Academic Edi o s: Ca mine Lub i o
and Ma ia Rosa Di Cicco
Recei ed: 17 Janua y 2025
Re ised: 25 Feb ua y 2025
Accep ed: 4 Ma ch 2025
Published: 7 Ma ch 2025
Ci a ion: de la Lama-Cal en e, D.;
Mancilla-Ley ón, J.M.; Ga ido-
Mu illo, I.; Rojas-Ca illo, J.; Bo ja, R.;
Fe nández-Rod íguez, M.J. In luence
o Ni ogen Bioa ailabili y on he
Anae obic Co-Diges ion o he
Aegag opiles o he Seag ass
Posidonia oceanica wi h Di e en
Ni ogen-Rich Subs a es: P ocess
Pe o mance and Kine ic Analysis.
Appl. Sci. 2025,15, 2880. h ps://
doi.o g/10.3390/app15062880
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/
licenses/by/4.0/).
A icle
In luence o Ni ogen Bioa ailabili y on he Anae obic
Co-Diges ion o he Aegag opiles o he Seag ass
Posidonia oceanica wi h Di e en Ni ogen-Rich Subs a es:
P ocess Pe o mance and Kine ic Analysis
Da id de la Lama-Cal en e 1, Juan Manuel Mancilla-Ley ón 2,* , I án Ga ido-Mu illo 3, Ja ie Rojas-Ca illo 4,
Ra ael Bo ja 1and Ma ía José Fe nández-Rod íguez 2
1Ins i u o de la G asa (CSIC), Campus Uni e sidad Pablo de Ola ide, Edi icio 46. C a. de U e a, km. 1,
41013 Se illa, Spain; [email p o ec ed] (D.d.l.L.-C.); [email p o ec ed] (R.B.)
2Depa amen o de Biología Vege al y Ecología, Facul ad de Biología, Uni e sidad de Se illa,
41080 Se illa, Spain; [email p o ec ed]
3Es ación de Ecología Acuá ica Albe o I de Mónaco, Emp esa Me opoli ana de Abas ecimien o y
Saneamien o de Aguas de Se illa (EMASESA), 41003 Se ille, Spain; [email p o ec ed]
4Depa men o Ag onomy, Food, Na u al Resou ces, Animals and En i onmen , Uni e si y o Padua,
35122 Padua, I aly; ja ie [email p o ec ed]
*Co espondence: [email p o ec ed]
Abs ac : The shedding o lea es by Posidonia oceanica (P. oceanica) in au umn esul s in he
accumula ion o sho eline deb is, con ibu ing o signi ican economic, social, and en i on-
men al p oblems. Due o he lack o al e na i e solu ions, his was e biomass is disposed o
in land ills, incu ing an economic cos o he disposal p ocess. In he con ex o he ci cula
economy, anae obic diges ion (AD) can se e as a highly e icien biological al e na i e o
ea ing and alo izing was es wi h a high o ganic load. The aim o his esea ch was o com-
pa a i ely e alua e he pe o mance and kine ics o he AD o asho e P. oceanica biomass and
i s anae obic co-diges ion (co-AD) wi h di e en ni ogen- ich co-subs a es. To e alua e
he e ec o he ni ogen sou ce in he co-AD sys em, pep one, casein, syn he ic casein, u ea,
and he mic oalgae Raphidocelis subcapi a a we e used as co-subs a es in biome haniza ion
es s a a mesophilic empe a u e (
35 ±2◦C
). The lowes me hane yield was achie ed o
he sole AD o P. oceanica (
79 ±3 NL CH4kg−1VS
), while he highes yields we e ound
o he h ee co-ADs o P. oceanica wi h p o eins (i.e., pep one, casein, and syn he ic casein),
showing no signi ican di e ences among hem (
380 ±30 −420 ±30 NL CH4kg−1VS
).
Addi ionally, he i s -o de kine ics and he ans e ence unc ion model we e p o en and
allowed o adequa ely i ing he expe imen al esul s o me hane p oduc ion wi h ime.
Keywo ds: ma ine angiospe m; biome haniza ion es s; me hane; Raphidocelis subcapi a a
1. In oduc ion
A ound 100 million yea s ago, e es ial monoco yledonous plan s, al eady ep oduc-
ing h ough a lo al sys em, began o colonize ma ine ecosys ems, o ming complex and
well-s uc u ed habi a s. Among hese ecosys ems, seag asses ha e become one o he mos
p oduc i e and aluable a eas on ou plane , p o iding a wide ange o essen ial se ices:
i al habi a o housands o ish and in e eb a e species, ood, nu ien cycling, ca bon
sink, e c. [1].
Posidonia oceanica (P. oceanica) is a ma ine angiospe m species ha is endemic o he
Medi e anean Sea. I o ms ex ensi e seag ass in shallow wa e , co e ing abou 1.5% o
Appl. Sci. 2025,15, 2880 h ps://doi.o g/10.3390/app15062880
Appl. Sci. 2025,15, 2880 2 o 18
he Medi e anean basin and playing an impo an ole in coas al ecosys em dynamics [
2
,
3
].
These meadows p o ide oxygen, ood, shel e , and p o ec ion o many ma ine species.
Addi ionally, hese ex ensi e meadows o m a na u al ba ie ha educes wa e ene gy,
p o ec ing agains e osion and s abilizing he coas al p o ile [
4
]. Cu en ly, P. oceanica beds
a e in dange o disappea ance and ha e been lis ed as p io i y habi a s by he Eu opean
au ho i ies [5].
P. oceanica p esen s a s em, long lea es g ouped in bunches, and a oo sys em ha
enables he indi idual specimens o cling o he subs a e. I s lea es a e emo ed om
he hizome in au umn a e losing hei pho osyn he ic capaci y. These loosed lea es a e
eleased in la ge quan i ies along he sho e and once hey each he sand, hey s a o
decompose, causing se ious en i onmen al p oblems (me hane elease o he a mosphe e,
p oli e a ion o mosqui oes, p oblems wi h na iga ion and ishing, e c.) [
6
,
7
]. F esh deposi s
o P. oceanica deb is usually appea a he coas du ing au umn and win e due o he
wea he and sea condi ions, making his ma ine plan a enewable esou ce. Some lea es
keep hei ib ous s uc u e o long pe iods a e he easily biodeg adable componen s a e
comple ely decomposed. These ibe s a e highly esis an o he mechanical and chemical
ac ion o he seawa e . They a e olled up by he wa e mo emen a ound a hizomic
nucleus, as a s a ing poin o he o ma ion o sphe ical agglome a es, om 3 o 12 cm
in diame e (Figu e 1). These agglome a es, called aegag opiles, a e e en ually le asho e
whe e hey emain and a e sun-d ied [8–10].
Appl. Sci. 2025, 15, x FOR PEER REVIEW 2 o 18
se ices: i al habi a o housands o ish and in e eb a e species, ood, nu ien cycling,
ca bon sink, e c. [1].
Posidonia oceanica (P. oceanica) is a ma ine angiospe m species ha is endemic o he
Medi e anean Sea. I o ms ex ensi e seag ass in shallow wa e , co e ing abou 1.5% o
he Medi e anean basin and playing an impo an ole in coas al ecosys em dynamics
[2,3]. These meadows p o ide oxygen, ood, shel e , and p o ec ion o many ma ine spe-
cies. Addi ionally, hese ex ensi e meadows o m a na u al ba ie ha educes wa e en-
e gy, p o ec ing agains e osion and s abilizing he coas al p o ile [4]. Cu en ly, P. ocean-
ica beds a e in dange o disappea ance and ha e been lis ed as p io i y habi a s by he
Eu opean au ho i ies [5].
P. oceanica p esen s a s em, long lea es g ouped in bunches, and a oo sys em ha
enables he indi idual specimens o cling o he subs a e. I s lea es a e emo ed om he
hizome in au umn a e losing hei pho osyn he ic capaci y. These loosed lea es a e e-
leased in la ge quan i ies along he sho e and once hey each he sand, hey s a o de-
compose, causing se ious en i onmen al p oblems (me hane elease o he a mosphe e,
p oli e a ion o mosqui oes, p oblems wi h na iga ion and ishing, e c.) [6,7]. F esh de-
posi s o P. oceanica deb is usually appea a he coas du ing au umn and win e due o
he wea he and sea condi ions, making his ma ine plan a enewable esou ce. Some
lea es keep hei ib ous s uc u e o long pe iods a e he easily biodeg adable compo-
nen s a e comple ely decomposed. These ibe s a e highly esis an o he mechanical and
chemical ac ion o he seawa e . They a e olled up by he wa e mo emen a ound a hi-
zomic nucleus, as a s a ing poin o he o ma ion o sphe ical agglome a es, om 3 o
12 cm in diame e (Figu e 1). These agglome a es, called aegag opiles, a e e en ually le
asho e whe e hey emain and a e sun-d ied [8–10].
Al hough in p o ec ed na u al a eas, he collec ion o P. oceanica and i s deb is is o -
bidden due o i s bene i s on he conse a ion o he local habi a (Dec ee 132/2005, 2005),
in ce ain loca ions, excep ionally, he esponsible au ho i ies allow and encou age he
collec ion o his biomass. Fo ins ance, on beaches o Fo men e a (Spain), his was e is
widely used as bedding o animals, c ops, and as insula ion in adi ional houses. In I aly,
he use as a co-subs a e in compos ing is also allowed, p o ided ha i does no exceed
20% o he inal mix u e in e ms o we ma e [11]. The emo al o ons o P. oceanica
emains is a common p ac ice in Medi e anean coas s o allow o ec ea ional use in he
summe mon hs. Due o he lack o al e na i e solu ions, his biomass is usually disposed
o in land ills, which p o okes an en i onmen al p oblem as a signi ican amoun o o -
ganic ma e is emo ed om he ecosys em. Mo eo e , i ca ies an economic cos due o
he disposal p ocess [4,12].
Figu e 1. De ail o he o ma ion o he Posidonia oceanica aegag opiles.
Figu e 1. De ail o he o ma ion o he Posidonia oceanica aegag opiles.
Al hough in p o ec ed na u al a eas, he collec ion o P. oceanica and i s deb is is
o bidden due o i s bene i s on he conse a ion o he local habi a (Dec ee 132/2005,
2005), in ce ain loca ions, excep ionally, he esponsible au ho i ies allow and encou age
he collec ion o his biomass. Fo ins ance, on beaches o Fo men e a (Spain), his was e is
widely used as bedding o animals, c ops, and as insula ion in adi ional houses. In I aly,
he use as a co-subs a e in compos ing is also allowed, p o ided ha i does no exceed
20% o he inal mix u e in e ms o we ma e [
11
]. The emo al o ons o P. oceanica
emains is a common p ac ice in Medi e anean coas s o allow o ec ea ional use in he
summe mon hs. Due o he lack o al e na i e solu ions, his biomass is usually disposed
o in land ills, which p o okes an en i onmen al p oblem as a signi ican amoun o o ganic
Appl. Sci. 2025,15, 2880 3 o 18
ma e is emo ed om he ecosys em. Mo eo e , i ca ies an economic cos due o he
disposal p ocess [4,12].
In he con ex o he ci cula economy, anae obic diges ion (AD) can be a highly
e icien biological al e na i e o he ea men and alo iza ion o was es wi h a high
o ganic load [
13
]. The in e es in AD acili ies has g own o e he pas ew yea s because o
he high me hane con en (a ound 50–60%) o he gene a ed biogas, making i a enewable
ene gy al e na i e o ossil uels. Fu he mo e, he diges a e can be used as an o ganic soil
amende [
13
,
14
]. Howe e , he iabili y o hese p ocesses is highly dependen on he ype
o subs a e used. The e o e, be o e hei implemen a ion on an indus ial o pilo scale, i
is necessa y o assess he me hane po en ial o he biomass h ough labo a o y-scale assays,
such as he Biochemical Me hane Po en ial (BMP) es , which allows he iden i ica ion o
subs a e biodeg adabili y, ac i i y o he inoculum, and me hane yield, p o iding some
insigh s abou he po en ial oxici y o he subs a e o he AD p ocess due o he p esence
o ce ain me aboli es [14].
The use o P. oceanica o me hane p oduc ion by AD has been sca cely in es iga ed in
he li e a u e. In an ea lie s udy, he me hane yield ob ained was a he low (
62 LCH4 kg−1
VS
Pos
), mos likely, due o he high lignin con en (>30% VS) and he p esence o mine als
and sal s [
4
]. Acco dingly, he e ec o he mal p e- ea men (132
◦
C; 2.3 ba ; 30 min)
coupled wi h he emo al o sal s and he use o acid (HCl) du ing hyd olysis was also
assessed by De Sanc is and Di Iaconi [
4
]. Howe e , despi e he applied p e- ea men s,
he me hane p oduc ion did no imp o e and e en he yield was some imes educed
by up o 73%. Ano he al e na i e could be he use o o he subs a es o an anae obic
co-diges ion (co-AD) p ocess. The co-AD p ocess has impo an ad an ages, such as:
(i) a oiding he use o chemicals o ex a ene gy; (ii) he mixed eeds ock could be p epa ed
o p esen an op imal C/N a io (25–30); (iii) mixing wo co-subs a es can dilu e he
p esence o oxican s o he anae obic p ocess, such as sal s o phenols; and (i ) i could
enhance he alo iza ion o o he biomasses o he wise was ed [
15
,
16
]. Lignocellulosic
biomasses, such as P. oceanica, a e usually subjec ed o co-AD p ocesses due o he lack
o he necessa y amoun o ni ogen and, hus, he balanced C/N a io o allow a s able
pe o mance. Howe e , he bioa ailabili y o he ni ogen sou ce p o ided may in luence
he inal me hane p oduc ion, al hough no sound conclusions can be d awn due o he
signi ican lack o speci ic s udies [17].
This s udy aimed o assess he in luence o di e en ni ogen- ich subs a es, which
ep esen di e en ni ogen bioa ailabili ies, as co-subs a es o he co-AD p ocess o
aegag opiles (asho e biomass) o he ma ine phane ogam P. oceanica, h ough BMP es s.
This s udy ep esen s a b eak h ough in esea ch in o he alo iza ion o P. oceanica was e,
as i is he i s ime his biomass has been p oposed o be used as a co-subs a e in a co-AD
p ocess. In addi ion, i aims o u he in es iga e he impo ance o he ype o ni ogen
sou ce in anae obic co-diges ion sys ems. Ano he objec i e o his s udy was o assess he
kine ics o anae obic diges ion o single P. oceanica biomass and mix u es o his biomass
wi h di e en co-subs a es ich in ni ogen, such as pep one, casein, syn he ic casein
(composed o a mix u e o amino acids), u ea, and he mic oalga Raphidocelis subcapi a a.
2. Ma e ials and Me hods
2.1. Collec ion and Main Cha ac e iza ion o he Ma e ial
D ied P. oceanica aegag opiles we e collec ed du ing he win e season along a 150 m
ansec a a dis ance o 5–7 m om he coas line a ‘Playa de las Hue as’, Alican e (Spain).
The samples (n= 72) we e anspo ed o he labo a o y, whe e hey we e d ied a oom
empe a u e (25
◦
C) and low humidi y o se e al days un il a cons an weigh was eached.
Subsequen ly, all samples we e weighed and hei olume es ima ed, assuming ha all
Appl. Sci. 2025,15, 2880 4 o 18
samples had an ellipsoidal shape. A e biome ic analyses, he samples we e g ound o
ob ain a homogeneous subs a e wi h a pa icle size o up o 3.5 mm.
The ollowing me hods we e used o de e mine he speci ic physicochemical pa ame-
e s o he P. oceanica biomass: APHA [
18
] s anda d me hods (2540B and 2540E) we e used
o de e mine o al solids (TS), ola ile solids (VS), and mine al solids (MS). Raposo e al. [
19
]
and APHA [
18
] s anda d me hod (5220D) we e used o de e mine he o al chemical oxygen
demand (COD ) and soluble chemical oxygen demand (CODs), espec i ely. pH and o al
alkalini y (TA) we e measu ed using a pH me e (model C ison 20 basic). The APHA [18]
s anda d me hod (2320B) wi h i a ion o pH 4.3 was used o de e mine he TA. A LECO
T uSpec
®
Mic o Elemen al Analyze om he Leco Co po a ion (S . Joseph, MI, USA) was
used o elemen al analysis. Me al elemen s we e de e mined by ollowing he U.S. EPA
3051A me hod [
20
]; b ie ly, samples we e diges ed wi h a Ma s X ac ion mic owa e (CEM,
Ma hews, NC, USA) and analyzed by induc i ely coupled plasma mass spec ome y
(ICP-MS, AGILENT 7800, Mad id, Spain).
2.2. Biome haniza ion Tes s
BMP es s we e pe o med in 250 mL eac o s as desc ibed by De la Lama-Cal en e
e al. [
21
]. A mix u e o anae obic inocula om wo indus ial anae obic diges e s, one
om a b ewe y (Heineken, Se ille) and he o he om a was ewa e ea men plan
(
EMASESA—EDAR
El Cope o, Se ille, Spain), was used o BMP es ing. Based on he
ola ile solids ac ion o each inoculum, he mixing a io was 1:1. The inal inoculum mix-
u e had alues o 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 achie e an app op ia e C/N a io (25) he ollowing ni ogen- ich co-subs a es
we e used (Table S1): (i) syn he ic u ea (Pan eac); (ii) casein om bo ine milk (Sigma-
Ald ich); (iii) gela in pep one o po cine o igin (Cul imed); (i ) a mix u e o amino
acids p epa ed in he labo a o y simila o comme cial casein [
22
]; and ( ) mic oalga
Raphidocelis subcapi a a
, g own in A non- ich medium. The used ac onyms along he ex o
iden i y he di e en subs a es we e: P.o.: P. oceanica; P.o.:U .: P. oceanica—U ea; P.o.:Cas.:
P. oceanica
—Casein; P.o.:Pep.: P. oceanica—Pep one; P.o.:Syn.Cas.: P. oceanica—Syn he ic
casein made o amino acids; P.o.:R.s.: P. oceanica—Raphidocelis subcapi a a.
The main physicochemical cha ac e is ics o he di e en subs a es used a e shown
in Table 1. All he eac o s we e se up wi h an inoculum- o-subs a e a io (ISR) o 2
(based on VS), dis illed wa e , and a mic o-nu ien solu ion. The composi ion o he
solu ion o ace elemen s was as ollows: (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 we e
ca ied ou in iplica e and we e pe o med a a mesophilic empe a u e (35
±
1
◦
C). In
addi ion, 3 eac o s wi hou added subs a e we e included as blanks o de e mine he
endogenous me hane p oduc ion o he inoculum. Addi ionally, 3 eac o s we e ed wi h
cellulose (Fluka A icel PH- 101) and ea ed as he posi i e con ol o con i m he good
ac i i y o he inoculum. Be o e sealing, ni ogen gas was applied o all he eac o s o
ensu e anae obic condi ions. The biogas p oduced in he eac o s was passed h ough a
NaOH 2N solu ion capable o apping he CO
2
om he biogas mix u e. The emained
gas was measu ed olume ically and assumed o be me hane, as suppo ed by se e al
publica ions [
23
,
24
]. The expe imen al accumula i e me hane alues a e gi en in s anda d
condi ions o p essu e and empe a u e (101.325 kPa and 273.15 K). Finally, he BMP es s
we e s opped when me hane p oduc ion was less han 1% o he accumula i e alue o
3 consecu i e days.
Appl. Sci. 2025,15, 2880 5 o 18
Table 1. Main physicochemical cha ac e is ics o he di e en s udied co-subs a es. Values a e gi en
by means ±s anda d de ia ions.
Pa ame e s Posidonia
Oceanica U ea Casein Pep one Syn he ic Casein Raphidocelis subcapi a a
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
* Calcula ed s oichiome ically om he chemical o mula. TS: To al solids; VS: Vola ile solids; MS: Mine al solids.
2.3. Kine ic S udy
The i s -o de kine ic model and he ans e ence unc ion model (TFM) we e applied
o pe o m he kine ic s udy o me hane p oduc ion om he di e en co-diges ion mix u es.
Fu he in o ma ion ega ding he used equa ions can be ound in he Appendix A.
2.4. S a is ical Analysis
Analysis o a iance (ANOVA) and Tukey’s HSD es we e used o compa e he esul s
o he di e en ea men s and o de e mine whe he he e we e signi ican di e ences.
Be o e his, a no mali y es (Shapi o–Wilk es ) was pe o med. A p- alue
≤
0.05 was
conside ed s a is ically signi ican . Fo he calcula ion o biodeg adabili y, he heo e ical
BMP es alues we e calcula ed using he o mula desc ibed by Niel a e al. [
25
] (Table S1).
All he s a is ical analyses we e ca ied ou by using he SPSS S a is ics so wa e .29 (IBM,
Business Machines Co p, New Yo k, NY, USA).
3. Resul s and Discussion
3.1. Posidonia Oceanica Cha ac e iza ion
The collec ed samples showed a no mal dis ibu ion in e ms o bio olume (Table 2),
anging om 10 o 850 cm
3
, wi h an a e age alue o 300
±
200 cm
3
and a mode o 50 cm
3
.
The aegag opiles we e milled and he pa icle size dis ibu ion was measu ed. The esul s
showed ha due o he mo phology o he ma e ial, all he ibe s passed h ough he 3.5 mm
mesh sie e. As sugges ed by he speci ic li e a u e [
26
], he ac ion ela ed o a pa icle
size be ween 1 mm and 3.5 mm was selec ed o u he analysis and he BMP es s.
The esul s showed a VS/TS a io abo e 0.8, which is conside ed a sui able alue
o AD and is wi hin he same ange as o he lignocellulosic biomasses [
27
]. Al hough
he samples we e kep a oom empe a u e un il a cons an weigh , he main humidi y
loss occu ed in he collec ed si e. Thus, i could be conside ed ha he aegag opiles we e
sun-d ied be o e u he analysis and use. This low humidi y (12%) was simila o o he
biomasses d ied na u ally. Fo example, di e en a ie ies o So ghum bicolo p esen ed
humidi y alues wi hin he ange o 7–9% when sun-d ied [
27
]. Simila ly, he sun-d ied
biomass o he asho e mac oalgae Rugulop e yx okamu ae showed humidi y alues be ween
28 and 7% [28].
Appl. Sci. 2025,15, 2880 6 o 18
Table 2. Biome ic and physicochemical cha ac e iza ion o he aegag opiles o Posidonia oceanica.
Values a e gi en by means ±s anda d de ia ions.
Pa ame e s Posidonia oceanica
Physical Bio-Pa ame e s
A e age weigh (g) 9±8
A e age wid h (cm) 4±2
A e age leng h (cm) 5±2
A e age heigh (cm) 3±1
Bio olume (cm3)300 ±200
Q1 (cm3)61
Q3 (cm3)450
Range (cm3)10–850
Mode (cm3)50
Elemen al 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
C (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: 1s qua ile o bio olume no mal dis ibu ion; Q3: 3 d qua ile o bio olume no mal dis ibu ion; LOD: Limi
o de ec ion.
Rega ding he elemen al analysis, he C/N a io (153
±
5) indica ed a low N con en
(Table 1), which may unbalance AD pe o mance by a ec ing he bac e ial g ow h and
subsequen ly hei hyd oly ic ac i i y [
29
]. This esul suppo ed he idea o ca ying ou a
co-AD p ocess using N- ich co-subs a es. Seag asses a e also cha ac e ized by hei po en-
ial o abso b hea y me als p esen in he seawa e and ac as bioindica o s [
30
]. Howe e ,
he hea y me al up ake capaci y is s ongly linked o he species and he loca ion [
30
,
31
].
Rega ding P. oceanica, s ong ela ionships be ween he loca ion, he plan pa , he collec-
ion season, and he hea y me al composi ion ha e been p e iously epo ed [
31
,
32
]. The
esul s ob ained in his s udy a e wi hin he ange o hose p e iously epo ed, wi h e y
ew excep ions [
31
–
33
]. Fo example, Zn is commonly de ec ed in he 100–1000 ppm ange,
while in he sample used in he p esen s udy his elemen could no be de ec ed. Simila ly,
Fe and Pb ha e been epo ed in he 100–3000 ppm and 10–100 ppm anges, espec i ely,
howe e , in he p esen s udy, Fe was ound a a concen a ion o 1.6
±
0.1 ppm and Pb a
3±2 ppm [31–33]
. While he da a epo ed in he li e a u e is ela ed o heal hy and ali e
Appl. Sci. 2025,15, 2880 7 o 18
specimens o P. oceanica, in he p esen s udy, he P. oceanica samples we e collec ed asho e,
sun-d ied, and a e a na u al decomposi ion p ocess. These di e ences in he biomass
o igin could signi ican ly al e he elemen al p o ile as i has been ecen ly epo ed [
28
].
Fu he mo e, hea y me als a e s ongly linked o a good pe o mance o he AD p ocess.
While i is well known ha he p esence o ace elemen s is essen ial o e icien AD
pe o mance, a ce ain concen a ions, hese may dis up he p ocess by inhibi ing he
mic obiome ac i i y [
34
]. The mos s udied and common elemen s a e Cu, Fe, Ni, Cd, C ,
Pb, and Zn. Al hough he e is a signi ican amoun o da a suppo ing hei impac on AD,
he e is s ill a lack o consensus ega ding speci ic limi s o iden i y he po en ial oxici y o
he subs a es. Ne e heless, he alues epo ed in Table 2we e no conside ed o majo
conce n when compa ed wi h hose epo ed in he li e a u e [34–37].
3.2. Co-Subs a e Cha ac e iza ion
The co-subs a es used in he p esen s udy we e comme cial p oduc s o chemical
deg ee, excep o he mic oalga R. subcapi a a, which was cul i a ed in an A non- ich
medium. The solid ac ion o hese compounds ep esen ed mo e han 90% o he sample,
while o he mic oalgae, he humidi y ep esen s 42% o he o al weigh (Table 1). In any
case, he VS/TS a io was abo e 0.94 ac oss he boa d, sugges ing ha he o ganic ac ion
o each co-subs a e could be conside ed quan i a i ely equal. Mo eo e , he elemen al
analysis e ealed ha he C/N a io anged om 11.78 (R. subcapi a a) o 0.43 (u ea), con-
i ming he sui abili y o hese co-subs a es as eeds ock o he co-AD wi h lignocellulosic
biomasses, such as P. oceanica aegag opiles.
3.3. Diges a e Main Cha ac e is ics
The main physicochemical cha ac e is ics o he diges a es esul ing om he BMP
es s ca ied ou in his s udy a e shown in Table 3. The pH anged om 7.77 o 8.22
ac oss he boa d, excep o he P.o.:U . es which showed a highe alue (9.2). pH
alues be ween 6.5 and 8.5 sugges a s able AD pe o mance, al hough a pH unde 6.3
o o e 7.8 he me hanogenesis s age may su e ailu es and be pa ially hinde ed [
38
].
A highe pH may be ela ed o he o ma ion o ammonia du ing he hyd olysis s age,
which is inhibi o y o me hanogens. Aqueous ammonia (NH
4+
) is in equilib ium wi h ee
ammonia (NH
3
); howe e , his equilib ium shi s o ee ammonia o ma ion as he pH
inc eases. Concen a ions o aqueous ammonia be ween 1.7 and 14 g NH
3
L
−1
ha e been
epo ed as inhibi o y o me hanogens, especially o ace oclas ic species [
38
]. Howe e , he
o al ammonium ni ogen (TAN) limi s, om which inhibi o y e ec s a e obse ed, a e
highly ela ed o o he ac o s, such as subs a e, inoculum, en i onmen al condi ions, and
mic obial acclima ion [
39
]. In any case, i is gene ally conside ed ha TAN alues below
2500 mg N-NH
4+
L
−1
do no indica e signi ican inhibi ion [
26
]. Ano he pa ame e di ec ly
linked o he s abili y o he AD p ocess is he bu e capaci y o he sys em, measu ed as
he TA. I is gene ally accep ed ha TA, as a measu e o ca bona e bu e capaci y, should
be abo e 3000 mg CaCO
3
kg
−1
[
26
]. Howe e , he e is e y li le in o ma ion abou a TA
uppe limi , which may indica e an inhibi ion p ocess. In ac , in his s udy, he TA alue o
he diges a e de i ed om he inhibi ed P.o.:U . es was as high as 15,100 mg CaCO
3
kg
−1
.
Appl. Sci. 2025,15, 2880 8 o 18
Table 3. Physicochemical pa ame e s o he diges a es de i ed om he biome haniza ion es s.
Values a e gi en as means
±
s anda d de ia ions. Values wi h di e en le e s in he same ow mean
signi ican di e ences.
P.o. P.o.:U . 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
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
BMP h (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;U .: U ea; Cas: Casein; Pep.: Pep one; Syn.Cas.: Syn he ic casein made o amino acids;
R.s.:Raphidocelis subcapi a a.TS: To al solids; VS: Vola ile solids; MS: Mine al Solids; TA: To al alkalini y;
CODs: Soluble chemical oxygen demand; TAN: To al Ammonium Ni ogen; BMP
h
: Theo e ical Biochemical
Me hane Po en ial; BMPexp: Expe imen al Biochemical Me hane Po en ial; BD: Biodeg adabili y.
Based on he physicochemical cha ac e is ics, he assayed diges a es could be oughly
sepa a ed in o h ee main g oups: (i) P.o. and P.o.:R.s.; (ii) P.o.:Cas., P.o.:Pep., and
P.o.:Syn.Cas.; (iii) P.o.:U . The i s g oup is cha ac e ized by showing he lowes pH,
TA, and TAN alues, al hough wi hin accep able alues o he adequa e pe o mance
o he AD p ocess. The expe imen al me hane yield was signi ican ly lowe han hose
achie ed om he second g oup, despi e he highe heo e ical alue (Table S2). This
could be ela ed o he lignocellulosic cha ac e o sun-d ied P. oceanica. Mo eo e , he
ela i ely low COD alue sugges ed ha he main limi ing s ep was he hyd oly ic s age,
al hough he di e ences be ween bo h P.o. and P.o.:R.s. es s migh also indica e ha he
me hanogenesis was mo e limi ed in he P.o. es , based on he highe COD alue and he
lowe me hane yield. These di e ences migh be ela ed o he mo e balanced C/N a io o
he P.o.:R.s. es , which allowed o mo e consis en and s able bac e ial and a chaea g ow h.
Simila esul s ha e been obse ed ecen ly when o he lignocellulosic biomasses, such as
he mo-chemical p e ea ed ice husk, we e co-diges ed wi h cow manu e a di e en C/N
a ios (13.8–75.7) [
40
]. The esul s showed ha he highes me hane yield was achie ed
a a C/N a io o 24.7, meaning a yield imp o emen o 76% agains he con ol [
40
]. In
he p esen s udy, an imp o emen o 146% in me hane yield o P.o.:R.s. compa ed o he
mono-diges ion o he sole P. oceanica was obse ed (Figu e 2). The e alua ion o
P. oceanica
as eeds ock o an AD p ocess is sca ce in he li e a u e. Only wo s udies epo ed speci ic
me hane yields using P. oceanica as a subs a e. De Sanc is and Di Iaconi [
4
] epo ed
alues om 29 o 62 L CH
4
kg
−1
VS, depending on he ope a ing condi ions. These esul s
we e sligh ly lowe han ha ob ained in his s udy om he sole diges ion o P. oceanica
(
79 ±3 NL CH4kg−1VS
). Addi ionally, De Sanc is and Di Iaconi [
4
] also e alua ed he e -
ec o some p e ea men s, such as washing, he mal, o acidi ica ion wi h HCl, on me hane
p oduc ion. The highes alue (94 L CH
4
kg
−1
VS) was obse ed o he unwashed biomass
he mally p e ea ed a 132
◦
C o 30 min a e sample acidi ica ion wi h HCl. Again, in he
p esen s udy, he co-AD o P. oceanica wi h a N- ich co-subs a e, such as he mic oalga
R. subcapi a a, led o highe alues (194 ±4 NL CH4kg−1VS, Figu e 2).
Appl. Sci. 2025,15, 2880 9 o 18
Appl. Sci. 2025, 15, x FOR PEER REVIEW 9 o 18
Figu e 2. Expe imen al and accumula i e me hane o he diffe en biome haniza ion (BMP) es s
ca ied ou e sus ime. P.o.: P. oceanica; U : u ea; Cas.: casein; Pep.: gela ine pep one; Syn.Cas.:
amino acids mix u e simula ing a syn he ic casein; R.s.: Raphidocelis subcapi a a. Values a e gi en as
means ± s anda d de ia ions.
The second g oup was cha ac e ized by he highes me hane yield (380 ± 30−420 ± 30
NL CH
4
kg
−1
VS), wi h no signi ican diffe ences be ween he h ee esul s. Mo eo e , bi-
odeg adabili y abo e 75% may indica e an almos comple e deg ada ion o he subs a e
[41]. COD alues simila o hose ound in he i s g oup (Table 3) also indica e ha he e
was no an accumula ion o solubilized o ganic ma e , sugges ing ha bo h he hyd o-
ly ic and he me hanogene ic s ages ope a ed p ope ly. Only he P.o.:Pep. es showed a
signi ican ly highe COD alue; howe e , i also showed he highes BD (97%). Pep one is
a pa ially deg aded p o ein, which makes i much mo e easily diges ible and bioa ailable
o mic oo ganisms, p o iding a quick sou ce o ni ogen. This could sugges ha he sub-
s a e was comple ely solubilized, unlike he o he wo es s whe e some pa s o he sub-
s a es may ha e emained in ac o no biodeg aded. Due o i s complex s uc u e, casein
is less soluble in wa e and akes longe o be diges ed by mic oo ganisms. In biological
sys ems, mic oo ganisms need o b eak down casein in o simple componen s be o e hey
can me abolize i . The co-diges ion o complex subs a es, such as casein p o eins and lig-
nocellulose can c ea e a mo e s able en i onmen o anae obic mic oo ganisms, he eby
op imizing biogas p oduc ion. The hyd olysis o p o eins, pa icula ly complex ones, such
as casein, is likely o be synch onized wi h he hyd olysis o complex lignocellulose, al-
lowing o a mo e balanced elease o ni ogen and ca bon compounds. This synch onized
elease can main ain an op imal soluble C/N a io, which is c ucial o he p ope de el-
opmen o anae obic mic oo ganisms. The pa allel hyd olysis o ni ogenous and ca bo-
naceous compounds p omo es a mo e efficien diges ion p ocess, suppo ing he mic o-
bial communi y’s s abili y and inc easing me hane p oduc ion. This is in line wi h he
p inciples es ablished in anae obic diges ion, whe e p ope synch oniza ion o subs a e
deg ada ion is key o maximizing biogas yields. In any case and despi e he appa en good
pe o mance, pH, TA, and TAN analyses o he diges a es indica ed ha he sys ems we e
close o ailu e due o inhibi ion p ocesses. In ac , when compa ed wi h he i s g oup,
he TAN alues we e a ound 2- old highe , he pH was highe a leas by 0.5 uni s, and
he TA also inc eased up o 9000 mg CaCO
3
kg
−1
(Table 3). Al hough hese alues we e s ill
Figu e 2. Expe imen al and accumula i e me hane o he di e en biome haniza ion (BMP) es s
ca ied ou e sus ime. P.o.: P. oceanica; U : u ea; Cas.: casein; Pep.: gela ine pep one; Syn.Cas.: amino
acids mix u e simula ing a syn he ic casein; R.s.: Raphidocelis subcapi a a. Values a e gi en as
means ±s anda d de ia ions.
Thesecondg oupwascha ac e izedby hehighes me haneyield(
380 ±30−420 ±30 NL
CH
4
kg
−1
VS), wi h no signi ican di e ences be ween he h ee esul s. Mo eo e ,
biodeg adabili y abo e 75% may indica e an almos comple e deg ada ion o he sub-
s a e [
41
]. COD alues simila o hose ound in he i s g oup (Table 3) also indica e
ha he e was no an accumula ion o solubilized o ganic ma e , sugges ing ha bo h he
hyd oly ic and he me hanogene ic s ages ope a ed p ope ly. Only he P.o.:Pep. es showed
a signi ican ly highe COD alue; howe e , i also showed he highes BD (97%). Pep one is
a pa ially deg aded p o ein, which makes i much mo e easily diges ible and bioa ailable
o mic oo ganisms, p o iding a quick sou ce o ni ogen. This could sugges ha he
subs a e was comple ely solubilized, unlike he o he wo es s whe e some pa s o he
subs a es may ha e emained in ac o no biodeg aded. Due o i s complex s uc u e,
casein is less soluble in wa e and akes longe o be diges ed by mic oo ganisms. In biolog-
ical sys ems, mic oo ganisms need o b eak down casein in o simple componen s be o e
hey can me abolize i . The co-diges ion o complex subs a es, such as casein p o eins
and lignocellulose can c ea e a mo e s able en i onmen o anae obic mic oo ganisms,
he eby op imizing biogas p oduc ion. The hyd olysis o p o eins, pa icula ly complex
ones, such as casein, is likely o be synch onized wi h he hyd olysis o complex ligno-
cellulose, allowing o a mo e balanced elease o ni ogen and ca bon compounds. This
synch onized elease can main ain an op imal soluble C/N a io, which is c ucial o he
p ope de elopmen o anae obic mic oo ganisms. The pa allel hyd olysis o ni ogenous
and ca bonaceous compounds p omo es a mo e e icien diges ion p ocess, suppo ing
he mic obial communi y’s s abili y and inc easing me hane p oduc ion. This is in line
wi h he p inciples es ablished in anae obic diges ion, whe e p ope synch oniza ion o
subs a e deg ada ion is key o maximizing biogas yields. In any case and despi e he
appa en good pe o mance, pH, TA, and TAN analyses o he diges a es indica ed ha
he sys ems we e close o ailu e due o inhibi ion p ocesses. In ac , when compa ed wi h
he i s g oup, he TAN alues we e a ound 2- old highe , he pH was highe a leas by
0.5 uni s, and he TA also inc eased up o 9000 mg CaCO
3
kg
−1
(Table 3). Al hough hese
alues we e s ill wi hin he no mal limi s o good AD pe o mance, hey we e close o
he uppe limi s which indica es inhibi ion due o he p esence o ammonia. This may
Appl. Sci. 2025,15, 2880 16 o 18
Re e ences
1.
Mu phy, G.E.P.; Dunic, J.C.; Adamczyk, E.M.; Bi ick, S.J.; Cô é, I.M.; C is iani, J.; Wong, M.C. F om coas o coas o coas : Ecology
and managemen o seag ass ecosys ems ac oss Canada. Face s 2021,6, 139–179. [C ossRe ]
2.
Ruiz, J.M.; Guillén, J.E.; Ramos Segu a, A.; O e o, M.M. A las de las P ade as Ma inas de España Ins i u o Español de Oceanog a ía.
2015. A ailable online: h ps://www.iucn.o g/es/con en /a las-de-las-p ade as-ma inas-de-espana (accessed on
12 Oc obe 2023
).
3.
Azcá a e-Ga cía, T.; Beca-Ca e e o, P.; B un, F.G. Plan and meadow s uc u e cha ac e isa ion o Posidonia oceanica in i s
wes e nmos dis ibu ion ange. Di e si y 2023,15, 101. [C ossRe ]
4.
De Sanc is, M.; Di Iaconi, C. E alua ion o posidonia oceanica esidues as eeds ock o anae obic diges ion. Bio esou . Technol.
Rep. 2019,8, 100317. [C ossRe ]
5.
Eu opean Union. Council Di ec i e 97/62/EC o 27 Oc obe 1997 adap ing Di ec i e 92/43/EEC on he conse a ion o na u al
habi a s and o wild auna and lo a. O . J. Eu . Communi ies 1997,L305, 42–65.
6.
Hachana, Y.; Jebba i, A.; El Mejdoub, H.; Yous i, W.; Fo ina, R. Seag ass deb is as sou ce o ibe and bioac i e compounds in
eed o dai y goa s. In . J. Ag ic. Biol. 2021,26, 722–730. [C ossRe ]
7.
Scho n, S.; Ahme kamp, S.; Bullock, E.; Webe , M.; Lo , C.; Liebeke, M.; La ik, G.; Kuype s, M.M.M.; G a , J.S.; Milucka, J.
Di e ese me hylo ophic me hanogenic a chaea cause high me hane emissions om seag ass meadows. P oc. Na l. Acad. Sci.
USA 2022,119, e2106628119. [C ossRe ]
8.
Le eb e, L.; Compè e, P.; Léona d, A.; Plougon en, E.; Vandewalle, N.; Gobe , S. Medi e anean aegag opiles om Posidonia
oceanica (L) delile (1813): A i s comple e desc ip ion om mac oscopic o mic oscopic s uc u e. Ma . Biol. 2021,168, 37.
[C ossRe ]
9.
Le eb e, L.; Compè e, P.; Gobe , S. The o ma ion o aegag opiles om he medi e anean seag ass Posidonia oceanica (L) delile
(1813): Plan issue sou ces and colonisa ion by melanised ungal mycelium. Ma . Biol. 2023,170, 19. [C ossRe ]
10.
Sanchez-Vidal, A.; Canals, M.; de Haan, W.P.; Rome o, J.; Veny, M. Seag asses p o ide a no el ecosys em se ice by apping
ma ine plas ics. Sci. Rep. 2021,11, 254. [C ossRe ]
11.
Pa en e, A.; Se io, F.; Mon esano, F.F.; Mininni, C.; San ama ia, P. The compos o posidonia esidues: A sho e iew on a new
componen o soilless g owing media. ISHS Ac a Ho icul . 2014,1034, 291–298. [C ossRe ]
12.
Pa en e, A.; Mon esano, F.F.; Lomo o, A.; Guido, M. Imp o emen o beached Posidonia esidues pe o mance o compos ing.
En i on. Eng. Manag. J. 2013,12, 81–84.
13.
Ma a-Al a ez, J.; Dos a, J.; Rome o-Güiza, M.S.; Fonoll, X.; Peces, M.; As als, S. A c i ical e iew on anae obic co-diges ion
achie emen s be ween 2010 and 2013. Renew Sus . Ene gy Re . 2014,36, 412–427. [C ossRe ]
14.
Raposo, F.; De La Rubia, M.A.; Fe nández-Ceg í, V.; Bo ja, R. Anae obic diges ion o solid o ganic subs a es in ba ch mode: An
o e iew ela ing o me hane yields and expe imen al p ocedu es. Renew. Sus . Ene g. Re . 2012,16, 861–877. [C ossRe ]
15.
Ma a-Al a ez, J.; Macé, S.; Llab és, P. Anae obic diges ion o o ganic solid was es an o e iew o esea ch achie emen s and
pe spec i es. Bio esou . Technol. 2000,74, 3–16. [C ossRe ]
16.
Xu, J.; Wang, X.; Sun, S.; Zhao, Y.; Hu, C. E ec s o in luen C/N a ios and ea men echnologies on in eg al biogas upg ading
and pollu an s emo al om syn he ic domes ic sewage. Sew. Sci. Rep. 2017,7, 10897. [C ossRe ]
17.
Ka abi, M.; Ranjba , F.M.; Shahna az, B.; Seyedi, S. A comp ehensi e e iew on biogas p oduc ion om lignocellulosic was es
h ough anae obic diges ion: An insigh in o pe o mance imp o emen s a egies. Fuel 2023,340, 127239. [C ossRe ]
18.
APHA. S anda d Me hods o he Examina ion o Wa e and Was ewa e , 23 d ed; Ame ican Public Heal h Associa ion: Washing on,
WA, USA, 2017.
19.
Raposo, F.; de la Rubia, M.A.; Bo ja, R.; Alaiz, M. Assessmen o a modi ied and op imised me hod o de e -mining chemical
oxygen demand o solid subs a es and solu ions wi h high suspended solid con en . Talan a 2008,76, 448–453. [C ossRe ]
20.
USEPA. Me hod 3051A (SW-846): Mic owa e Assis ed Acid Diges ion o Sedimen s Sludges and Oils, 1s ed.; Uni ed S a es En i on-
men al P o ec ion Agency: Washing on, WA, USA, 2007.
21.
De la Lama-Cal en e, D.; Cube o, J.; Fe nández-Rod íguez, M.J.; Jiménez-Rod íguez, A.; Bo ja, R. Anae obic co-diges ion o
mic oalgae and indus ial was es: A c i ical and bibliome ic e iew. In P og ess in Mic oalgae Resea ch—A Pa h o Shaping
Sus ainable Fu u es; Zepka, Q., o Jacob-Lopes, E., Cos a Dep á, M., Eds.; In ech Open: London, UK, 2022; pp. 1–26.
22.
Go don, W.G.; Semme , W.F.; Cable, R.S.; Mo is, M. Amino acid composi ion o
α
-casein and
β
-Casein2. J. Am. Chem. Soc. 1949,
71, 3293–3297. [C ossRe ]
23.
Fe nández-Rod íguez, M.J.; Pun ano, N.F.; Mancilla-Ley ón, J.M.; Bo ja, R. Ba ch mesophilic anae obic co-diges ion o spen goa
s aw bedding and goa cheese whey: Compa ison wi h he mono-diges ion o he wo sole subs a es. J. En i on. Manag. 2021,
280, 111733. [C ossRe ]
24.
Casallas-Ojeda, M.; Meneses-Beja ano, S.; U ueña-A go e, R.; Ma molejo-Rebellón, L.F.; To es-Lozada, P. Techniques o
quan i ying me hane p oduc ion po en ial in he anae obic diges ion p ocess. Was e Biomass Valo i. 2022,13, 2493–2510. [C ossRe ]
25.
Niel a, A.; Cano, R.; Fe nández-Polanco, M. Theo e ical me hane p oduc ion gene a ed by he co-diges ion o o ganic ac ion
municipal solid was e and biological sludge. Bio echnol. Rep. 2015,5, 14–21. [C ossRe ] [PubMed]
Appl. Sci. 2025,15, 2880 17 o 18
26.
Hollige , C.; Al es, M.; And ade, D.; Angelidaki, I.; As als, S.; Baie , U.; Boug ie , C.; Bu i‘e e, P.; Ca balla, M.; de Wilde, V.; e al.
Towa ds a s anda diza ion o biome hane po en ial es s. Wa e Sci. Technol. 2016,74, 2515–2522. [C ossRe ] [PubMed]
27.
De la Lama-Cal en e, D.; Fe nández-Rod íguez, M.J.; Gandullo, J.; Desena, I.; de la Osa, C.; Fe ia, A.B.; Jiménez-Rod íguez, A.;
Bo ja, R. Valo iza ion o di e en land ace and comme cial so ghum (So ghum bicolo (L) moench) s aw a ie ies by anae obic
diges ion. Glob. Change Biol. 2022,15, 332–345. [C ossRe ]
28.
De la Lama-Cal en e D, Fe nández-Rod íguez MJ, Ga cía-Gómez JC, Bo ja R, Impac o na u al deg ada ion o he in asi e
alga Rugulop e yx okamu ae on anae obic diges ion: Hea y me al pollu ion and kine ic pe o mance. Ma . Pollu . Bull. 2023,
192, 115005. [C ossRe ]
29.
Khalid, A.; A shad, M.; Anjum, M.; Mahmood, T.; Dawson, L. The anae obic diges ion o solid o ganic was e. Was e Manag. 2011,
31, 1737–1744. [C ossRe ]
30.
Naznee, S.; Mish a, A.K.; Raju, N.J.; Mehmood, G. Coas al mac ophy es as bioindica o s o ace me als in he Asia’s la ges
lagoon ecosys em. Ma . Pol. Bull. 2022,178, 113576. [C ossRe ]
31.
Wa nau, M.; Leden , G.; Tema a, A.; Bouquegneau, J.M.; Jangoux, M.; Dubois, P. Hea y me als in Posidonia oceanica and
Pa acen o us li idus om seag ass beds o he no h-wes e n. Medi e . Sci. To al En i on. 1995,171, 95–99. [C ossRe ]
32.
Wa d, T.J. Tempo al a ia ion o me als in he seag ass Posidonia aus alis and i s po en ial as a sen inel accumula o nea a lead
smel e . Ma . Biol. 1987,95, 315–321. [C ossRe ]
33.
Öz ü k, R.Ç.; Gedik, K.; ¸Sahin, A.; Öz a ol, Y.; Mu lu, E. Compa a i e ace elemen ends in Posidonia oceanica and i s
sedimen s along he Tu kish-Medi e anean Sea coas . En i on. Sci. Pollu . Res. 2021,28, 31697–61407. [C ossRe ]
34.
Guo, Q.; Majeed, S.; Xu, R.; Zhang, K.; Kakade, A.; Kahn, A.; Ha eez, H.Y.; Mao, C.; Liu, P.; Li, X. Hea y me als in e ac wi h
he mic obial communi y and a ec biogas p oduc ion in anae obic diges ion: A e iew. J. En i on. Manag. 2019,240, 266–272.
[C ossRe ]
35.
an Hullebusch, E.D.; Guibaud, G.; Simon, S.; Lenz, M.; Yek a, S.S.; Fe moso, F.G.; Jain, R.; Dues e , L.; Roussel, J.; Guillon, E.; e al.
Me hodological app oaches o ac iona ion and specia ion o es ima e ace elemen bioa ailabili y in enginee ed anae obic
diges ion ecosys ems: An o e iew. C i . Re . En i on. Sci. Technol. 2016,46, 1324–1366. [C ossRe ]
36.
Al awashdeh, K.A.; Gul, E.; Yang, Q.; Yang, H.; Ba occi, P.; Fan ozzi, F. E ec o hea y me als in he pe o mance o anae obic
diges ion o oli e mill was e. P ocesses 2020,8, 1146. [C ossRe ]
37.
Zhang, J.; Qi, C.; Wang, Y.; Li, Y.; Han, T.; Gong, X.; Shan, M.; Li, G.; Luo, W. Enhancing biogas p oduc ion om li es ock manu e
in solid-s a e anae obic diges ion by so ghum- inega esidues. En i on. Technol. Inno . 2022,26, 102276. [C ossRe ]
38.
Sa ke , S.; Lamb, J.J.; Hjelme, D.R.; Lien, K.M. A e iew o he ole o c i ical pa ame e s in he design and ope a ion o biogas
p oduc ion plan s. Appl. Sci. 2019,9, 1915. [C ossRe ]
39.
Chen, Y.; Cheng, J.J.; C eame , K.S. Inhibi ion o anae obic diges ion p ocess: A e iew. Bio esou . Technol. 2008,99, 4044–4067.
[C ossRe ]
40.
Hassan, M.; Masud, S.F.B.; Anwa , M.; Zhao, C.; Singh, R.S.; Meh ya , E. Me hane enhancemen by he co-diges ion o he mo-
chemical alkali solubilized ice husk and cow manu e: Lignocellulosics decomposi ion pe spec i es. Biomass Con e s. Bio e in
2023,13, 13963–13975. [C ossRe ]
41.
Raposo, F.; Fe nández-Ceg í, V.; De la Rubia, M.A.; Bo ja, R.; Béline, F.; Ca ina o, C.; Demi e , G.; Fe nández, B.; Fe nández-
Polanco, M.; F igon, J.C.; e al. Biochemical me hane po en ial (BMP) o solid o ganic subs a es: E alua ion o anae obic
biodeg adabili y using da a om an in e na ional in e labo a o y s udy. J. Chem. Technol. Bio echnol. 2011,86, 1088–1098.
[C ossRe ]
42.
Rhee, C.; Pa k, S.G.; Yu, S.I.; Dalan ai, T.; Shin, J.; Chae, K.J.; Shin, S.G. Mapping mic obial dynamics in anae obic diges ion
sys em linked wi h o ganic composi ion o subs a es: P o ein and lipid. Ene gy 2023,275, 127411. [C ossRe ]
43.
Malik, R.K.; Singh, R.; Tau o, P. E ec o ino ganic ni ogen supplemen a ion on biogas p oduc ion. Biol. Was es 1987,21, 139–142.
[C ossRe ]
44.
Liu, L.; Du, Z.; Li, Y.; Han, R. E ec s o u ea addi ion on anae obic diges ion cha ac e is ics o hulless ba ley s aw p e ea ed
wi h KOH. J. Ma e . Cycles Was e Manag. 2023,25, 479–489. [C ossRe ]
45.
Guo, Y.; Xiao, F.; Yan, M.; Tang, S.; Duan, Z.; Sun, Y.; Li, Y. E ec o ammonia on anae obic diges ion: Focusing on ene gy low
and elec on ans e . Chem. Eng. J. 2023,471, 144638. [C ossRe ]
46.
Li, C.; S ömbe gc, S.; Liu, G.; Nges, I.A.; Liu, J. Assessmen o egional biomass as co-subs a e in he anae obic diges ion o
chicken manu e: Impac o co-diges ion wi h chicken p ocessing was e seag ass and Miscan hus. Biochem. Eng. J. 2017,118, 1–10.
[C ossRe ]
47.
Mo e i, E.R.; Ros on, D.M.; da Sil a, A.J.; Pe eda-Reyes, I. Biogas om aqua ic plan s: A bioene ge ics incen i e o cons uc ed
we lands usage. Heliyon 2023,9, e12537. [C ossRe ] [PubMed]
48.
Du a, J.C.F.; Passos, M.F.; da Sil a, T.F.; Mockai is, G. Py olysis and anae obic diges ion o he Ege ia Densa biomass in di e en
p e ea men condi ions o po en ial use as a subs a e in he gene a ion o biop oduc s. J. Wa e P ocess Eng. 2021,40, 101772.
[C ossRe ]
Appl. Sci. 2025,15, 2880 18 o 18
49.
Li, L.; Kong, X.; Yang, F.; Li, D.; Yuan, Z.; Sun, Y. Biogas p oduc ion po en ial and kine ics o mic owa e and con en ional he mal
p e ea men o g ass. Appl. Biochem. Bio echnol. 2012,166, 1188–1191. [C ossRe ]
50.
Fe nández-Rod íguez, M.J.; De La Lama-Cal en e, D.; Ga cía-González, M.; Mo eno-Fe nández, J.; Jiménez-Rod íguez, A.; Bo ja,
R.; Rincón-Llo en e, B. In eg al alo iza ion o wo-phase oli e mill solid was e (OMSW) and ela ed washing wa e s by anae obic
co-diges ion o OMSW and he mic oalga Raphidocelis subcapi a a cul i a ed in hese e luen s. J. Ag ic. Food Chem. 2022,70,
3219–3227. [C ossRe ]
51.
Sa o, S.; Hildaya , R.; Syaichu ozi, I. E ec o chemical p e ea men using sul u ic acid on biogas p oduc ion om wa e
hyacin h and kine ics. Renew. Ene g. 2019,132, 335–350. [C ossRe ]
52.
Sca celli, P.G.; Se ejo, M.L.; Paulo, P.L.; Boncz, M.Á. E alua ion o biome haniza ion du ing co-diges ion o he mally p e ea ed
mic oalgae and was e ac i a ed sludge, and es ima ion o i s kine ic pa ame e s. Sci. To al En i on. 2020,706, 135745. [C ossRe ]
53.
Wang, M.; Lee, E.; Dilbeck, M.P.; Liebel , M.; Zhang, Q.; E gas, S.J. The mal p e- ea men o mic oalgae o biome hane
p oduc ion: Expe imen al s udies, kine ics and ene gy analysis. J. Chem. Technol. Bio echnol. 2017,92, 399–407. [C ossRe ]
54.
Donoso-B a o, A.; Pe ez-El i a, S.I.; Fe nández-Polanco, F. Applica ion o simpli ied models o anae obic biodeg adabili y es s.
E alua ion o p e- ea men p ocesses. Chem. Eng. J. 2010,160, 607–614. [C ossRe ]
55.
Fe nández-Rod íguez, M.J.; De la Lama-Cal en e, D.; Jiménez-Rod íguez, A.; Bo ja, R.; Rincón-Llo en e, B. In luence o he cell
wall o Chlamydomonas einha d ii on anae obic diges ion yield and on i s anae obic co-diges ion wi h a ca bon- ich subs a e.
P ocess Sa . En i on. P o . 2019,128, 167–175. [C ossRe ]
Disclaime /Publishe ’s No e: The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual
au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .
