Sus ainable Ma e ials and Technologies 31 (2022) e00369
A ailable online 29 No embe 2021
2214-9937/© 2021 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license
(h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
Impac o he lignin ype and sou ce on he cha ac e is ics o physical
lignin hyd ogels
Amaia Mo ales
a
, Jalel Labidi
a
,
*
, Pa icia Gull´
on
b
a
Chemical and En i onmen al Enginee ing Depa men , Uni e si y o he Basque Coun y UPV/EHU, Plaza Eu opa 1, 20018 San Sebas i´
an, Spain
b
Cen o de Apoio Cien í ico e Tecnol´
oxico ´
a In es igaci´
on, Uni e sidade de Vigo, Technology Pa k o Galicia- Tecnopole, CTC Building, 32901, San Cib ao das Vi˜
nas,
Ou ense, Spain
ARTICLE INFO
Keywo ds:
Lignin
Poly ( inyl alcohol)
Almond shells
Walnu shells
Physical hyd ogels
ABSTRACT
Mul iple na u al polyme s ha e been in es iga ed o he syn hesis o hyd ogels o he p esen da e, bu lignin has
demons a ed o be a p omising one o his pu pose o he mul iple ad an ages i o e s. Lignin can be isola ed
om lignocellulosic ma e ial such as nu shells, which a e usually unde alued was es, and would be a g ea s ep
o wa d on ci cula economy. Thus, in he p esen wo k, lignin was ex ac ed om almond and walnu shells
ollowing a single-s ep (deligni ica ion) and double-s ep (au ohyd olysis and deligni ica ion) bio e ine y scheme.
A e he chemical composi ion and s uc u es o hese lignins we e de e mined, hyd ogels we e syn hesized
combining hem wi h poly ( inyl alcohol) by he means o eeze- hawing cycles so as o s udy he in luence o
he di e en lignins on hei inal p ope ies. Addi ionally, he las hawing cycle o he syn hesis p ocess was
leng hened in o de o con i m p e ious assump ions abou i s e ec on he cha ac e is ics o he syn hesized
ma e ials. The ob ained esul s showed signi ican a ia ion be ween he 8 lignin samples, especially in hei
pu i y, molecula weigh s and o al phenolic con en s. The a ia ion on he lignins leaded o se e al hyd ogel
mo phologies, which di ec ly a ec ed hei p ope ies, p ima ily hei swelling capaci y, glass ansi ion em-
pe a u es and comp ession s eng hs. I was also demons a ed he g ea e ec ha he du a ion o he las
hawing had on he mo phology and, hence, on he cha ac e is ics o he ob ained ma e ials. The syn hesized
samples we e success ully employed as dye adso ben s and he e alua ion o hei an i ungal ac i i y showed
posi i e esul s in some o he samples, which could be applied o ood packaging.
1. In oduc ion
Syn he ic polyme -based i ems like hyd ogels ha e comple ely s i -
ed up ou e e yday li es. These ma e ials a e comp ised by idimen-
sionally en angled polyme ic chains ha exhibi high wa e e en ion
capaci y due o he mul iple hyd ophilic g oups hey con ain [1]. In
addi ion o hei g ea swelling p ope ies, hei in e nal s uc u e
oge he wi h hei mechanical p ope ies ha e made hem gain
conside able a en ion in he las yea s [2]. Hyd ogels can be a anged
acco ding o he ypes o c osslinks ha hei polyme ic chains p esen ,
which can be physical o chemical [2]. The chemical ones o en demand
he p esence o oxic and cos ly c osslinking eagen s whe eas he
physical ones do no . Thus, physical c osslinking makes he syn hesis
p ocess g eene and mo e economical [3]. Hyd ogels can be p esen ed in
many subs a es and o ms, which, oge he wi h he a o emen ioned
ea u es, make hem use ul in a wide ange o applica ions such as
ag icul u e and en i onmen , pe sonal hygiene o biomedicine, among
o he s [1].
The beginning o he 21s cen u y has been ma ked by a signi ican
economic boos coupled wi h high plas ic pollu ion which ha e leaded
o se e al i eco e able en i onmen al conce ns. In his con ex , he
possibili y o in oducing biodeg adable and enewable polyme s o
p oduce new ma e ials such as hyd ogels is nowadays one o he p in-
cipal goals o esea ch. Biopolyme s, which a e polyme s coming om
plan s, animals and mic oo ganisms, seem o be he solu ion o he
s a ed p oblem since, apa om helping o ace ecological p oblems [4],
hey would also con ibu e o sus ainabili y and ci cula economy [5].
Biopolyme s and o he co-p oduc s (e.g. bio uels and biochemicals)
can be ex ac ed om biomass h ough bio e ine y p ocesses [6].
Lignocellulosic biomass, o ins ance, has eme ged as a po en ial,
enewable and a ailable sou ce o biopolyme s and biochemicals. This
ype o biomass in ol es o es y, u ban and alimen a y esidues [7].
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (J. Labidi).
Con en s lis s a ailable a ScienceDi ec
Sus ainable Ma e ials and Technologies
jou nal homepage: www.else ie .com/loca e/susma
h ps://doi.o g/10.1016/j.susma .2021.e00369
Recei ed 5 July 2021; Recei ed in e ised o m 14 Sep embe 2021; Accep ed 26 No embe 2021
Sus ainable Ma e ials and Technologies 31 (2022) e00369
2
The main cons i uen s o lignocellulosic biomass a e cellulose, hemi-
celluloses and lignin, which can be employed as backbones on he
o mula ion o hyd ogels and o he ma e ials.
Among hese na u al polyme s, lignin is he mos boun i ul a oma ic
one on Ea h [8]. I s amo phous and complex s uc u e is p incipally
cons i u ed by a andom combina ion o h ee kinds o phenyl p opane
monome s (coni e yl, p-couma yl and sinapyl alcohols) connec ed
h ough se e al s able linkages [8]. The isola ion me hod in luences he
p ope ies o he ex ac ed lignin such as i s eac i i y. The ypical lignin
ex ac ion me hods include K a , sulphi e, alkaline and o ganosol
deligni ica ions [9]. Lignin is commonly ob ained as a by-p oduc o
pulp and pape indus ies; ne e heless, a small pe cen age o he en i e
quan i y (<2%) o lignin gene a ed e e y yea has an added- alue [8].
Hence, employing i o he syn hesis o hyd ogels, o ins ance, would
con ibu e o co e he u gen need o sa e and g eene al e na i e
ma e ials.
Almond and walnu shells a e an abundan was e all o e he wo ld.
The low pe cen age o edible ke nel in bo h nu s leads o an eno mous
p oduc ion o shells wi h no ecognized indus ial o comme cial
applica ion [7,10]. Howe e , hese shells belong o he lignocellulosic
biomass and a e e y ich in lignin and o he biopolyme s. The e o e,
hese esidues could be u he alo ised hough a bio e ine y s a egy so
as o use hem as inpu eeds ocks o he p oduc ion o added- alue
ma e ials [7,10].
In he las yea s, se e al esea che s ha e desc ibed he syn hesis o
hyd ogels wi h lignin om di e en sou ces and wi h di e en ex ac-
ion me hods [11,12]. None heless, a g ea pa o he published wo ks
epo s he p oduc ion o chemically c osslinked hyd ogels, gene ally
in ol ing he use o oxic c osslinke s as well as he need o emo ing
he esidual pa o i [3].
In his ligh , he objec i e o he p esen s udy was o analyse he
in luence o he ype o lignin and i s ea u es on he inal cha ac e is ics
o physically c osslinked lignin-hyd ogels. Fo his aim, lignin was
ex ac ed h ough alkaline and o ganosol deligni ica ions om walnu
and almond shells (wi h and wi hou p io hyd o he mal p e ea men )
[7,10] and hyd ogels we e o mula ed combining lignin and poly ( inyl
alcohol) ollowing he syn hesis ou e epo ed p e iously [13]. In
addi ion, hyd ogels we e also syn hesized h ough a a ia ion o he
ini ial syn hesis ou e, which consis ed in leng hening he las hawing
s age. In his way, he p ope ies o hese hyd ogels we e compa ed o
he o he ones, highligh ing he mos signi ican di e ences. The iso-
la ed lignins we e cha ac e ised ia pu i y, Py-GC/MS, HPSEC, FTIR,
XRD, TGA and TPC analysis. The lignin was e and swelling capaci y o
all he syn hesized hyd ogels we e measu ed and hey we e u he
cha ac e ised by FTIR, SEM, DSC and comp ession es s. Finally, he
adso p ion capaci y o he hyd ogels was e alua ed by employing
me hylene blue as wa e pollu an , and hei an i ungal capaci y agains
Aspe gillus nige was also s udied so as o explo e hei applicabili y in
ood packaging, o example.
2. Ma e ials and me hods
2.1. Ma e ials
Almond shells (AS) we e supplied by local a me s (Ma cona a ie y)
and walnu shells (WNS) we e kindly supplied by Olagi cide house
(Al zaga, Gipuzkoa). The shells we e milled and sie ed (pa icle size
be ween 2 and 1 mm) and s o ed in a da k and d y place a oom em-
pe a u e un il use.
Poly ( inyl alcohol) (M
w
=83,000–124,000 g/mol, 99 +% hyd o-
lyzed), phospha e bu e saline (PBS) able s, ypan blue solu ion and
me hylene blue powde we e pu chased om Sigma Ald ich. Sodium
hyd oxide (NaOH, analysis g ade, ≥98%, pelle s) was supplied by
PanReac Química SLU. Po a o dex ose aga (PDA) was acqui ed om
Scha lab S.L. and DMSO om Fishe Scien i ic S.L. All eagen s we e
employed as supplied.
2.2. Lignin ex ac ion
The ope a ional condi ions we e chosen in acco dance o p io
knowledge [7,10]. The alkaline and o ganosol deligni ica ion s ages
we e ca ied ou wice o each ype o shells: he i s one wi hou a
p io hyd o he mal ea men and he second one wi h a p io hyd o-
he mal ea men (au ohyd olysis). Table 1 summa izes he condi ions
employed o each lignin ex ac ion.
2.3. Hyd ogel syn hesis
Lignin-hyd ogels we e syn hesized acco ding o a p e ious wo k
[13]. The concen a ions o he blends (9.87 w.% PVA and 9.12 w.%
lignin) we e also de e mined on he basis o p e ious s udies. B ie ly, he
co esponding amoun o PVA (5.92 g) was added o 60 mL o a 2% (w/
w) NaOH aqueous solu ion and i was magne ically s i ed and hea ed o
90 ◦C. Once he PVA was dissol ed, lignin (5.47 g) was added while
s i ing un il comple el dissolu ion. The blends we e pou ed in o silicon
moulds. The in e nal bubbles we e elimina ed by ul asounds and he
emaining supe icial ai bubbles we e hen poked manually.
The blends we e ozen o 2.5 h a −20 ◦C and hey we e hen
hawed a 28 ◦C o 1.5 h. This cycle was epea ed i e imes, lea ing he
samples eezing o e nigh du ing he second and i h cycles. A e -
wa ds, he hyd ogels we e washed as i was done p e iously [13] in
o de o emo e he esidual lignin and NaOH. Finally, he hyd ogels
we e d ied a oom empe a u e.
Based on p io expe imen s (da a no shown), i was hough ha he
du a ion o he las hawing s ep could al e he ea u es o he syn he-
sized hyd ogels. Fo his eason, hyd ogels wi h a longe 5 h hawing
s age (24 h inside he hea e ) we e also p epa ed. These samples we e
agged as LC samples (long cycle) and he p e ious ones as SC (sho
cycle).
2.4. Cha ac e iza ion me hods
The cha ac e iza ion me hods and equipmen s used in his wo k bo h
o he ex ac ed lignins and he syn hesized hyd ogels a e desc ibed in
Table 2.
2.5. Me hylene blue adso p ion es s
The emo al o me hylene blue (MB) was in es iga ed acco ding o a
Table 1
Ope a ional condi ions o each o he ex ac ed lignins.
Abb e ia ion Desc ip ion Au ohyd olysis Deligni ica ion
AAL AS alkaline lignin – 121 ◦C, 90 min, 7.5
w.% NaOH, LSR 6:1
AOL AS o ganosol lignin –
200 ◦C, 90 min, 70/
30 ( / ) E OH/H
2
O,
LSR 6:1
WAL WNS alkaline lignin – 121 ◦C, 90 min, 7.5
w.% NaOH, LSR 6:1
WOL WNS o ganosol
lignin –
200 ◦C, 90 min, 70/
30 ( / ) E OH/H
2
O,
LSR 6:1
AAAL Au ohyd olysed AS
alkaline lignin
179 ◦C
(iso he mal), 23
min, LSR 8:1
121 ◦C, 90 min, 7.5
w.% NaOH, LSR 6:1
AAOL Au ohyd olysed AS
o ganosol lignin
179 ◦C
(iso he mal), 23
min, LSR 8:1
200 ◦C, 90 min, 70/
30 ( / ) E OH/H
2
O,
LSR 6:1
AWAL Au ohyd olysed
WNS alkaline lignin
200 ◦C (non-
iso he mal), LSR
8:1
121 ◦C, 90 min, 7.5
w.% NaOH, LSR 6:1
AWOL
Au ohyd olysed
WNS o ganosol
lignin
200 ◦C (non-
iso he mal), LSR
8:1
200 ◦C, 90 min, 70/
30 ( / ) E OH/H
2
O,
LSR 6:1
A. Mo ales e al.
Sus ainable Ma e ials and Technologies 31 (2022) e00369
3
p e ious s udy [13]. In b ie , i s , mul iple solu ions o ce ain con-
cen a ions (5–0.25 mg/L) o MB we e p epa ed. Then, hei abso bance
was measu ed a 665 nm by a V-730 UV-Jasco spec opho ome e , and
ela ing each concen a ion wi h i s co esponding abso bance a cali-
b a ion cu e was designed. Fo he adso p ion expe imen s, a solu ion
con aining 1 mg/L o MB was p epa ed, and a ound 0.5 g o d y samples
we e in oduced in 15 mL o his solu ion, keeping hem a oom em-
pe a u e and s a ic egimen o 24 h. The pe cen age o MB emo al was
also calcula ed by means o Eq. (1) a e he concen a ions o he ini ial
and inal dissolu ions we e de e mined by he calib a ion cu e:
P(%) = C0−Ceq
C0
⋅100 (1)
whe e P is he equilib ium adso p ion a e o he hyd ogel, C
0
is he
ini ial dye concen a ion, C
eq
is he dye concen a ion a equilib ium.
The adso p ion pe o mance was simila ly calcula ed by Eq. (2).
Qe(mgMB
gHG )=C0−Ceq
m⋅V(2)
whe e V is he o al olume o dye employed o each sample and m is
he d y weigh o he hyd ogel and he es o he a iables a e he same
as he ones de ined o Eq. (1) [13].
2.6. An i ungal s udies
The an i ungal ac i i y o he ex ac ed lignins and hyd ogels was
measu ed acco ding o he me hods p e iously epo ed by Salabe ia
e al. (2017) and Da Sil a e al. (2018) wi h sligh modi ica ions [14,15].
B ie ly, a e cul u ing he mould ungus Aspe gillus nige (CBS 554.65)
on Pe i dishes co e ed wi h PDA o 7 days a 25 ◦C ±1.5 ◦C in a cli-
ma ic chambe , some spo es we e dilu ed in a PBS dissolu ion and i s
concen a ion was adjus ed o a ound 1.21 ×10
6
spo es/ml using an
au oma ic cell coun e o he measu emen s (Cellome e ® Mini, Nex-
celom Bioscience LLC). Then, he p ocedu e was a ied acco ding o he
ype o sample.
The lignins we e i s ly dissol ed in DMSO (a ound 75–100 mg/mL)
and hen, 40
μ
L o he sample dissolu ion was pou ed on a Pe i dish
co e ed wi h PDA and he ungal s ain was sp ayed a ound. The blank
was pe o med wi h DMSO [15].
In he case o he hyd ogels, squa e po ions (app oxima ely 1 cm ×
1 cm) o each sample we e in oduced in o he PDA co e ed Pe i dishes
a e ha ing inocula ed hem wi h ungal s ain. The blank was pe -
o med using nea PVA hyd ogel po ions.
All he es s we e done by duplica e. A e 7 days o incuba ion a
25 ◦C ±1.5 ◦C, he lignins we e e alua ed isually using a nume ical
scale epo ed by da Sil a e al. (2018) in acco dance wi h ISO 846,
whe eas he hyd ogels we e ex ac ed om he aga and washed wi h 1
mL o PBS in o de o collec he spo e solu ion in o an Eppendo . A -
e wa ds, hese solu ions we e inged blue wi h 5
μ
L ypan blue solu ion
and a e shaking hem, hei spo e concen a ion was de e mined wi h
he abo emen ioned cell coun e [14]. The ungal g ow h inhibi ion
(FGI) o he samples we e calcula ed h ough he equa ion gi en by
Salabe ia e al. (2017) [14].
3. Resul s and discussion
3.1. Lignin cha ac e iza ion
3.1.1. Pu i y
So as o analyse he selec i i y o he lignin ex ac ion p ocesses, he
pu i y o he lignin samples oge he wi h he amoun o impu i ies ha
had p ecipi a ed wi h hem was de e mined by quan i a i e acid hy-
d olysis. The measu ed pu i ies o he ob ained lignins we e calcula ed
aking he Klason lignin and acid soluble lignin (ASL) in o accoun .
As shown in Table 3, he pu i y esul s we e, in gene al, highe o
o ganosol lignins han o alkaline ones. These esul s a e ela ed o he
selec i i y o o ganosol ex ac ions [16]. I was also obse ed ha he
o ganosol lignins wi hou au ohyd olysis (AOL and WOL) had pu i ies
o e 90%, whe eas he ones o alkaline lignins we e lowe han 59%.
Howe e , a e au ohyd olysis, he pu i ies o he la es inc eased up o
88–95% while o he o ganosol ones his inc emen was jus o a 3–5%.
The e o e, i can be said ha au ohyd olysis g ea ly imp o es he pu i y
o he ex ac ed lignins, especially in alkaline p ocesses, because o he
e ec i e p io elimina ion o hemicelluloses, which a e he main im-
pu i ies a e lignin ex ac ion as demons a ed by D´
a ila e al. (2017).
The ob ained esul s a e consis en wi h hose epo ed p e iously
[7,10].
3.1.2. Composi ion o he lignins
Py olysis-Gas Ch oma og aphy/Mass Spec ome y (Py-GC/MS) an-
alyses we e pe o med in o de o s udy he composi ion o he eigh
di e en lignins. The esul ing py og ams a e shown in Fig. 1. The
numbe ed peaks co espond o lignin-de i ed phenolic compounds wi h
Table 2
Cha ac e iza ion me hods and equipmen s o he ex ac ed lignins and syn-
hesized hyd ogels.
Analysis Sample Equipmen Re e ence
Pu i y Lignin
HPLC-RI/PDA (Jasco LC-Ne II/
ADC, 300 ×7.8 mm Aminex HPX-
87H column)
[21]
Acid soluble
lignin Lignin UV–Vis spec opho ome e (Jasco
V-630, JASCO) [18]
Composi ion Lignin
Py-GC/MS (Py: 5150 Py op obe,
GC: Agilen 6890, MS: Agilen
5973)
[21]
A e age
molecula
weigh
Lignin
GPC (JASCO LC-Ne ll/ADC,
de ec o : RI-2031Plus,Two Pola
Gel-M columns: 300 mm ×7.5 mm)
[18]
To al phenolic
con en (TPC) Lignin UV–Vis spec opho ome e (Jasco
V-630, JASCO) [18]
The mal
deg ada ion Lignin TGA/DTG (TGA/SDTA RSI analyse
851 Me le Toledo) [13]
C ys allini y Lignin XRD (Phillips X'Pe P o Au oma ic
mul ipu pose di ac ome e ) [13]
Chemical
s uc u e Lignin ATR-FTIR (Pe kinElme Spec um
Two FTIR Spec ome e ) [22]
Lignin Was e Hyd ogels UV–Vis spec opho ome e (Jasco
V-630, JASCO) [13]
Swelling Hyd ogels Swelling (%) = mswollen −md y
md y
x100 [13]
Mo phology Hyd ogels SEM (Hi achi S-4800, 5 kV, 20 nm
Au co e ing) [56]
Glass ansi ion
empe a u e Hyd ogels DSC (Me le Toledo DSC 822) [13]
Comp ession
es s Hyd ogels Mechanical es machine (Ins on
5967, 500 N load cell) [13]
Table 3
Summa y o he pu i y, GPC, TPC and TGA esul s o he isola ed lignins.
Lignin
sample
Pu i y
(%)
M
wa
(g/
mol)
M
nb
(g/
mol)
M
w
/
M
nc
TPC (%
GAE
d
)
T
maxe
(◦C)
AAL 58.2 4770 1109 4.3 13.8 307
AOL 90.4 8301 1072 7.8 15.6 389
WAL 49.4 4761 1054 4.5 10.6 295
WOL 92.7 6371 1246 5.1 16.5 388
AAAL 88.2 12,793 1528 8.4 33.1 355
AAOL 95.2 9020 1520 5.9 26.2 357
AWAL 95.7 16,670 1604 10.4 33.8 354
AWOL 95.2 7644 1359 5.6 27.2 365
a
M
w
: weigh a e age molecula weigh .
b
M
n
: numbe a e age molecula weigh .
c
M
w
/M
n
: polydispe si y index.
d
% GAE: pe cen age o gallic acid equi alen s.
e
T
max
: maximum deg ada ion empe a u e om TG/DTGA cu es.
A. Mo ales e al.
Sus ainable Ma e ials and Technologies 31 (2022) e00369
4
la ge a ea han 1% in a leas one o he lignin samples, and hei
iden i ica ion is displayed in Supplemen a y da a. The un agged peaks a
ea ly imes (3–7 min) mainly co esponded o deg ada ion compounds
om impu i ies. Fu u al, o ins ance, was one o hese compounds
coming om ca bohyd a es and i appea ed a minu e 5 [10]. These
impu i ies we e especially isible in AAL and WAL samples, which also
p esen ed he lowes pu i y alues in Table 3. F om minu e 23 on, he
majo i y o he compounds wi h la ge a eas we e a y acids, which a e
cha ac e is ic o nu s [17]. Thus, he iden i ied compounds we e
de ec ed in he ange o 7–23 min and hey we e g ouped based on he
o igin o hei a oma ic s uc u e (p-hyd oxyphenyl (H), guaiacol (G)
and sy ingol (S)) [7,10].
Among he iden i ied compounds, guaiacol (peak #5), 4-me hyl-
guaiacol (peak #9), sy ingol (peak #13) and 4-me hylsy ingol (peak
#16) we e he mos abundan ones in all he lignin samples, cons i u ing
he 15%, 13.7%, 11.8% and 10.8% o he agged compounds, subse-
quen ly. 4-e hylguaiacol and 4- inylguaiacol (peaks #11 and #12,
espec i ely) we e also p esen in la ge amoun s in all he samples. The
compounds coming om he p-hyd oxyphenyl uni such as phenol, p-
c esol and p-e hylphenol seemed o be mo e abundan in WNS lignins
(peaks #1, #3 and #4).
Rega ding he es ima ed S/G a ios, all he samples p esen ed mo e G
uni s excep o WOL and AWOL samples. This means ha almos all he
S/G a ios we e below 1. Howe e , an inc ease o his a io was obse ed
o he samples om he double-s ep p ocess, which will be u he
s udied so as o ind a consis en explana ion Howe e , hese alues a e
qui e di e en o he ones epo ed p e iously o AS and WNS lignins
[7,10], which could be ela ed o any possible modi ica ion on he
composi ion o he eeds ock o in he ex ac ion p ocess.
3.1.3. A e age molecula weigh analysis
F om he GPC analyses, based on he molecula weigh dis ibu ions
o he lignin samples, hei numbe -a e age (M
n
) and weigh a e age
(M
w
) molecula weigh s we e de e mined and hei polydispe si y in-
dexes (M
w
/M
n
) we e es ima ed. These alues a e displayed in Table 3.
The lignins a e au ohyd olysis p esen ed highe weigh and numbe
a e age molecula weigh s in all cases. In addi ion, he o ganosol lig-
nins wi hou au ohyd olysis p esen ed highe M
w
han he alkaline ones,
whe eas o he lignins wi h au ohyd olysis, he opposi e beha iou was
obse ed. A simila end was pe cei ed o he polydispe si y indexes. I
should also be no iced ha he M
w
o all he o ganosol lignins was e y
alike, whe eas o he alkaline lignins his di e ence was huge.
Howe e , and looking a he pu i y pe cen ages and p e ious wo ks
[18], hese esul s make us belie e ha he analysed lignin ac ions o
AAL and WAL we e no ep esen a i e o he whole lignin samples; in
o he wo ds, he e we e p obably bigge chains han 0.40
μ
m (which is
he po e size o he used il e ) ha we e excluded om he analysis.
These would explain such eno mous di e ence in M
w
epo ed o
alkaline lignins wi h and wi hou au ohyd olysis.
Some au ho s also epo ed small a ia ions on he molecula
weigh s o o ganosol lignins om solids wi h and wi hou an au ohy-
d olysis p ocess [19,20]. In addi ion, hey epo ed ha au ohyd olysis
pe mi ed he ob aining o lowe polydispe si y indexes o o ganosol
lignins [19,20], as happened in he p esen wo k.
Al hough i is ha d o ind p e ious esea ch on he compa ison o
alkaline deligni ica ion ea men s wi h and wi hou a p io hyd o-
he mal ea men , he esul s o alkaline lignins in Table 3 a e aligned
wi h hose epo ed in li e a u e [10,16,21].
3.1.4. To al phenolic con en (TPC)
The phenolic hyd oxyl g oups in he s uc u e o lignins a e usually
ela ed o i s an ioxidan capaci y as well as o i s sui abili y o he
syn hesis o new ma e ials [22]. The e o e, i is impo an o s udy he
TPC o he lignin samples. Looking a he esul s, i was obse ed ha he
alues o he lignin samples coming om he di ec deligni ica ion o
he aw ma e ial (AAL, AOL, WAL and WOL) we e conside ably lowe
han hose epo ed o he ones coming om he wo-s ep p ocess
(AAAL, AAOL, AWAL and AWOL), which could be ela ed o hei pu-
i ies [22]. I was also app ecia ed ha AAL and WAL p esen ed sligh ly
lowe TPC alues han AOL and WOL, and hey we e aligned wi h he
esul s ob ained by Ga cía e al. (2012, 2017) and Sequei os e al. (2014)
o soda lignin [23–25]. Ne e heless, he TPC o o ganosol lignins
(AOL and WOL) we e signi ican ly lowe han hose epo ed by o he
au ho s [23,24,26]. As commen ed, he lignin samples ob ained a e a
p io au ohyd olysis p ocess, p esen ed signi ican ly g ea e pe cen ages
o GAE (26.2–33.8% GAE). A compa able beha iou was epo ed by
D´
a ila e al. (2019) o alkaline lignin ex ac ed om p e- ea ed and
non- ea ed ine shoo s [22], sugges ing ha a p io hyd o he mal
ea men can be c ucial o ob ain high o al phenolic con en s in lignin.
In his case, alkaline lignin samples (AAAL and AWAL) p esen ed highe
TPC alues han he ones ob ained o o ganosol lignins. These esul s
a e in ag eemen wi h he ones epo ed p e iously o almond shells
[10]. O he au ho s pu i ied lignins coming om he di ec deligni i-
ca ion o he eeds ock wi h an acid hyd olysis; none heless, hey e-
po ed much lowe GAE pe cen ages in he pu i ied samples han in he
o iginal ones [23,24,27]. This ac also suppo s he idea o subjec ing
he aw ma e ial o a hyd o he mal p e- ea men i high TPC alues
wan o be achie ed.
3.1.5. The mal deg ada ion analysis (TGA)
The he mal s abili y o lignins is also an impo an cha ac e is ic o
ake in o accoun , especially o hei use in he p oduc ion o composi e
ma e ials. Thus, he he mal deg ada ion o he ex ac ed lignins was
s udied. The TGA cu es a e shown in Supplemen a y da a. The
maximum deg ada ion empe a u es o he samples a e displayed in
Table 3. All he samples had a common ini ial deg ada ion s ep below
100 ◦C, co esponding o mois u e e apo a ion. The second deg ada ion
s ep was he maximum deg ada ion s age o all he samples. Howe e ,
he empe a u es di e ed depending on he lignin ex ac ion. In ac , o
he alkaline lignins ob ained hough a single-s ep p ocess (AAL and
WAL), he maximum deg ada ion happened a ound 300 ◦C, whe eas o
he o ganosol lignins his empe a u e was signi ican ly highe
(≈390 ◦C). This could be asc ibed o hei high amoun o impu i ies as
well as o he ac ions wi h low molecula weigh [21,28], as epo ed
in Sec ion 3.1.3. The lignins coming om he double-s ep p ocesses
p esen ed simila maximum deg ada ion empe a u es (354–365 ◦C),
which we e wi hin he ones epo ed o single-s ep p ocess lignins and
we e in acco dance wi h p e ious esul s [10]. In his empe a u e
Fig. 1. Py og ams o he ex ac ed lignins.
A. Mo ales e al.
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5
ange, he scission o β–O–4 e he bondages end o occu , ollowed by
he di ision o C
–
C linkages and a oma ic ings [28].
A hi d deg ada ion s age was obse ed a ound 420 ◦C, al hough his
empe a u e was again lowe o AAL and WAL (≈390 ◦C) samples and
highe o AOL and WOL samples (≈470 ◦C). The la es hen p esen ed a
cons an weigh loss un il 37% o hei ini ial weigh . The es o he
samples p esen ed a ou h deg ada ion s ep a ound 700 ◦C, leading o a
inal esidue be ween 20 and 29% o hei ini ial weigh . This las s age
could be ela ed o he deme hoxyla ion o condensa ion eac ions o he
ola ile p oduc s o lignin [28], and he amoun s o esidue we e aligned
wi h hose epo ed p e iously [10].
3.1.6. C ys allini y
The XRD pa e ns o he lignin samples a e depic ed in Fig. 2. As
shown, all he o ganosol lignins and he alkaline ones coming om he
double-s ep p ocesses (AAAL and AWAL), p esen ed he ypical b oad
signal a ound 2θ =22◦, ep esen ing i s amo phous s uc u e [29]. AAL
and WAL samples p esen ed na owe peaks a he same di ac ion
angle. Mo eo e , hey also p esen ed sha p signals a 11, 12, 19, 25 and
31◦, which a e mo e cha ac e is ic o he c ys alline domains o cellulose
[30,31] and hemicelluloses [32]. The e o e, an impo an p esence o
impu i ies in hese lignins was again con i med by XRD analysis, leading
o a modi ica ion on he o de ed domains.
3.1.7. Chemical s uc u e
The main unc ional g oups o he isola ed lignins we e de e mined
by FTIR echnique (Figu e 3). Al hough all he eco ded spec a p e-
sen ed he ypical lignin bands [7,22,26], he in ensi ies o some o hem
changed om sample o sample. Some o hese a ia ions we e obse ed
in he ange 2827–2998 cm
−1
, which co esponded o he C
–
H (CH
3
,
CH
2
and CH) s e ching ib a ion o lignin and polysaccha ides [19],
and we e mo e no able o alkaline lignin samples, p obably due o he
lowe elimina ion o suga s du ing he au ohyd olysis s ep. Be o e he
oo p in ange, a 1650 cm
−1
, a peak co esponding o conjuga ed C
–
–
O
s e ching ib a ion [19] was de ec ed o he single-s ep lignins, bu i
disappea ed a e he au ohyd olysis s ep. Al hough some au ho s ha e
p e iously ela ed his band o alkaline p ocesses [33], i may be
a ibu ed o he p esence o impu i ies in AAL, WAL, AOL and WOL
lignins. On he oo p in ange (1500–600 cm
−1
) he main changes we e
seen on he in ensi y o he bands. AAL and WAL samples, o ins ance,
p esen ed a clea dec ease on he in ensi y o he bands asc ibed o he
condensed sy ingil o guaiacyl uni b ea hing (1325 cm
−1
) [34,35] and
a oma ic me hyl e he s o lignin (1215 cm
−1
) [19]. Con e sely, he
peaks a 1157, 1080, 975 and 896 cm
−1
, co esponding o C
–
O
s e ching ib a ion in es e g oups [34,35], C
–
O de o ma ion in sec-
onda y alcohol and alipha ic e he s,
–
HC
–
–
CH ou -o -plane and C
–
H
de o ma ion ib a ions [35], subsequen ly, go in ensi ied in he a o e-
men ioned samples. The emaining bands we e simila o all he lignin
samples, con i ming he exis ence o sy ingyl and guaiacyl uni s in all
he lignins, al hough hei a ios we e di e en , as demons a ed in
Sec ion 3.1.2.
3.2. Hyd ogel cha ac e iza ion
3.2.1. Lignin was e
The lignin was e o he samples was analysed so as o de e mine hei
inal lignin con en . The esul s a e shown in Table 4. Fo he samples
made om alkaline lignin, he ones con aining lignins om he single-
s ep p ocesses (AA and WA) p esen ed highe lignin was es han he
ones con aining lignins coming om double-s ep p ocesses (AAA and
AWA). A simila end was obse ed o o ganosol lignins om WNS
(WO and AWO), whe eas o he ones coming om AS (AO and AAO) he
epo ed beha iou was he opposi e. This migh be ela ed o he
polidispe si y o he lignins, since excep o AOL and AAOL, he poly-
dispe si y o he es o he lignins inc eased om single o double-s ep
lignins. This ac sugges s ha highes molecula weigh lignin chains
migh ha e been able o in e ac wi h he PVA ma ix, whe eas lowes
molecula weigh ac ions we e elimina ed. Howe e , u he s udies
should be done in o de o e i y his s a emen . Mo eo e , lowe was e
pe cen ages we e accoun ed o AS lignins han o WNS lignins in he
case o he single-s ep lignins (56.2–66.5% s. 60.0–68.5%), while o
he double-s ep lignins he con a y was app ecia ed (44.2–59.9% s.
59.6–71.1%). These alues a e in g ea acco dance wi h hose epo ed
p e iously [13]. I should also be no ed ha among he single-s ep lignin
con aining samples he ones wi h alkaline lignin showed g ea e lignin
was es ha he ones con aining o ganosol lignin, whe eas he samples
wi h double-s ep lignins exhibi ed he in e se end.
In addi ion, he samples wi h a long las hawing s ep, in gene al,
p esen ed lowe lignin was e alues, sugges ing ha he leng hening o
he las cycle may ha e enhanced he in e ac ions be ween he PVA and
he lignin. Fu he mo e, he samples wi h single-s ep alkaline WNS
lignin (WA) showed a signi ican educ ion on hei lignin was e. On he
con a y, he samples wi h double-s ep alkaline WNS lignin (AWA)
p esen ed much g ea e lignin was e alues han hose epo ed be o e.
I can also be said ha , excep o AWA sample, all he alkaline lignin
con aining hyd ogels los less lignin han he ones con aining o ganosol
lignin.
3.2.2. Swelling
As he swelling capaci y o he hyd ogels is de e mining o assessing
hei applica ions, his p ope y was measu ed o all he samples and i
is displayed in Fig. 4.
The samples wi h sho las hawing cycle p esen ed swelling alues
be ween 336 and 505%, which co esponded o AWO and WA samples,
espec i ely. Mo eo e , he samples con aining alkaline lignin showed
in all cases highe swelling capaci ies, which is in acco dance wi h he
da a epo ed p e iously [13], which could be ela ed o he a achmen
o he ac ions wi h highes molecula weigh s o he ma ix. Compa ing
he a o emen ioned samples wi h he ones wi h a long las hawing
s age, a huge enhancemen o his p ope y was clea ly seen. The mos
signi ican imp o emen (80% mo e ega ding i s p io swelling abili y)
was seen on AWO sample, which was he one ha had p e iously p e-
sen ed he lowes swelling capaci y. On he con a y, he sample ha
exhibi ed he sligh es enhancemen was AWA, whose swelling abili y
was jus imp o ed abou 22%. Agudelo e al. (2018) s udied he in lu-
ence o a ious syn hesis pa ame e s such as he ime o he eeze-
hawing cycles on he swelling capaci y o nea PVA hyd ogels [36].
Acco ding o hei wo k, he excessi e leng hening o he hawing cycles
Fig. 2. XRD pa e ns o he ex ac ed lignins.
A. Mo ales e al.
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6
leads o he dissolu ion o he o med c ys alli es, which educes o hei
quan i y and size and, hence, also he c osslinking densi y and he
swelling capaci y o he hyd ogel [36]. Thus, as in he p esen wo k he
leng hened hawing s ep was he las one, he mol en c ys alli es migh
ha e e apo a ed, educing he c osslinking densi y o he samples and
esul ing in a no able enhancemen o hei swelling capaci y. Ne e -
heless, his phenomenon should be u he in es iga ed in o de o ind
a mo e eliable explana ion.
I should also be men ioned ha he samples con aining lignins
coming om single-s ep p ocesses, p esen ed highe swelling capaci ies
han he ones coming om he double-s ep p ocesses, p obably due o
he in e ac ions o hei impu i ies wi h he polyme ic ma ix. Al hough
Wu e al. (2019) ela ed he swelling p ope ies o he con en o
phenolic hyd oxyl g oups and o he molecula weigh o he employed
lignins, hei hypo hesis would no explain he beha iou o he
hyd ogels in his wo k [12]. In ac , he samples con aining lignin wi h
he lowes o al phenolic con en s and lowes a e age molecula weigh s
(AA and WA) p esen ed he highes swelling capaci ies. Howe e , his
migh suppo he p e ious s a emen abou he non- ep esen a i e
a e age molecula weigh s de e mined o hese lignins (Sec ion 3.1.3)
and also he one abou he in e ac ions wi h non-lignin componen s
(Sec ion 3.2.1), since hese samples p esen ed high lignin was es.
3.2.3. Mo phology
The mo phology o he syn hesized hyd ogels was s udied by Scan-
ning Elec on Mic oscopy (SEM). The co esponding mic og aphs o he
samples con aining AS lignins a 500×and 5000×magni ica ions a e
shown in Fig. 5 and he ones o WNS lignin samples in Supplemen a y
da a.
A i s sigh , i was seen ha all he hyd ogels p esen ed di e en
appea ances. Al hough all he samples p esen ed po ous s uc u es a
di e en le els, hei dis ibu ion, size and densi y was dis inc . In ac ,
all he samples ha co esponded o hyd ogels con aining lignins om
single-s ep p ocesses we e qui e simila and p esen ed a highly po ous
honeycomb s uc u e, as expec ed o lignin con aining PVA hyd ogels
[13,37–39]. Thei po e sizes and dis ibu ions we e qui e homogeneous,
bu he walls be ween he mic o oids we e smoo he and mo e b i le in
he case o AA and WA samples han in AO and WO samples, which
p esen ed hicke walls. These s uc u es we e esponsible o hei high
wa e abso p ion capaci ies [40] and could ha e been c ea ed due o he
in e ac ions o PVA wi h he highes molecula weigh lignin ac ions.
When lignins om he double-s ep p ocesses we e employed, he
syn hesized hyd ogels displayed much dense and con inuous s uc u es
wi h ha dly ecognizable po es, which may be ela ed o a highe
Fig. 3. FTIR spec a o he ex ac ed lignins.
Table 4
Lignin was e (%) o he analysed sho (SC) and long (LC) las hawing s ep
samples.
Sample SC (%) LC (%)
AA 66.5 ±2.9 48.5 ±3.0
AO 56.2 ±2.4 53.9 ±3.4
WA 68.5 ±0.7 26.0 ±6.4
WO 60.0 ±1.8 53.9 ±2.1
AAA 59.6 ±2.8 36.3 ±2.0
AAO 71.1 ±3.0 48.1 ±3.3
AWA 44.2 ±1.6 74.3 ±3.7
AWO 59.9 ±4.0 51.7 ±2.2
Fig. 4. Swelling capaci y o he syn hesized hyd ogels wi h sho (SC) and long
(LC) las hawing s eps.
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c osslinking densi y wi h hese lignins due o hei highe con en s in
phenolic hyd oxyl g oups [12]. Rachip e al. (2013) also epo ed a -
iable hyd ogel mo phologies acco ding o he
–
OH and
–
COOH g oups
in lignin [41]. These mic os uc u es would explain he d op on he
swelling abili y o he p esen samples compa ed o he a o emen ioned
ones.
I was also app ecia ed ha when he las hawing s ep was leng h-
ened, he e was an e iden c ea ion o mac o po es in all cases. As
a o emen ioned, his migh be asc ibed o he e apo a ion o mol en
c ys alli es du ing he las s ep o he syn hesis, which pe mi ed he
o ma ion o hese s uc u es and imp o ed he swelling pe o mance o
all he hyd ogels, ega dless o hei o igin o ex ac ion ype.
Al hough he in luence o he numbe o cycles has been p e iously
s udied by many au ho s o eeze- hawed PVA hyd ogels
[13,36,40,42,43], he du a ion o las hawing s ep has no been
in es iga ed. So, he p esen s udy could pa e he way o new syn hesis
ou es since i o e s a simple way o enhancing he swelling capaci y o
he samples.
3.2.4. Glass ansi ion empe a u e
The glass ansi ion empe a u e (T
g
) is an impo an cha ac e is ic
o polyme ic ma e ials since i de e mines hei applicabili y. Thus, he
syn hesized hyd ogels we e subjec ed o Di e en ial Scanning Calo-
ime y (DSC) analyses and he esul s a e p esen ed in Table 5.
The samples con aining alkaline lignins p esen ed highe T
g
alues
anging om 72 o 91 ◦C whe eas he ones con aining o ganosol lignins
showed lowe T
g
alues (69–86 ◦C). The highes T
g
alues we e ound
o AA and AAA samples (≈91 ◦C), which migh be because he impu-
i ies could ha e ac ed as b idges be ween he PVA and he lignin chains,
enhancing hei in e ac ions and hinde ing he elaxa ion and a ange-
men o he chains [41,44]. On he con a y, al hough WA sample
con ained lignin wi h a highe amoun o impu i ies, pa o he la es
migh ha e dissol ed in he aqueous phase, being washed ou du ing he
washing s ep and, hence, being unable o enhance he in e ac ions
be ween PVA and lignin. The e o e, in his case, he T
g
alue was lowe .
Excep o AWA sample, he hyd ogels con aining double-s ep lignins
p esen ed highe T
g
alues han he ones wi h single-s ep lignins, which
also ma ched wi h he p e iously epo ed d op on hei swelling ca-
paci y due o a highe c osslinking deg ee be ween lignin and PVA.
Howe e , his could also be a ibu ed o he inc emen on he a e age
molecula weigh s o he lignins as well as o hei o al phenolic con-
en . These wo ac o s a ec he o al
–
OH g oups o lignin, which
al hough acco ding o Raschip e al. (2013) seemed o dec ease he T
g
o
pu e lignin, hey could ha e enhanced he hyd ogen bonding wi h he
ma ix polyme , making i s chains low a highe empe a u es [41].
When he las hawing s ep was leng hened, all he T
g
alues d opped
excep o WO and AWO samples. This migh ha e occu ed due o an
inc emen on he in e ac ions be ween lignin and PVA a e wa e
e apo a ion due o he ea angemen o WO and AWO chains, which
p esen ed low a e age molecula weigh s and he lowes poly-
dispe si ies. Thus, he esul ing hyd ogels we e mo e he mally s able.
Ne e heless, o he bes o ou knowledge he e is no li e a u e ha
suppo s and explains his ac .
Fig. 5. SEM mic og aphs o he hyd ogels con aining AS lignins a 500×and 5000×magni ica ions.
Table 5
Glass ansi ion empe a u es (T
g
) and comp ession modules o he syn hesized
hyd ogels a e sho (SC) and long (LC) las hawing s eps.
Sample T
g
(◦C) Comp ession module
(MPa)
MB emo al (%)
SC LC SC LC SC LC
AA 91.0 77.3 16.3 ±1.8 10.8 ±3.2 79.5 ±3.6 81.3 ±1.8
AO 73.0 66.6 5.0 ±1.9 2.1 ±0.7 90.3 ±0.6 83.4 ±2.8
WA 79.0 64.0 14.9 ±1.9 13.6 ±0.8 75.3 ±3.7 82.3 ±3.0
WO 69.7 77.7 6.0 ±1.0 2.0 ±0.8 89.6 ±1.0 86.9 ±4.4
AAA 91.1 66.6 13.1 ±0.8 7.5 ±2.7 86.6 ±3.2 93.3 ±1.1
AAO 85.8 82.8 2.1 ±1.0 2.0 ±0.8 90.1 ±2.2 87.1 ±6.7
AWA 72.1 66.4 6.6 ±2.9 6.4 ±1.3 87.9 ±3.6 86.9 ±5.5
AWO 70.8 92.2 5.4 ±2.8 2.88 ±1.2 93.0 ±0.6 88.6 ±1.9
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3.2.5. Comp ession es s
The mechanical pe o mance o he hyd ogels was s udied com-
p essing hem up o he 80% o hei ini ial hickness. F om he ob ained
s ess-s ain diag ams, he comp ession modules we e calcula ed, as
displayed in Table 5. As in he p e ious wo k, a he end o he es s none
o he es ed hyd ogels was ac u ed and showed excellen eco e -
abili y. Ne e heless, he samples con aining alkaline lignin om he
single-s ep p ocesses we e sligh ly damaged due o hei he e ogeneous
appea ance.
The a o emen ioned samples (AA and WA) we e he ones p esen ing
he highes comp ession modules (14.8 and 16.3 MPa, espec i ely)
al hough hey we e also he ones wi h he highes swelling abili y, which
migh be ela ed o he o med honeycomb s uc u es due o he in-
e ac ions o he ma ix wi h lignin and i s impu i ies, as shown in
Sec ion 3.2.3. In spi e o hei analogous mo phology, he samples
con aining single-s ep o ganosol lignins (AO and WO) p esen ed much
lowe comp ession modules (4.95 and 6.01 MPa, espec i ely). A simila
beha iou was obse ed o he samples composed o double-s ep lig-
nins: he ones wi h alkaline lignin p esen ed highe comp ession mod-
ules (6.63–13.07 MPa) han he o ganosol ones (2.05–5.4 MPa).
The modules o all he samples p esen ed a sligh d op when
inc easing he du a ion o he las hawing s ep, p obably due o he
c ea ion o he mac o po es, as a o emen ioned. This end was also
aligned o he one obse ed o T
g
alues. All he es ima ed modules
we e in g ea acco dance wi h p e ious esul s [13], and we e highe
han hose epo ed by o he au ho s o lignin-based hyd ogels [1,45].
3.3. Me hylene blue adso p ion s udies
So as o s udy he applicabili y o he designed hyd ogels as dye
adso ben s, me hylene blue (MB) adso p ion es s we e pe o med
ollowing he p ocedu e desc ibed be o e [13]. I is known ha hanks o
in e ac ions o he mul iple nega i e cha ges on he su ace o lignin-
hyd ogels, hese ma e ials a e able o cap u e posi i ely cha ged com-
pounds such as ca ionic dyes [12,13], which a e also employed in many
medical applica ions [46].
As displayed in Table 5, i was demons a ed ha he syn hesized
samples p esen ed g ea po en ial o MB adso p ion o he es ed so-
lu ion, being he lowes emo al alue 75% and he highes 93% o he
pollu an , which exceeded in bo h cases p e ious esul s [13]. In gen-
e al, he samples con aining double-s ep lignins we e able o ap la ge
quan i ies o dye, and he alkaline lignin-based hyd ogels emo ed
sligh ly lowe alues o MB han he o ganosol -based ones, which is
also in line wi h he end epo ed o he TPC o he lignins.
Dominguez-Robles e al. (2018) also epo ed lowe MB adso p ion
alues o hei samples con aining soda lignin han o he ones wi h
o ganosol lignin, which could be ela ed wi h he alues hey epo ed
o he phenolic hyd oxyl g oups in hese lignins [11]. In con as , when
he las hawing cycle was leng hened, he MB adso p ion capaci y o he
samples con aining alkaline lignins was enhanced whe eas he MB
emo al o o ganosol lignin-based hyd ogels go sligh ly educed. This
ac sugges ed ha al hough he las hawing cycle in ensi ied he
po osi y o he samples, he a ailabili y o he nega i e cha ges on he
su ace was al e ed; ne e heless, his s a emen canno be
demons a ed.
In spi e o he emo al alues being e y high, i is ue ha he yield
o he adso p ion es s was much lowe han in o he s udies. Whe eas
alues om 2 o 200 mg dye/g hyd ogel ha e been p e iously epo ed
o lignin hyd ogels [11,47–49], in his wo k none o he syn hesized
hyd ogels su passed 0.1 mg dye/g hyd ogel. Fu he mo e, in ecen
yea s biocha s ha e been used o he emo al o hese dyes, which a
exceed he yields epo ed in his wo k [11,50]. Thus, he p esen
hyd ogels could be used o dilu ed MB en i onmen s, and modi ica ions
could be s udied oge he wi h he combina ion wi h o he compounds
in o de o enhance he adso p ion yields.
3.4. An i ungal es s
The u gen need o sea ching o al e na i e eco- iendly ma e ials
has also swayed he ood-packaging sec o . In his con ex , bio-based
hyd ogels ha e eme ged as po en ial abso ben s o hese sys ems
[51]. Howe e , hese ma e ials should ex end he shel -li e o he
packaged p oduc s, which in ol es hinde ing he g ow h o mic oo -
ganisms and ungi on hem [51,52]. Thus, he an i ungal p ope ies
agains Aspe gillus nige (b own- o ungi), one o he mos common
ungi in ood spoilage, we e s udied o all he syn hesized hyd ogels.
Fi s o all, a isual e alua ion o lignin's an i ungal capaci y was
pe o med (see Supplemen a y da a). Al hough mainly whi e- o ungi
a e able o depolyme ise lignin [53], some au ho s ha e also seen ha
b own- o ungi a e also capable o be lignin deg ade s. Ne e heless, in
his case he es ed ungi did no seem o ha e such abili y. In spi e o he
g ow h o ungi he aga su ace o all he samples, he deposi ed lignin
d ops could be clea ly obse ed a e he pe iod o he es . This showed
he an i ungal abili y o lignin, as also demons a ed by o he au ho s
be o e. Among he s udied samples, he alkaline ones seemed o be able
o inhibi mo e e ec i ely he ungal g ow h han he o ganosol ones,
leading o a g ow h in ensi y o 3 (GI =3), whe eas mos o he es o
he samples p esen ed g ea e in ensi ies (GI ≈4), acco ding o ISO 846
[15].
On he o he hand, he same es was pe o med o he hyd ogels. In
all cases, he whole hyd ogel po ion was isible a e he es (see
Supplemen a y da a). A i s sigh , he samples wi h alkaline WNS lig-
nins (WA and AWA) as well as he samples con aining o ganosol AS
lignins (AO and AWO) seemed o hinde he ungal de elopmen mo e
han he samples con aining o he lignins. Mo eo e , he e was almos
no app eciable ungal g ow h on he op su ace o he samples. In his
case, he g ow h in ensi y would be be ween 3 and 4 o e e y sample
[15].
The samples we e washed wi h PBS and he su ounding spo es we e
quan i ied as a o emen ioned. F om he es ima ed FGI alues (Fig. 6), i
was seen ha among he s udied samples, he ones p esen ing he lowes
ungal ac i i y we e WO and AAO (58 and 56.5% FGI, espec i ely), and
he one p esen ing he highes g ow h was su p isingly AWA (almos
40% mo e han he con ol sample). I was obse ed ha he samples
con aining single-s ep AS lignins p esen ed lowe FGI alues han he
ones wi h double-s ep AS lignins. Con e sely, he samples con aining
double-s ep WNS lignins p esen ed wo se an i ungal ac i i y han he
ones wi h single-s ep WNS lignins. Al hough he es ima ed alues we e
app oxima e, hese esul s helped making an idea o he an i ungal
p ope ies ha he syn hesized samples p esen ed. I is also wo h o
men ion ha none o he s udied samples los weigh du ing he
Fig. 6. Fungal g ow h inhibi ion (FGI, %) o he samples wi h espec o he
con ol hyd ogels (y =0).
A. Mo ales e al.
Sus ainable Ma e ials and Technologies 31 (2022) e00369
9
an i ungal es , which suppo s hei e ec i eness agains A. nige .
Se e al au ho s ha e epo ed simila FGI alues o o he ma e ials
o ood packaging applica ions. Fo ins ance, Salabe ia e al. (2017)
epo ed 52–62% o inhibi ion o PLA ilms con aining unc ionalized
chi in nanoc ys als [14]. In he s udy o Fe nandez-Ma in e al. (2021),
hese alues we e be ween 72 and 86% o hei chi osan/β-chi in
nano ibe s nanocomposi es due o he inco po a ion o de e pena ed
O iganum majo ana L. essen ial oil [54]. Con e sely, Dey e al. (2021)
epo ed alues be ween 51 and 56% o nea PVA ilms, which we e
nega i ely a ec ed by he addi ion o cellulose nanoc ys als and chi o-
san nanopa icles [55]. Thus, aking hese da a in o accoun , i can be
concluded ha he esul s epo ed o WO and AAO hyd ogels we e in
o al ag eemen wi h he ones epo ed by o he au ho s and could be
employed in ood packaging.
4. Conclusions
In his s udy, alkaline and o ganosol lignins we e ex ac ed om
almond and walnu shells h ough wo di e en bio e ine y s a egies
and success ully employed o he syn hesis o lignin-based physical
hyd ogels. The cha ac e iza ion o he ex ac ed lignins showed signi -
ican di e ences be ween he lignin samples, especially on hei
composi ion, a e age molecula weigh s and o al phenolic con en s. As
expec ed, he au ohyd olysis enhanced he pu i y o he lignins, espe-
cially in he case o alkaline ones, and i also p omo ed he ex ac ion o
lignins wi h highe a e age molecula weigh s. These changes al e ed
he mo phology o he p oduced hyd ogels and, hence, hei p ope ies
such as hei swelling capaci y, glass ansi ion empe a u es and
comp ession modules. In ac , hyd ogels con aining lignins om he
single-s ep p ocess esul ed in o a mo e honeycomb po ous s uc u e
and, hus, a highe wa e abso p ion capaci y. In addi ion, when he las
hawing s ep was leng hened, la ge po es we e c ea ed, which also
leaded o a no able enhancemen o he swelling pe cen age o he
hyd ogels. The syn hesized ma e ials showed good me hylene blue
emo al (75–93%) and, in some cases, an i ungal p ope ies (up o 58%
o FGI), demons a ing hei e sa ili y on a ious applica ion ields.
Decla a ion o Compe ing In e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Acknowledgemen s
The au ho s would like o acknowledge he inancial suppo o he
Depa men o Educa ion o he Basque Go e nmen (IT1008-16). A.
Mo ales would like o hank he Uni e si y o he Basque Coun y
(T aining o Resea che S a , PIF17/207). P. Gull´
on would like o
acknowledge he G an s o he ec ui men o echnical suppo s a
(PTA2019-017850-I) unde he S a e Plan o Scien i ic and Technical
Resea ch and Inno a ion 2017–2020. The au ho s hank SGIke (UPV/
EHU/ERDF, EU) o hei echnical and human suppo .
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j.susma .2021.e00369.
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