 575 Pa ame e s ONS-Z ONS-T INS Pa ame e s 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) Conduc i i y (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) Alkalini y (mg CaCO3 kg-1) 6730 (30) 6800 (100) - C (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 a io 210:2.3:25 210:2.2:26 210:40:229 Cd (ppb)1 50 (20) 40 (20) C/N a io 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)