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
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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
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a Spanish Scien i ic Resea ch Council (CSIC) - Ins i u o de la G asa (IG), Depa men o
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Food Bio echnology, Campus Uni e sidad Pablo de Ola ide, Edi icio 46. C a. de U e a,
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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
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Se illa, 41080 Se ille, Spain.
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Abs ac
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The apid and agg essi e coloniza ion o he in asi e mac oalgae Rugulop e yx
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okamu ae on he sou he n coas o Spain has caused an unp eceden ed en i onmen al,
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heal h and economic impac . A p esen , he scien i ic communi y has ocused on he
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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
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iabili y o he R. okamu ae diges a e as bio e ilize o g ow h he Medi e anean o age
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species A ena s igosa (black oa ). Two anae obic diges a es, de i ed om he anae obic
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diges ion o he he mally p e- ea ed (120 ºC; 45 min) and he zeoli e-assis ed
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mechanically p e- ea ed in asi e mac oalgae, we e used in his s udy. Bo h diges a es
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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
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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
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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
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in he cul i a ion o black oa s, ob aining p oduc ion e iciency alues simila (diges a e
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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 .
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Keywo ds: Ci cula economy model; Anae obic diges a e; Zeoli e; Rugulop e yx
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okamu ae; A ena s igosa; Pho osyn he ic ac i i y.
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1. In oduc ion
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An in asi e alien species is ha which h ea ens o ad e sely impac he biodi e si y and
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he ela ed ecosys em o an a ea whe e i was ecen ly in oduced (EU, 2014). Among
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hese species, seaweeds had been poin ed as he main eason o he biodi e si y loss in
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he Medi e anean Sea (Coll e al., 2010), whe e mo e han 60 di e en mac oalgae ha e
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been in oduced (Piazzi and Bala a, 2009), o which only one specie is cu en ly included
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in he lis o in asi e alien species o Union conce n: he b own mac oalgae Rugulop e yx
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okamu ae (R.o.) (EU, 2022).
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The apid and agg essi e coloniza ion o his mac oalgae on he sou he n coas o Spain,
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mo e speci ically in he S ai o Gib al a , has cos a ound 1.2 million eu os pe season
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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,
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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
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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,
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he mac oalgae R.o. ha e been p oposed as a eeds ock o he eco e y o di e pene
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compounds wi h pha maceu ical applica ions; he p oduc ion o bioplas ics; compos ; o
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he gene a ion o biogas (Casal-Po as e al., 2021; Cue as e al., 2021; De la Lama-
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Cal en e e al., 2021; Pa ón e al., 2022; San ana e al., 2022).
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Thus, he anae obic diges ion (AD) o mac oalgae has been poin ed ou as a easible
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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
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ha he heo e ical biome hane p oduc ion o mac oalgae is wi hin he ange o 500 –
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1000 NLCH4 kgVS-1, wi h ac ual epo ed alues eaching up o 90% o i s heo e ical yield,
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which con i ms he po en ial o hese biomasses as eeds ock o an AD p ocess (Da ko
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e al., 2020; Pa dilhó e al., 2022). Addi ionally, he AD p ocess, besides he biogas,
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gene a es a diges a e en iched in mine als and o he mac o and mic onu ien s which has
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he po en ial o being used as a bio e ilize (Be gs and 2022; Thompson e al., 2020). In
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o de o educe he consump ion o mine al esou ces and ossil uels, he e is a end
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owa d he use o o ganic e ilize s as opposed o mine al e ilize s. I is known ha he
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use o diges a e as a bio e ilize has many bene i s on he soils whe e i is applied, helping
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o imp o e en i onmen al, social and p oduc i e p oblems (Liu and Lal, 2015).
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Fe nández-Rod íguez e al. (2021) epo ed ha he diges a e om he AD o oli e mill
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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
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he g ass Lolium igidum. Fu he mo e, Bus aman e e al. (2019) indica ed ha he
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composi ion o Rosma inus o icinalis L. was dependen on he e ilize used when
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applying he compos ed diges a e de i ed om he AD o ca le and pig slu y. Thus, he
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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
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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,
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a e he mos impo an pa ame e s ha could impac he plan quali y (Bus aman e e al.,
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2019; Fe nández-Rod íguez e al., 2021; Golo ko e al., 2022; Simioni e al., 2022). As
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mos o he po en ially oxic and ecalci an compounds a e p esen in he solid ac ion
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o he diges a e, some au ho s ha e e alua ed he use o bo h liquid and solid ac ions
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sepa a ely and in combina ion, being he mos used he liquid pa a di e en
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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).
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Ne e heless, he li e a u e shows ew s udies whe e anae obic diges a es ha e been used
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as soil amendmen s o bio e ilize s. I is known ha diges a e e ilize cha ac e is ics
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a e dependen on he subs a e used in he anae obic p ocess. Recen and cu en li e a u e
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on he use o diges a e as a e ilize o soil amendmen s mainly uses as subs a e ca le
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o pig slu y, mic oalgae, o ood was e as subs a e (Bus aman e e al., 2019; Fe nández-
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Rod íguez e al., 2022; Golo ko e al., 2022). Al hough some s udies ha e analyzed he
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diges a es in o de o compa e hem agains he applicable egula ions o hei use as
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e ilize s (De la Lama-Cal en e e al., 2023; Thompson e al., 2020), o he bes o ou
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knowledge, no ac ual expe imen s ha e been ca ied ou up o now in o de o assess he
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iabili y o anae obic diges a e de i ed om he AD o ma ine mac oalgae.
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The aims o his s udy was o assess, o he i s ime, he iabili y o he anae obic
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diges a es, de i ed om he ea men o he in asi e R. okamu ae, as bio e ilize du ing
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he g ow h o he Medi e anean o age species A ena s igosa (black oa ). The speci ic
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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
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a ailabili y on he g ow h, seedling es ablishmen , p oduc i i y o black oa and
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pe o mance o cul i a ed plan s, speci ically ega ding hei in luence on gas exchange
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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
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e ilize s and nu ien concen a ions in plan issues.
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2. Ma e ials and Me hods
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2.1. Anae obic diges a e
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De la Lama-Cal en e e al. (2023) ecen ly e alua ed he iabili y o R.o. was e biomass
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as he eeds ock o an AD p ocess in o de o p oduce enewable ene gy. In hei s udy,
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he highes me hane yields we e ob ained when he algal biomass was p e ea ed wi h a
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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
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35% o e he un ea ed mac oalgae. The anae obic diges a es de i ed om he AD
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p ocess o he wo p e ea men s we e il e ed and he liquid ac ions selec ed o his
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esea ch o p o ide he o ganic nu ien solu ions (ONS): i) om he mally p e ea ed R.o.
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a 120 ºC du ing 45 min (ONS-T); ii) om mechanically p e ea ed R.o. assis ed by
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zeoli e (ONS-Z).
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The anae obic diges a es used in his expe imen we e de i ed om biochemical me hane
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po en ial (BMP) es s ca ied ou a mesophilic empe a u e (35 ± 2 ºC) using R.o. as
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subs a e. The ini ial anae obic inoculum was collec ed om a nea by b ewe y indus y
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which used an up- low sludge blanke eac o as a sys em o ea he gene a ed
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was ewa e . Once he BMP assays inished, he selec ed diges a es we e cen i uged and
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he liquid phase was s o ed a -20 ºC un il u he use.
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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
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eac o s ollowing he same condi ions as p e iously epo ed. The accumula i e me hane
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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).
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128
2.1.1. Diges a e cha ac e isa ion
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The liquid ac ions o he diges a es used in he p esen wo k we e analyzed in
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acco dance wi h in e na ional s anda d me hods (De la Lama-Cal en e e al., 2023). A
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po able pH me e model Hach sensION MM150 was used o de e mine pH and
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conduc i i y. To al alkalini y (TA) was de e mined by using a pH me e model C ison by
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i a ion o pH 4.3 as shown in he 2320B s anda d me hod (APHA, 2017). The solid
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con en ( o al solids (TS) and ola ile solids (VS)) was analyzed ollowing he s anda d
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me hod 2540B & 2540E (APHA, 2017). C and N analyses o he lyophilized liquid
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ac ions we e pe o med by using a LECO CN828 Elemen al Analyze (Leco
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Co po a ion, USA). The U.S. EPA 3051A me hod (USEPA, 2007) was ollowed in o de
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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,
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AGILENT 7800, Spain).
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2.2. C op species selec ion
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The selec ed species o his s udy was A. s igosa Sch eb (also known as black oa ), a
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high-yielding annual g ass wi h apid es ablishmen in he ield and high o age
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p oduc ion. I is e y in e es ing o he clima ic and soil condi ions o he Ibe ian
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Peninsula due o i s excellen adap a ion (i g ows adequa ely in mode a e clima ic and
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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
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in he Ibe ian Peninsula, p esen s a high nu i ional alue and is easily diges ed by ca le
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(Ug eno ić e al., 2021). A. s igosa seeds we e supplied by he company Fe ip ado
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España.
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153
2.3. Expe imen al se -up
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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
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comme cial ino ganic nu i i e solu ion (INS) was also se up as a posi i e con ol and in
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o de o assess he iabili y o he p oposed bio e ilize s agains a comme cial ino ganic
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solu ion. The selec ed INS was he 50% Hoagland nu ien solu ion (Hoagland and
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A non, 1950) as i has been widely used in hese ypes o expe imen s (Fe nández-
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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
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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
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co espond o a educ ion o an inc ease o 30% o he a e age alues o p ecipi a ion o
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he cul i a ion a eas o he oa in he Sou h o Eu ope (Rod íguez-Be bel e al., 2022).
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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).
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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
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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
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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
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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
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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
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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,
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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
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Re e ences
383
384
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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)