biomolecules
A icle
P oduc ion o A oma ic Compounds by Ca aly ic
Depolyme iza ion o Technical and Downs eam
Bio e ine y Lignins
Al onso Co nejo 1,* , Fe nando Bimbela 1,* , Rui Mo ei a 2, Ka ina Hablich 1,
Íñigo Ga cía-Yoldi 1, Mai ane Mais e a 1, An ónio Po ugal 2, Luis M. Gandía1and
Víc o Ma ínez-Me ino 1
1Ins i u e o Ad anced Ma e ials and Ma hema ics (InaMa 2) and Depa men o Sciences,
Uni e sidad Pública de Na a a, E31006 Pamplona, Spain; [email p o ec ed] (K.H.);
[email p o ec ed] (Í.G.-Y.); [email p o ec ed] (M.M.); [email p o ec ed] (L.M.G.);
[email p o ec ed] (V.M.-M.)
2CIEPQPF, FCTUC, Depa men o Chemical Enginee ing, Uni e si y o Coimb a, Rua Síl io Lima,
Pólo II—Pinhal de Ma ocos, 3030-790 Coimb a, Po ugal; [email p o ec ed] (R.M.); [email p o ec ed] (A.P.)
*Co espondence: [email p o ec ed] (A.C.); [email p o ec ed] (F.B.);
Tel.: +34-948-166304 (A.C.); +34-948-166259 (F.B.)
Recei ed: 31 July 2020; Accep ed: 11 Sep embe 2020; Published: 18 Sep embe 2020


Abs ac :
Lignocellulosic ma e ials a e p omising al e na i es o non- enewable ossil sou ces when
p oducing a oma ic compounds. Lignins om Populus salicaceae.Pinus adia a and Pinus pinas e om
indus ial was es and bio e ine y e luen s we e isola ed and cha ac e ized. Lignin was depolyme ized
using homogenous (NaOH) and he e ogeneous (Ni-, Cu- o Ni-Cu-hyd o alci es) base ca alysis and
ca aly ic hyd ogenolysis using Ru/C. When homogeneous base ca alyzed depolyme iza ion (BCD)
and Ru/C hyd ogenolysis we e combined on popla lignin, he a oma ics amoun was ca. 11 w .%.
Monome dis ibu ions changed depending on he eeds ock and he eac ion condi ions. Aqueous
NaOH p oduced clea age o he alkyl side chain ha was p ese ed when using modi ied hyd o alci e
ca alys s o Ru/C-ca alyzed hyd ogenolysis in e hanol. Depolyme iza ion using hyd o alci e ca alys s
in e hanol p oduced monome s bea ing ca bonyl g oups on he alkyl side chain. The analysis o
he eac ion mix u es was done by size exclusion ch oma og aphy (SEC) and di usion o de ed
nuclea magne ic esonance spec oscopy (DOSY NMR).
31
P NMR and he e onuclea single quan um
cohe ence spec oscopy (HSQC) we e also used in his s udy. The con en in poly-(hyd oxy)-a oma ic
e he s in he eac ion mix u es dec eased upon he mal ea men s in e hanol. I was concluded ha
he mo-sol olysis is key in lignin depolyme iza ion, and ha he syne gis ic e ec o Ni and Cu
p o ided monome s wi h oxidized alkyl side chains.
Keywo ds:
lignin; depolyme iza ion; homogeneous and he e ogeneous ca alysis; bio-based
a oma ic compounds
1. In oduc ion
Lignin is a e y complex na u al amo phous polyme , mainly composed o h ee di e en
phenylp opanoid uni s: p-couma yl alcohol, sinapyl alcohol and coni e yl alcohol [
1
,
2
]. In spi e o he
ela i ely simple s uc u e o hese h ee monome ic uni s, hei layou is highly complex, con o ming
an in ica e idimensional s uc u e o dis inc and chemically di e en mo i s and na u al al e na i e
monome s as a consequence o hei andom adical polyme iza ion [
3
]. These moie ies and dis inc i e
bonds a e mos esponsible o he unique s uc u es and p ope ies o lignins a he han he h ee main
Biomolecules 2020,10, 1338; doi:10.3390/biom10091338 www.mdpi.com/jou nal/biomolecules
Biomolecules 2020,10, 1338 2 o 30
phenolic building blocks, and hey a e o en he key o elucida e lignin mo phology, physicochemical
p ope ies and eac i i y.
The s uc u e and p ope ies o na i e lignin a e al e ed upon lignin isola ion, as can be seen in
Table 1, which p esen s di e en me hodologies o lignin isola ion. G ea amoun s o lignin- ich s eams
a e p oduced, o emos in he pulp and pape indus y by K a pulping [
4
]. Mo e ecen ly, la ge-scale
p ocesses o p oducing bio uels, e.g., second-gene a ion cellulosic bioe hanol, ha e gene a ed la ge
amoun s o downs eam bio e ine y e luen s [
5
,
6
]. Due o he inhe en ecalci ance o lignins,
hei main indus ial use is as low-g ade uel o p oduce ene gy [
7
], whils allowing o he eco e y o
K a cooking eagen s [
8
]. Howe e , he la ge amoun s o lignin p oduced in bio e ine ies gene a e
su plus s ocks ha o en exceed he ene gy demands o hose plan s [
5
]. The s uc u e and p ope ies
o bo h K a and downs eam bio e ine y lignins a e e y di e en o hose o he na i e lignins.
O he al e na i es o ob aining lignins include di e en o ganosol (O g) me hods which use
o ganic sol en s, ei he pu e o in aqueous solu ions [
9
,
10
], some imes combined wi h o he eac an s,
as in he soda-o ganosol me hod (Seol) [
11
]. Due o he mild condi ions used, he s uc u e o
o ganosol lignins is e y simila o hei na i e s uc u e. These isola ion me hods yield lignins o en
e e ed o as echnical lignins.
Biomolecules 2020,10, 1338 3 o 30
Table 1. Compa ison o a ious lignin isola ion me hods. Adap ed om e e ence [12].
Isola ion Me hod Lignin Name Typical P ocess Cha ac e is ics
Klason me hod [13] Klason 2% H2SO4
Ex ensi e s uc u e change, ha dwood lignin is pa ly dissol ed
K a p ocess [4] K a Na2S/NaOH Highly modi ied, pa ially agmen ed. High S con en
Sul i e pulping p ocess Lignosul ona e Ex ac lignin om was e liquo o he sul a e
pulping p ocess o so wood.
Highly modi ied, high a e age molecula weigh s, clea age o
e he linkages, loss o me hoxyl g oups and o ma ion o new
C–C bonds
Bjö kman p ocess [14,15] Milled Wood Lignin (MWL) Ball milling, hen ex ac ed by aqueous dioxane. Simila o he na i e s uc u e, possible depolyme iza ion due
o ex ensi e milling
O ganosol p ocess O ganosol [9,10] Using o ganic sol en s o ex ac lignin.
Mild condi ions, esul s in mo e unal e ed lignin, sol en could
be eco e ed by dis illa ion
Soda e hanosol [11] Basic eac an (NaOH, KOH . . . )
Reduc i e ca aly ic ac iona ion [16,17] Pd/C, Ru/C, Ni/Al2O3Phenolic monome s, low molecula weigh
poly(-hyd oxy)-a oma ic e he s
Alkaline we oxida ion [18] Alkaline we oxida ion Oxida i e p e ea men unde alkaline condi ions. Pa ial deg ada ion ia β-O-4 clea age
S eam explosion p ocess [19] S eam explosion lignin High empe a u e s eam explosion o he ibe s. Requi e li le o no chemical inpu , sho ea men ime, low
ene gy equi emen , changes o ce ain unc ional g oups
Mechano-ca aly ic p ocess [20] Mechano-ca aly ic lignin Mechano- ca aly ic depolyme iza ion. Sul u - ee lignin
Biomolecules 2020,10, 1338 4 o 30
Gi en he chemical ichness de i ed om i s highly complex s uc u e, lignin depolyme iza ion is
seen as an al e na i e o pe ochemical indus y o he p oduc ion o a oma ic compounds. A oma ics
including monome ic phenols and o he ela ed a oma ic compounds ha e been ex ensi ely sough a e
by di e en s udies on ca aly ic depolyme iza ion o lignin [
15
,
16
], some o which da e back o 1963 [
21
].
Much e o has been made o de elop ou es o alo izing lignin in o highe alue-added chemicals
and p oduc s, as can be deduced om he comp ehensi e e iews on he opic [
2
–
4
,
7
,
12
,
22
–
35
], many o
which ha e been success ul and he numbe o p o en comme cial applica ions and p oduc s om
echnical lignin is as [36].
Mos o he s udies on ca aly ic echnical lignin depolyme iza ion ely on he he e ogeneous
ca alysis o ob aining bioa oma ics and o he alue-added compounds. In his con ex , Ru suppo ed
on ca bon (Ru/C) has become he mos widely used ca alys o he hyd ogenolysis o echnical lignins
owing o i s supe io selec i i y o clea ing C
a yl
-O bonds while main aining a mode a e ac i i y o
he hyd ogena ion o a oma ic ings in compa ison o o he ca alys s [
37
]. Ru/C is commonly used in
combina ion wi h alcohols, mainly me hanol and e hanol unde supe c i ical condi ions [
28
]. The main
easons o his is he apid hea ans e , he high solubili y o echnical lignins and he eac ion
mechanism i sel : alcohols no only can ac as hyd ogen dono s bu also as nucleophiles ini ia ing
he C-O-C bond clea age [
38
]. Fu he mo e, e hanol also ac s as a capping agen and o maldehyde
sca enge h ough he o ma ion o n-p opanol hus p e en ing epolyme iza ion eac ions [
39
,
40
].
The combina ion o Ru/C ca alys s and NaOH unde me hanol sol en medium has p o en o be
e ec i e in he depolyme iza ion o o ganosol pine-de i ed lignin owing o he syne gis ic ca aly ic
e ec o NaOH and Ru/C [41].
Reduc i e ca aly ic ac iona ion (RCF) using Ru/C ca alys s, including ca aly ic hyd ogenolysis
in he liquid phase [
2
,
16
,
17
], is an in e es ing app oach o ac iona e lignocellulosic esidues. To his
end, deligni ica ion is conduc ed ia he mo-sol olysis eadily ollowed by educ i e s abiliza ion o
lignin. Using a combina ion o a Ru/C ca alys , me hanol as sol en and p essu izing wi h hyd ogen a
30 ba , deligni ica ion o ha dwoods, such as bi ch sawdus , can be success ully a ained. In his way,
depolyme iza ion o lignin agmen s e ec i ely leads o high yields o me hoxyphenolic monome s,
dime s and oligome s [
16
]. Howe e , ecen s udies ha e e ealed ha , in he p esence o bases such
as KOH, RCF o bi ch wood su e s om pa ial epolyme iza ion o lignin. This lowe s he yield o
phenolic monome s, bu he selec i i y o desi able C2- agmen ed phenols can be a o ed o e ha o
C3 phenols [
17
]. The e o e, adding bases o he eac ion medium may hampe lignin depolyme iza ion
bu enhances selec i i y owa d he desi ed p oduc s; hence, a ade-o has o be achie ed by an
app op ia e selec ion o eac ions condi ions and p ocess design.
Amids he di e en p oposed ou es, bio e ine y s a egies ha comp ise ca aly ic
depolyme iza ion o lignin o downs eam bio e ine y lignins a e o g ea in e es , because hey
allow p oducing di e en o ganic compounds ha can ha e po en ial applica ions and indus ial use.
Howe e , he high ecalci ance o hese lignins es ic s mos o he esea ch o homogeneous BCD [
42
],
whe eas he e ogeneous ca alysis has been conside ably much less in es iga ed. Ru/C was selec ed,
among o he Ru-based ca alys s, o conduc ing he educ i e depolyme iza ion o an indus ial
lignin-con aining s illage de i ed om lignocellulosic bioe hanol p oduc ion [
5
]. Ru/C yielded
he highes amoun o monome ic phenols, bu he yields we e somewha low, p obably due o
epolyme iza ion eac ions. In a p e ious wo k [
6
], samples om lignin- ich bio e ine y s eams
om popla and pine woodchips we e success ully depolyme ized unde BCD condi ions, ob aining
ema kable monome yields by doing a ca e ul selec ion o ope a ing condi ions.
He e ogeneous BCD using double-laye ed hyd oxides known as hyd o alci es (HTC) has eme ged
as a p omising al e na i e in ecen yea s. Ni- unc ionalized HTCs ha e p o en o be e ec i e in
clea ing C-O bonds o model compounds and in lignin depolyme iza ion [
43
]. Up o 7–9 w .%
monome yield was ob ained due o he e ec o ni a es in e cala ed as coun e ions be ween he
b uci e-like laye s ha cons i u e he HTCs s uc u e [
44
]. The e iciency o Ni-modi ied HTC ca alys s
is a ibu ed bo h o hei capabili y o speci ically hyd olyzing he ca bon-hyd oxyl linkage a he side
Biomolecules 2020,10, 1338 5 o 30
chain in o alkanes and o he p esence o s ong binding si es o e he linkages, hus achie ing hei
speci ic clea age [
45
]. HTCs con aining coppe (Cu-HTCs) ha e also p o en o be e ec i e in lignin
depolyme iza ion unde supe c i ical e hanol (scE OH) a 573 K [
46
] and 613 K [
47
], gi ing monome
yields a ound 20 w .% and 30 w .%, espec i ely. Howe e , hese agmen s a e u he alkyla ed
o hyd odeoxygena ed by he sol en [
39
], which e ec i ely educes he a oma ic monome s yield.
No ewo hy, HTC-Cu ca alys s unde scE OH a 653 K p oduced up o 86 w .% alkyla ed monome s in
lignin depolyme iza ion, wi h high deg ee o hyd o-deoxygena ion and hyd ogena ion o he a oma ic
ing [
39
]. I has been also epo ed ha HTCs-Cu p omo e lignin depolyme iza ion in supe c i ical
me hanol (scMeOH), p e en ing cha o ma ion [48].
Bime allic ca alys s combining Ni wi h noble me als and ansi ion me als like Fe, Mo and Ti
ha e been p oposed in di e en s udies o explo e syne gis ic e ec s, hus seeking o inc ease he
eac i i y and selec i i y [
31
]. Zhai e al. ound ha bime allic NiFe suppo ed on ac i e ca bons
could e ec i ely clea e e he linkages whils supp essing hyd ogena ion o a oma ic ings [
49
]. I was
p oposed ha he o ma ion o NiFe alloys uned he eac i i y o he ac i e si es. HTC-Ni ca alys s
ha e al eady been used in he clea age o model lignin dime ic compounds as well as in lignin- ich
bio e ine y s eams de i ed om co n s o e [
44
]. Mo e speci ically, enzyma ically hyd olyzed lignins,
some o which had been p e ea ed wi h dilu ed acids, we e subjec ed o depolyme iza ion, inding
ha he ca alys was e ec i e in clea ing e he linkages, as e idenced by he o ma ion o subs an ial
amoun s o 4- inylphenol (4-VP) p oduced by he deca boxyla ion o p-couma ic acid. To he bes o
ou knowledge, bime allic HTC-NiCu ca alys s ha e no been used in he depolyme iza ion o lignins.
In his con ibu ion, homogeneous and he e ogeneous ca aly ic me hods we e de eloped o
depolyme ize downs eam bio e ine y and echnical lignins. The esul an eac ion mix u es we e
ho oughly cha ac e ized o accu a ely de e mine he a oma ic monome yield and selec i i y,
he poly-(hyd oxy)-a oma ic ac ions appa en masses and he ex en o side- eac ions.
2. Ma e ials and Me hods
All high-pu i y liquid and solid chemicals we e pu chased om Ca lo E ba, Fishe Scien i ic
and Sigma-Ald ich and used as ecei ed. High-pu i y gases we e supplied by Nippon Gases Spain.
Syn he ic hyd o alci e (Mg
6
Al
2
(CO
3
)(OH)
16·
4H
2
O), used as suppo o p epa ing he ca alys s,
was pu chased om Sigma Ald ich (P.N. 652288). A comme cial 5% Ru/C (50% wa e we ) ca alys
was pu chased om Al a Aesa (P.N. 044338.14).
Popla (Populus sp.) was an indus ial esidue kindly p o ided as sawdus by Ga nica Plywood
Inc. (Baños de Rio Tobia, Spain). Popla sawdus was ai d ied a oom empe a u e (~295 K) o
72 h, hen i was sie ed o ob ain a pa icle size dis ibu ion comp ised be ween 0.5 and 1.0 mm
(35–18 mesh). A e sie ing, he ai -d ied ma e ial was u he d ied in an o en a 376 K o 24 h.
Bio e ine y downs eam lignins om pine (P. adia a) and popla (Populus sp.), named as BioA and
BioB, espec i ely, we e kindly p o ided by CENER’s Bio e ine y and Bioene gy Cen e (Bio2C, Aoiz,
Spain). Bo h P. adia a and Populus sp. we e used as eeds ocks in he p oduc ion o 2 G bioe hanol in a
bio e ine y, ob aining BioA and B as byp oduc s. The whole p ocess o ob aining BioA and B s a ing
solids is de ailed in a p e ious s udy [
50
], al hough succinc ly desc ibed below (please see Sec ion 2.1).
BioA and B lignins we e ecei ed in powde o m and used wi hou u he ea men s. P. pinas e
wood (PPW) chips we e kindly supplied by Eu opac (Deoc is e, Po ugal). P io o ac iona ion,
as ecei ed PPW chips we e ai -d ied and g ound in a Re sch Cu ing Mill SM 100 wi h a sie e o
squa e holes o 4 mm. Fu he de ails can be ound elsewhe e [51].
2.1. Lignin Isola ion and Cha ac e iza ion
Di e en s a egies we e used in his s udy o ob aining lignin om he ou eeds ocks desc ibed
abo e (see Scheme 1), namely au ohyd olysis ollowed by soda e hanosol (SeolA and B lignins o
pine and popla espec i ely), di ec o ganosol (O gB, o popla o ganosol ) using isop opanol,
and he use o bio e ine y downs eam lignins (BioA and B lignins o pine and popla espec i ely).

Biomolecules 2020,10, 1338 6 o 30
Biomolecules 2020, 10, x FOR PEER REVIEW 6 o 30
Scheme 1. S a egies o he isola ion o lignins om he eeds ocks selec ed in his wo k.
Bio e ine y downs eam lignins, BioA and B, we e ob ained a e acidic p e ea men a 465 K
o 5 min o he pine and popla eeds ocks, espec i ely, which allowed o sepa a e hei
co esponding hemicellulosic ac ions and o alo ize hem he ea e . The esul ing solids
p esen ed high accessibili y o he cellulosic ac ion ha was hyd olyzed o glucose using an
enzyma ic cock ail, whils yielding BioA and B as downs eam bio e ine y lignins [6,50].
Technical lignins, O gB, SeolA and B, we e isola ed in he labo a o y. SeolA and B we e ob ained
om P. pinas e and was e popla sawdus , espec i ely. Feeds ocks we e subjec ed o au ohyd olysis
a 448 K du ing 30 min o emo e hemicelluloses, ollowed by soda e hanosol diges ion a 443 K
du ing 90 min, using a NaOH load o 30 w .% (on a wood basis) and a biomass o sol en a io o 1:8
(w/w). SeolA isola ion expe imen s we e pe o med in a 1 L au ocla e eac o (Pa Ins umen s),
agi a ed by a double six-blade p opelle while SeolB isola ion expe imen s we e pe o med in a 150
mL au ocla e (Pa ins umen s) agi a ed by a ou -blade p opelle . On he o he hand, O gB was
di ec ly ob ained ollowing a di ec o ganosol p ocedu e using se e al combina ions o alcohols and
acidic ca alys s.
Amongs hem, o ganosol diges ion wi h e hanol and isop opanol a 463 K
p o ided he bes lignin yields. Howe e , DOSY (see Figu e S8 in Suppo ing In o ma ion) e idenced
ha O gB ob ained wi h e hanol p esen ed mo e impu i ies, including some cellulosic esidues.
The e o e, O gB ob ained wi h isop opanol was selec ed o conduc ing he depolyme iza ion assays.
Lignin con en was de e mined acco ding o NREL/TP 510-42618. The a io o sy ingyl (S) and
guaiacyl (G) uni s, he S/G a io, was de e mined as desc ibed in he li e a u e in BioA and B (see
Suppo ing In o ma ion SI.1) whe eas
31
P NMR was used in he es o lignins (see Suppo ing
In o ma ion SI.2).
A e age molecula weigh (M
w
) and numbe molecula weigh (M
n
) we e de e mined using Size
Exclusion Ch oma og aphy (SEC, see de ails in Suppo ing In o ma ion SI.2), in lignins and
depolyme iza ion p oduc s. Addi ionally, he appa en masses
we e es ima ed using Di usion
O de ed Spec oscopy NMR (DOSY, see Suppo ing In o ma ion SI.3 o de ails). DOSY spec oscopy
has been used in his wo k o moni o depolyme iza ion eac ions and o selec i ely de e mine he
Scheme 1. S a egies o he isola ion o lignins om he eeds ocks selec ed in his wo k.
Bio e ine y downs eam lignins, BioA and B, we e ob ained a e acidic p e ea men a 465 K o
5 min o he pine and popla eeds ocks, espec i ely, which allowed o sepa a e hei co esponding
hemicellulosic ac ions and o alo ize hem he ea e . The esul ing solids p esen ed high accessibili y
o he cellulosic ac ion ha was hyd olyzed o glucose using an enzyma ic cock ail, whils yielding
BioA and B as downs eam bio e ine y lignins [6,50].
Technical lignins, O gB, SeolA and B, we e isola ed in he labo a o y. SeolA and B we e ob ained
om P. pinas e and was e popla sawdus , espec i ely. Feeds ocks we e subjec ed o au ohyd olysis a
448 K du ing 30 min o emo e hemicelluloses, ollowed by soda e hanosol diges ion a 443 K du ing
90 min, using a NaOH load o 30 w .% (on a wood basis) and a biomass o sol en a io o 1:8 (w/w).
SeolA isola ion expe imen s we e pe o med in a 1 L au ocla e eac o (Pa Ins umen s), agi a ed by
a double six-blade p opelle while SeolB isola ion expe imen s we e pe o med in a 150 mL au ocla e
(Pa ins umen s) agi a ed by a ou -blade p opelle . On he o he hand, O gB was di ec ly ob ained
ollowing a di ec o ganosol p ocedu e using se e al combina ions o alcohols and acidic ca alys s.
Amongs hem, o ganosol diges ion wi h e hanol and isop opanol a 463 K p o ided he bes lignin
yields. Howe e , DOSY (see Figu e S8 in Suppo ing In o ma ion) e idenced ha O gB ob ained wi h
e hanol p esen ed mo e impu i ies, including some cellulosic esidues. The e o e, O gB ob ained wi h
isop opanol was selec ed o conduc ing he depolyme iza ion assays.
Lignin con en was de e mined acco ding o NREL/TP 510-42618. The a io o sy ingyl (S)
and guaiacyl (G) uni s, he S/G a io, was de e mined as desc ibed in he li e a u e in BioA and B
(see Suppo ing In o ma ion SI.1) whe eas
31
P NMR was used in he es o lignins (see Suppo ing
In o ma ion SI.2).
A e age molecula weigh (M
w
) and numbe molecula weigh (M
n
) we e de e mined using
Size Exclusion Ch oma og aphy (SEC, see de ails in Suppo ing In o ma ion SI.2), in lignins and
depolyme iza ion p oduc s. Addi ionally, he appa en masses we e es ima ed using Di usion O de ed
Biomolecules 2020,10, 1338 7 o 30
Spec oscopy NMR (DOSY, see Suppo ing In o ma ion SI.3 o de ails). DOSY spec oscopy has been
used in his wo k o moni o depolyme iza ion eac ions and o selec i ely de e mine he appa en
mass o he poly-(hyd oxy)-a oma ic ac ion in he eac ion mix u es. Appa en masses we e es ima ed
using wo calib a ion cu es ha co ela ed he a e age log D o he di usion aces and hei log MW
(see e e ence [
6
] o de ails). The calib a ion cu es we e calcula ed using wo di e en amilies o
s anda ds. The i s was polys y ene (PS) s anda ds ha co esponded o dispe sion o ces, and he
second was polye hylene glycol (PEG) and dime ic and monome ic phenolic s anda ds— e e ed o as a
PEG calib a ion cu e)— ha co esponded o Van de Waals and e en hyd ogen bond in e ac ions wi h
he sol en . Lignin and depolyme iza ion p oduc s ha e been also analyzed using He e onuclea Single
Quan um Cohe ence Spec oscopy NMR, HSQC, and To al Co ela ion HSQC NMR spec oscopy,
HSQC-TOCSY (see Suppo ing In o ma ion SI.3 o expe imen al de ails).
2.1.1. O ganosol Me hod (O gB)
The ex ac ion o lignin om was e popla sawdus was ca ied ou in a 200 mL high-p essu e
s ainless-s eel eac o (Be gho BR-300) equipped wi h a magne ic s i e and a Te lon line . In a ypical
isola ion eac ion, a 10% (w/ ) suspension o popla sawdus in isop opyl alcohol was p essu ized
wi h ni ogen (20 ba ), s i ed a 463 K o 3 h, and hen allowed o cool down o oom empe a u e.
The eac ion mix u e was il e ed, and he solid esidue was washed h ice wi h isop opyl alcohol
(3 ×20 mL)
. The il a e was concen a ed o ca. 20 mL in a o a y e apo a o , hen pou ed on o
dis illed wa e (200 mL) a 273 K unde igo ous s i ing. The mix u e was allowed o se le o e nigh ,
and he esul ing b own p ecipi a e was collec ed by il a ion and acuum d ied a oom empe a u e
o e nigh , hus yielding O gB.
2.1.2. Au ohyd olysis and Soda Alkaline E hanosol Me hod (SeolA and B)
SeolA and B lignins we e ob ained by ac iona ion o P. pinas e wood and popla sawdus ,
espec i ely. Aqueous suspensions o he eeds ocks in Milli-Q H
2
O (10% w/ ) we e p epa ed and
eadily pu in o he eac o . The suspensions we e s i ed a 448 K o 30 min and hen allowed o
cool down o oom empe a u e. The eac ion mix u e was hen il e ed and he solid was washed
wi h H2O.
The solid was e-suspended in a 35% e hanol-wa e mix u e (biomass- o-sol en mass a io o 1:8)
and eac ed wi h 30 w .% NaOH (on solid basis) a 443 K o 90 min, and hen allowed o cool down
again. The eac ion mix u e was hen il e ed, and he solids we e washed wi h H
2
O. The combined
il a es we e concen a ed by o a y dis illa ion. The esul ing solu ion was b ough o pH 5 upon
addi ion o H
2
SO
4
and allowed o se le down. The esul ing solids we e collec ed by il a ion,
e-suspended in H
2
O and collec ed again by il a ion. The as-ob ained lignin was d ied a 313 K o
7 days.
2.2. P epa a ion and Cha ac e iza ion o Hyd o alci e-Suppo ed Me al Ca alys s (HTC-M)
A se o i e HTC-M solids we e p epa ed by imp egna ion wi h a o al nominal me al con en o
5 w .%. Va ying amoun s o Ni and Cu we e used in he p epa a ion o he di e en HTC-M ca alys s,
esul ing in HTC0-5, whe e he igu e co esponds o he nominal nickel loading.
The hyd o alci e-suppo ed me al ca alys s we e p epa ed by we imp egna ion o a comme cial
hyd o alci e. The suppo was calcined a 723 K o 24 h in a mu le u nace, hus ob aining he calcined
suppo (HTC). Then, he HTC was ehyd a ed wi h H
2
O o 1.5 h and subjec ed o ul asonic ib a ion
o 5 min. Known amoun s o s ock solu ions o Ni(NO
3
)
2·
6H
2
O and Cu(NO
3
)
2·
3H
2
O in e hanol we e
hen added o he ehyd a ed HTC, o ob ain ca alys s wi h di e en nominal me al loadings: 5 w .%
Ni, (HTC-5), 4 w .% Ni—1 w .% Cu, (HTC-4), 2.5 w .% Ni—2.5 w .%-Cu (HTC-2.5), 1 w .% Ni—4 w .%
Cu (HTC-1) and 5 w .% Cu (HTC-0). The esul ing suspensions we e hen s i ed a oom empe a u e
o 5 min. The sol en was hen e apo a ed, and he esul ing solids we e g inded in o powde o m
o ob aining he di e en me al-HTC ca alys s.
Biomolecules 2020,10, 1338 8 o 30
The esul ing solids we e cha ac e ized by N
2
physiso p ion, Induc i ely Coupled Plasma-Op ical
Emission Spec ome y (ICP-OES) and X- ay Di ac ion (XRD). N
2
adso p ion-deso p ion iso he ms
we e de e mined in a Mic ome i ics Gemini V 2380 s a ic olume ic analyze a 77 K. The samples
we e p e iously degassed a 473 K o 2 h unde a N
2
gas low. The speci ic su ace a ea (m
2
/g)
o he samples was de e mined by he B unaue -Emme -Telle (BET) me hod, whe eas he speci ic
po e olume and he a e age po e size we e calcula ed by he Ba e –Joyne –Halenda (BJH) me hod.
The elemen al analysis o he ca alys s was done by means o op ical emission spec ome y wi h
induc i ely coupled plasma (ICP-OES, The mo Elemen al IRIS INTREPID RADIAL, equipped wi h
a Timbe line IIS au oma ic). XRD analyses we e done using a D-Max 2500 Rigaku di ac ome e
wi h CuK
α
adia ion a 40 kV and 80 mA and scanning 2
θ
om 5 o 95
◦
. Bo h ICP-OES and XRD
analyses we e ca ied ou a he “Se icio de Apoyo a la In es igaci
ó
n” o he Uni e sidad de Za agoza
(Za agoza, Spain).
2.3. Lignin Depolyme iza ion
Di e en s a egies we e planned o each o he isola ed lignins depending on hei physico-
chemical p ope ies (see Scheme 2). These will be desc ibed in de ail in he ollowing subsec ions,
including homogeneous base ca alyzed depolyme iza ion using aqueous NaOH, NaOH in e hanol-wa e
mix u es, and he e ogeneously ca alyzed depolyme iza ion using Ru/C o Ni-, Cu- and NiCu-HTCs.
Biomolecules 2020, 10, x FOR PEER REVIEW 8 o 30
iso he ms we e de e mined in a Mic ome i ics Gemini V 2380 s a ic olume ic analyze a 77 K. The
samples we e p e iously degassed a 473 K o 2 h unde a N
2
gas low. The speci ic su ace a ea
(m
2
/g) o he samples was de e mined by he B unaue -Emme -Telle (BET) me hod, whe eas he
speci ic po e olume and he a e age po e size we e calcula ed by he Ba e – Joyne –Halenda (BJH)
me hod. The elemen al analysis o he ca alys s was done by means o op ical emission spec ome y
wi h induc i ely coupled plasma (ICP-OES, The mo Elemen al IRIS INTREPID RADIAL, equipped
wi h a Timbe line IIS au oma ic). XRD analyses we e done using a D-Max 2500 Rigaku di ac ome e
wi h CuKα adia ion a 40 kV and 80 mA and scanning 2θ om 5 o 95°. Bo h ICP-OES and XRD
analyses we e ca ied ou a he “Se icio de Apoyo a la In es igación” o he Uni e sidad de
Za agoza (Za agoza, Spain).
2.3. Lignin Depolyme iza ion
Di e en s a egies we e planned o each o he isola ed lignins depending on hei physico-
chemical p ope ies (see Scheme 2). These will be desc ibed in de ail in he ollowing subsec ions,
including homogeneous base ca alyzed depolyme iza ion using aqueous NaOH, NaOH in e hanol-
wa e mix u es, and he e ogeneously ca alyzed depolyme iza ion using Ru/C o Ni-, Cu- and NiCu-
HTCs.
Scheme 2. Lignin depolyme iza ion s a egies.
2.3.1. Base Ca alyzed Depolyme iza ion Using Aqueous NaOH o NaOH aq. (BioA473, BioB473,
O gB463)
Depolyme iza ion eac ions we e ca ied ou using aqueous solu ions o NaOH in a 100 mL
ba ch s i ed p essu ized s ainless-s eel essel eac o (Au ocla e Enginee s, EZ100RXR) equipped
wi h a PID con olle . Following his, 50 mL o lignin suspension in 0.25 M NaOH aq. (1% w/ ; pH
Scheme 2. Lignin depolyme iza ion s a egies.
2.3.1. Base Ca alyzed Depolyme iza ion Using Aqueous NaOH o NaOH aq. (BioA473, BioB473, O gB463)
Depolyme iza ion eac ions we e ca ied ou using aqueous solu ions o NaOH in a 100 mL ba ch
s i ed p essu ized s ainless-s eel essel eac o (Au ocla e Enginee s, EZ100RXR) equipped wi h a
PID con olle . Following his, 50 mL o lignin suspension in 0.25 M NaOH aq. (1% w/ ; pH 13.4,
NaOH/solid a io 25:1) was p essu ized wi h N
2
(20 ba ) and s i ed a 463 K (O gB) o 473 K (BioA o B)
du ing 240 min. The esul ing solu ions we e il e ed, and he liquid was acidi ied o pH 2 upon addi ion
Biomolecules 2020,10, 1338 9 o 30
o 1 M HCl. Then, 25 mL o a solu ion o b omobenzene (0.0015 M) in e hyl ace a e, and addi ional e hyl
ace a e when necessa y, was hen added and he o ganic phase was sepa a ed. 1.0 mL o he o ganic
phase was aken o he quan i ica ion o monome ic phenols by GC-FID. The iden i y o he monome ic
phenols was con i med by GC-MS (see
Suppo ing In o ma ion SI.2
). The es was e apo a ed o
d yness and he yield o he oily ac ion eco e ed (he ein e e ed o as bio-oil) was g a ime ically
calcula ed and used o u he cha ac e iza ion (Suppo ing In o ma ion SI.2 and SI.3).
2.3.2. Base Ca alyzed Depolyme iza ion Using NaOH in Aqueous E OH o E OH aq. (O gB523,
SeolA493, SeolA523 and SeolB523)
Lignin (1.900 g) was suspended in a NaOH (1.800 g) solu ion in E OH/H
2
O (35 w .%, 50 mL)
.
The mix u e was p essu ized wi h ni ogen (20 ba ), eadily s i ed a 493 K o 523 K in an Au ocla e
Enginee s eac o o 1 h and hen allowed o cool down. The eac ion mix u e was il e ed, and he
solids we e washed. The e hanol in he combined il a e was emo ed by o a y e apo a ion and
he esul ing solu ion was b ough o pH 2 upon addi ion o 1 M HCl. The subsequen p ocedu e o
quan i ying monome ic phenols in he samples by GC-FID and o de e mine he bio-oil yield is he
same as ha de ailed in he p e ious subsec ion (see abo e).
2.3.3. Depolyme iza ion Using Ru/C o HTC-M Ca alys s
A solu ion o lignin o solids SeolA
493
o A
523
(250 mg) in e hanol (50 mL) and a load o ca alys
(250 mg o HTC-M o 500 mg o Ru/C -50% wa e we -) we e placed in he au ocla e eac o , which was
hen sealed. Ai was pu ged wi h ni ogen h ice and he eac o was subsequen ly p essu ized wi h
ni ogen (20 ba ) o hyd ogen (30 ba ). The eac ion medium was hen s i ed a 523 K du ing 30 min
(Ru/C ca alys ) o 543 K du ing 60 min (HTC-M ca alys ). The eac o was hen allowed o cool down,
he eac ion mix u e was il e ed and he solid was washed wi h e hanol (20 mL). The combined il a es
we e concen a ed by o a y e apo a ion and hen dis illed wa e (50 mL) was added. The nex s eps
o quan i ying monome ic phenols in he samples a e he same as hose de ailed in he wo p e ious
subsec ions (see abo e).
3. Resul s
3.1. Cha ac e iza ion o he Di e en Lignins
Table 2shows he main cha ac e is ics o he lignins used in his s udy. As expec ed, he pu i y
o he echnical lignins SeolA, and O gB was much highe (98.0 w .% and 96.1 w .%, espec i ely)
han hose o downs eam bio e ine y lignins, BioA and B (51.1 w .% and 69.1 w .%, espec i ely).
I is impo an o no e ha BioA and B p esen ed a ela i ely high sul u con en coming om he
acidic he mal p e ea men wi h sul u ic acid [
50
], which causes some sul ona ion in he a oma ic
ings. Mos o he impu i ies in bio e ine y lignins, 38.8 w .% in BioB and 23.2 w .% in BioA,
co esponded o glycans ha we e no ully emo ed upon enzyma ic hyd olysis. Opposi e o his,
nei he hemicelluloses no celluloses o glycans we e de ec ed in SeolA and B, whe eas only 0.4 w .%
o glycans we e ound in O gB. In he case o SeolB, lignin con en in he isola ed solid was su p isingly
low, 68.7 w .%, wi h a high ash con en , 30.1 w .%. The eason o his high con en in ashes could be a
non-e icien emo al o sal s o med du ing lignin isola ion.
Acco ding o he SEC analyses, downs eam bio e ine y lignins (see Figu e S2 in Suppo ing
In o ma ion) p esen ed much b oade mass dis ibu ions han echnical lignins. BioB p esen ed i s
maximum a 1038 Da and BioA a 228 Da. Howe e , ac ions beyond 32000 Da we e ound in bo h
cases. Al hough SEC o bio e ine y lignins was ca ied ou using 0.1% LiB in N,N-dime hyl o mamide,
nei he BioA no B could be comple ely solubilized. This is p obably he eason o he high dispe si y
index ound o hese solids. P esumably, low molecula weigh ac ions a e much mo e soluble
and p o ide he ela i ely low M
n
in bo h cases, 535 Da and 414 Da, espec i ely. Size dis ibu ions
in SeolA, B and O gB we e much na owe , as i is poin ed by he dispe si y index, which is, in all
Biomolecules 2020,10, 1338 16 o 30
and A
523-Ru
, bu he e is a s ong inc ease in 10 ( om 20% o 3%) oge he wi h a dec ease in 18 (10% o
2%, a 30 min o eac ion ime), and gigan ol, 19, (6% o 2%, a 30 min o eac ion ime).
Nex , SeolB was eac ed ollowing he op imized p ocedu e o SeolA. Monome yields o
SeolB
523
(9.7 w .%) we e, howe e , much highe han o SeolA
523-Ru
, because o lowe c oss-linking in
he s a ing lignin. The mos abundan monome s we e 4-hyd oxy-3-me hoxyphenylace one (36%),
15, ollowed by sy ingol, 20 (17%). Fu he hyd ogenolysis in he p esence o Ru/C p oduced a sligh
inc ease in he a oma ics yield up o 11.2 w .% wi h 15 being he mos abundan bioa oma ic (45%)
ollowed by 12 (16%) and 20 (15%). Finally, ea men o O gB wi h NaOH a 523 K in E OH/H
2
O,
O gB
523
, p o ided 7.5 w .% yield o phenolic monome s, being 20 (34%) he mos abundan ollowed by
phenols, while 15 was no de ec ed and 26 accoun ed o jus 11% o he bioa oma ics. Hyd ogena ion
o O gB
523
also p oduced a sligh inc ease in a oma ic monome s, 8.7 w .%, wi h 15 (36%) and 20 (19%)
being he mos abundan monome s. This sugges ed ha mos o he depolyme iza ion in SeolB and
O gB occu ed unde BCD in E OH/H
2
O a 523 K, while Ru/C ca alyzed hyd ogenolysis had li le
e ec in monome yields.
3.4. Poly-(Hyd oxy)-A oma ic F ac ions
BioB was subjec ed o BCD in he p esence o NaOH a 473 K, BioB
473
. DOSY spec a (see Figu e 1a)
clea ly showed he di e ence in appa en mass be ween he a oma ic hyd ogen a oms in he
s a ing lignin and hose co esponding o he BCD eac ion mix u e, wi h ac ions in he ange o
360 Da–1060 Da. Mo eo e , some di usion aces we e de ec ed in he alipha ic egion (see Figu e S5
in Suppo ing In o ma ion), which co espond o deg ada ion p oduc s om he peeling eac ions
o saccha ides in he basic medium [
58
]. A simila obse a ion can be made when O gB is eac ed
unde simila eac ion condi ions a 463 K (see Figu e 1c), O gB
463
. The appa en mass dec eased om
1454 Da o 709 Da (see Table 6) acco ding o DOSY measu emen s in he a oma ic egion, al hough he
SEC analyses did no show any no iceable change bu a sligh shi o lowe masses (see Figu e 1d).
Howe e , when he same eac ion was ca ied ou in e hanol unde he same condi ions, he mass
dis ibu ion changed d as ically and he size dis ibu ion na owed wi h a p ominen peak a ca. 530 Da,
al hough he monome yield dec eased o 2.1 w .% (see Table 4, Table 6and Figu e S9). The maximum
monome yield in he depolyme iza ion o O gB was ob ained using HTC-1 in scE OH (543 K), wi h an
inc ease in he monome s yield up o 6.8 w .%. M
n
de e mined by SEC was 694 Da while he appa en
mass de e mined by DOSY was 428 Da, which ep esen s a no iceable dec ease in appa en mass om
he s a ing O gB and B
463
in H
2
O. Size dis ibu ion was clea ly shi ed o lowe masses when BCD was
ca alyzed by HTC-M, O gB
HTCx,
wha e e he Ni and/o Cu loading. In addi ion, DOSY measu emen s
(see Figu e 1) showed aces wi h no iceable lowe appa en masses han O gB
463
, which e idenced
ha depolyme iza ion is much mo e ex ensi e when he eac ion is ca ied in e hanol han when using
H2O as sol en , which can be due o he highes solubili y o O gB in e hanol han in H2O.
SeolA and B depolyme iza ion eac ions we e conduc ed using a di e en s a egy ha consis ed
o a wo-s ep app oach. Fi s ly, NaOH ea men in E OH/H
2
O was ca ied ou a 493 K o 523 K
o 60 min uns, SeolA
493
and A
523
. The monome yields in bo h cases we e low, 0.6 w .% and
1.0 w .%, espec i ely (see Table 5). Howe e , DOSY o SeolA
493
and A
523
showed in bo h cases
ha depolyme iza ion o SeolA did occu , being mo e ex ensi e in SeolA
523
, 660 Da, han in SeolA
493
1039 Da (see Table 6). The mos ep esen a i e di usion ace o SeolA
493
was cen ed a 800 Da bu
ep esen a i e aces we e p esen in he 800–1600 Da ange, whe eas in he case o SeolA
523
, he mos
ep esen a i e di usion aces appea ed cen ed a 630 Da wi h some small di usion aces a ound
1024 Da (see Figu e 2).
Hyd ogenolysis o SeolA
493
and A
523
was done in scE OH a 523 K in he p esence o Ru/C
(20 ba )
.
As expec ed, he monome yields we e highe han in s a ing SeolA
493
and A
523
, eaching 4.2 w .%
in SeolA
493-Ru
and 7.3 w .% in SeolA
523-Ru
. DOSY spec a showed in his case a mo e p onounced
e ec o he ea men in SeolA
493-Ru
, since i s appa en mass dec eased om 1039 Da o 691 Da
(see Table 6)
. In he case o SeolA
523-Ru
he appa en mass was simila o ha o SeolA
523
(ca. 660 Da)

Biomolecules 2020,10, 1338 17 o 30
and, al hough DOSY spec a showed some sligh di e ences be ween SeolA
523
and A
523-Ru
, i can be
app ecia ed ha an impo an ac ion o he di usion in SeolA523 o e laps wi h hose o SeolA523-Ru
(see Figu e 2). No ewo hy, he es ima ed appa en masses, 746 Da, we e only sligh ly highe when
SeolA was di ec ly ea ed wi h Ru/C a 523 K in e hanol and di usion aces we e essen ially simila o
hose o SeolA
523-Ru
(see Table 6and Figu e 3) al hough i s chemical monome yield was much lowe .
Biomolecules 2020, 10, x FOR PEER REVIEW 18 o 30
SeolA was di ec ly ea ed wi h Ru/C a 523 K in e hanol and di usion aces we e essen ially simila
o hose o SeolA
523-Ru
(see Table 6 and Figu e 3) al hough i s chemical monome yield was much
lowe .
Figu e 1. (a) A oma ic egion o he DOSY spec a o BioB (black) and BioB
473
(blue); (b) SEC o BioB
( ed) and BioB
473
(blue); (c) DOSY spec a and (d) SEC o O gB (black), O gB
463
( ed) and O gB
HTC2.5
(blue). Figu es in black, ed and blue co espond o polys y ene, PS, calib a ion.
Da a p o ided by DOSY spec oscopy shed ligh abou he somewha s ange SEC
ch oma og ams ob ained a e depolyme iza ion o SeolA (see Figu es 2 and 3). Appa en masses
de e mined by DOSY do no seem o ma ch wi h he mos p ominen peaks de e mined by SEC. Thus,
in all analysed samples, a peak a ca. 130 Da appea ed. This peak is he mos in ense in he samples
ea ed wi h Ru/C in E OH, bu i is also impo an in O gB samples depolyme ized using HTC in
E OH. This is consis en wi h he de ec ion o di usion aces in he alipha ic egion o he DOSY
spec a ha migh co espond o compounds a ising om e hanol ia hyd ogen ans e mechanism
(see Figu e 4 and Figu e S7).
Besides his peak, he es o SEC ch oma og ams p esen ed a b oad dis ibu ion which is
cen ed a simila mass alues han hose es ima ed by DOSY o he a oma ic egion (i.e., poly-
(hyd oxy)-a oma ic ac ion). In he case o SeolA
493
and A
493-Ru
, b oad mass ac ions we e p esen ,
which we e mo e impo an in SeolA
493,
cen ed a 1360 Da, and a 570 Da in SeolA
493-Ru.
In SeolA
523
,
A
523-Ru
and A
Ru
mass dis ibu ions we e much na owe and p esen ed a p ominen peak a ca. 360 Da
(see Figu e 2). O gB
HTC
p esen ed also a b oad dis ibu ion wi h wo majo peaks cen ed a 526 Da
and 758 Da ha may co espond o h ee and ou -uni a oma ic compounds.
Figu e 1.
(
a
) A oma ic egion o he DOSY spec a o BioB (black) and BioB
473
(blue); (
b
) SEC o BioB
( ed) and BioB
473
(blue); (
c
) DOSY spec a and (
d
) SEC o O gB (black), O gB
463
( ed) and O gB
HTC2.5
(blue). Figu es in black, ed and blue co espond o polys y ene, PS, calib a ion.
Da a p o ided by DOSY spec oscopy shed ligh abou he somewha s ange SEC ch oma og ams
ob ained a e depolyme iza ion o SeolA (see Figu es 2and 3). Appa en masses de e mined by
DOSY do no seem o ma ch wi h he mos p ominen peaks de e mined by SEC. Thus, in all analysed
samples, a peak a ca. 130 Da appea ed. This peak is he mos in ense in he samples ea ed wi h Ru/C
in E OH, bu i is also impo an in O gB samples depolyme ized using HTC in E OH. This is consis en
wi h he de ec ion o di usion aces in he alipha ic egion o he DOSY spec a ha migh co espond
o compounds a ising om e hanol ia hyd ogen ans e mechanism (see Figu e 4and Figu e S7).
Besides his peak, he es o SEC ch oma og ams p esen ed a b oad dis ibu ion which is cen ed a
simila mass alues han hose es ima ed by DOSY o he a oma ic egion (i.e., poly-(hyd oxy)-a oma ic
ac ion). In he case o SeolA
493
and A
493-Ru
, b oad mass ac ions we e p esen , which we e mo e
impo an in SeolA
493,
cen ed a 1360 Da, and a 570 Da in SeolA
493-Ru.
In SeolA
523
, A
523-Ru
and A
Ru
mass dis ibu ions we e much na owe and p esen ed a p ominen peak a ca. 360 Da (see Figu e 2).
O gB
HTC
p esen ed also a b oad dis ibu ion wi h wo majo peaks cen ed a 526 Da and 758 Da ha
may co espond o h ee and ou -uni a oma ic compounds.
Biomolecules 2020,10, 1338 18 o 30
Table 5. Dis ibu ion and yields (Y%) o monome s a om di e en depolyme iza ion expe imen s ca ied a 523 K.
Lignin Ca bSol en (Min) Y (w .%). 1–9 10 11 12 13 15 16 17 18 19 20 21 24 25 26
SeolA cNaOH E OH/H2O 60 0.6 - 18 3 18 0 1 13 13 14 0 - - 5 5 -
SeolA493 Ru/C E OH 0 2.4 - 12 10 54 5 - - - 7 5 - - 4 0 -
30 d2.2 4 9 10 42 4 1 - - 11 6 - - 7 2 -
60 d3.7 - 17 11 46 5 - - - 7 6 - - 4 - -
120 d4.2 2 19 11 36 3 3 - - 9 10 - - 3 - -
240 d4.2 - 20 10 43 3 1 - - 6 8 - - 1 2 -
SeolA NaOH E OH/H2O60 d1.1 1 45 8 29 3 0 - - 5 2 - - - 5 -
SeolA523 Ru/C E OH 0 5 - 30 12 46 4 0 - - 2 4 - - 0 0 -
30 d7.3 3 31 8 43 4 0 - - 2 2 - - - 4 -
60 5.1 - 32 11 44 4 2 - - - 0 - - - - -
120 6.7 - 33 11 42 4 0 - - 3 4 - - 0 0 -
240 d6.6 - 32 12 44 4 - - - 2 4 - - - 0 -
SeolB NaOH E OH/H2O 60 9.7 2 11 3 14 9 36 - - - - 17 3 - 1 3
SeolB523 Ru/C E OH 30 11.2 1 7 3 16 1 45 - - - - 15 8 - 2 1
O gB NaOH E OH/H2O 60 7.5 27 7 0 12 0 - - - - - 34 9 - - 11
O gB523 Ru/C E OH 30 8.7 8 5 3 9 1 36 - - - - 19 7 - 7 2
SeolA Ru/C E OH 30 d3.5 2 7 8 51 15 1 - - 5 6 - - - - -
SeolB Ru/C E OH 30 3.9 - - - 11 4 32 - - - - 14 11 - 5 20
O gB Ru/C E OH 30 6.3 5 4 1 - 8 31 - - - - - - 5 - 37
a
(1) Phenol; (2) 2-e hylphenol; (3) o-c esol; (4-5) m-c esol/p-c esol; (6) Ca echol; (7) 4-me hylca echol; (8) 4-e hylca echol; (10) Guaiacol; (11) 4-me hylguaiacol; (12) 4-e hylguaiacol;
(13) 4-P opylguaiacol; (14) Vanillin; (15) 4-hyd oxy-3-me hoxyphenylace one; (16) Homo anillyl alcohol; (17) Homo anillic acid; (18) Ace o anillone; (19) Gigan ol; (20) 2,6 dime hoxyphenol
(Sy ingol); (21) 4-me hylsy ingol; (24) Sy ingaldehyde; (25) Ace osy ingone; (26) Homosy ingaldehyde. bCa .: ca alys ; c eac ion ca ied a 493 K; d eac ions we e made by iplica e.
Biomolecules 2020,10, 1338 19 o 30
Table 6. Appa en masses es ima ed by SEC and NMR DOSY o he s a ing lignins and samples.
SEC DOSY NMR
Sample Mw(Da) Mn(Da) A oma ic Region (Da) Alipha ic Regions (Da) A oma ic-OH (mmol/g) A oma ic-C (%)
BioB473 1051 719 658 482 418 3.30 58
BioA473 1173 720 764 494 520 n.m. 54
SeolA 3005 2296 2200 279 553 4.57 97
SeolA493 1798 891 1039 285 463 3.58 83
SeolA493-Ru 811 523 691 441 502 2.97 61
SeolA523 1250 587 660 322 401 4.51 66
SeolA523-Ru 818 468 665 433 540 3.74 56
SeolARu 956 752 746 562 509 2.77 65
SeolB 1791 1206 1477 869 n.m. 1.69 98
SeolB523 1794 1194 742 492 447 2.54 68
SeolB523-Ru 1464 962 671 406 527 2.11 51
SeolBRu 1434 980 720 539 425 n.m. 71
O gB 1636 1230 1454 548 950 4.72 90
O gB (E OH) 1650 1195 1529 286 764 n.m. 85
O gB463 1578 1186 709 245 515 5.10 73
O gB463-E OH 948 702 770 414 480 2.20 62
O gB543-E OH 1515 924 267 191 273 n.m. 16
O gBE OH-543 920 668 723 365 605 n.m. 65
O gBHTC0 1328 724 420 348 421 n.m. 38
O gBHTC1 908 694 428 396 459 n.m. 38
O gBHTC2.5 821 6478 583 381 462 2.61 44
O gBHTC4 1003 725 437 406 396 nm 49
O gBHTC5 968 681 478 276 381 nm 57
O gB523 1163 764 601 320 450 n.m. 61
O gB523-Ru 836 593 638 319 457 n.m. 44
O gBRu 888 682 559 316 559 n.m. 68
Biomolecules 2020,10, 1338 20 o 30
Biomolecules 2020, 10, x FOR PEER REVIEW 19 o 30
Figu e 2. (a) DOSY in he a oma ic egion and (b) SEC o SeolA (black), A
493
(blue) and A
493-Ru
( ed).
(c) DOSY in he a oma ic egion and (d) SEC o SeolA (black), A
523
(blue) and A
523-Ru
( ed). Figu es in
ed and blue co espond o PS calib a ion.
I is impo an o no e ha in all depolyme iza ion eac ions o O gB, ca ied ou in E OH in he
p esence o ei he NaOH o HTCs wi h di e en Ni-Cu loadings, no signi ican di e ences in M
n
(ca.
700 Da) could be ound among he di e en Ni-Cu loadings o in he blank eac ion. Howe e , when
BCD was ca ied ou in H
2
O in he p esence o NaOH, M
n
was much highe , ca. 1200 Da, which
sugges s ha ac iona ion o lignin is caused by he e hanolic sol en a high empe a u es ( iz.
he mos-sol olysis). Simila ly, di ec hyd ogenolysis in he p esence o Ru/C in e hanol and BCD in
e hanol/H2O a 523 K o SeolA (see Figu e 3), B and O gB, M
n
was in he ange o 600–700 Da, ha
also sugges he essen ial ole o he mos-sol olysis in lignin depolyme iza ion.
Di usion aces in he ange o 300–330 Da using he PS calib a ion cu e o in he ange o 185–
300 Da using PEG cu es, could be de ec ed in all he DOSY spec a. This ag ees wi h he peak ha
can be obse ed in mos SEC ch oma og ams in he ange o 275–350 Da. These di usion aces in
he a oma ic egion can be a ibu ed o he p esence o dime s (e.g., guaiacylglyce ol-β-guaiacyl
e he , gigan ol, e c.). I mus be highligh ed ha in all he s udied samples de i ed om echnical
lignins, SeolA, B and O gB, di usion aces in his appa en mass ange could be de ec ed in he
a oma ic egion, which sugges s ha mos o his peak may come om he p esence o wo-uni
a oma ic compounds. Howe e (see Figu e 4 and below), di usion aces co esponding o hese
ela i ely low appa en masses we e much mo e in ense in he alipha ic egions.
Figu e 2.
(
a
) DOSY in he a oma ic egion and (
b
) SEC o SeolA (black), A
493
(blue) and A
493-Ru
( ed).
(
c
) DOSY in he a oma ic egion and (
d
) SEC o SeolA (black), A
523
(blue) and A
523-Ru
( ed). Figu es in
ed and blue co espond o PS calib a ion.
Biomolecules 2020, 10, x FOR PEER REVIEW 20 o 30
Figu e 3. (a) DOSY spec a and (b) SEC o SeolA (black), A
523-Ru
( ed) and A
Ru
(blue). Figu es on he
le co espond o PS calib a ion.
Figu e 4. DOSY spec um o SeolA
523-Ru
. Figu es on he le co espond o PS calib a ion and igu es
on he igh o PEG calib a ion. Red do ed squa es co espond o he in eg a ion egions in he
a oma ic and in he alipha ic egions.
DOSY spec a p esen ed in mos cases a ema kable di e ence be ween he di usion coe icien s
associa ed o he a oma ic and he alipha ic egions, being he la e signi ican ly highe . Di usion
aces in he DOSY spec a (see Figu e 4) associa ed o he a oma ic egion a e ela ed o appa en
masses abo e 400 Da, and cen e ed in he ange o 486–630 Da, whe eas he alipha ic egions a e
below 500 Da, acco ding o PS calib a ion, and below 300 Da acco ding o PEG calib a ion ( o he
compa ison o a e age appa en masses in bo h egions see Table 6). In a p e ious wo k [6], hese
aces in he alipha ic egion we e associa ed o he peeling eac ion o saccha ide impu i ies in he
s a ing lignins, as is he case o BioA and B. Howe e , di usion aces a low appa en masses we e
also obse ed in he depolyme iza ion o echnical lignins, as i can be clea ly seen o SeolA
523-Ru
(see
Figu e 4). GC-MS analysis om he depolyme iza ion eac ions ca ied in SeolA, B and O gB
p esen ed p oduc s whose o igin can be associa ed o side eac ions om e hanol ( iz. hexanol, 2-
e hyl-bu an-1-ol, 4-me hyl e ahyd o-2H-py an-2-one, e c.). Bo h Ru/C and Cu-con aining HTC
Figu e 3.
(
a
) DOSY spec a and (
b
) SEC o SeolA (black), A
523-Ru
( ed) and A
Ru
(blue). Figu es on he
le co espond o PS calib a ion.
Biomolecules 2020,10, 1338 21 o 30
Biomolecules 2020, 10, x FOR PEER REVIEW 20 o 30
Figu e 3. (a) DOSY spec a and (b) SEC o SeolA (black), A
523-Ru
( ed) and A
Ru
(blue). Figu es on he
le co espond o PS calib a ion.
Figu e 4. DOSY spec um o SeolA
523-Ru
. Figu es on he le co espond o PS calib a ion and igu es
on he igh o PEG calib a ion. Red do ed squa es co espond o he in eg a ion egions in he
a oma ic and in he alipha ic egions.
DOSY spec a p esen ed in mos cases a ema kable di e ence be ween he di usion coe icien s
associa ed o he a oma ic and he alipha ic egions, being he la e signi ican ly highe . Di usion
aces in he DOSY spec a (see Figu e 4) associa ed o he a oma ic egion a e ela ed o appa en
masses abo e 400 Da, and cen e ed in he ange o 486–630 Da, whe eas he alipha ic egions a e
below 500 Da, acco ding o PS calib a ion, and below 300 Da acco ding o PEG calib a ion ( o he
compa ison o a e age appa en masses in bo h egions see Table 6). In a p e ious wo k [6], hese
aces in he alipha ic egion we e associa ed o he peeling eac ion o saccha ide impu i ies in he
s a ing lignins, as is he case o BioA and B. Howe e , di usion aces a low appa en masses we e
also obse ed in he depolyme iza ion o echnical lignins, as i can be clea ly seen o SeolA
523-Ru
(see
Figu e 4). GC-MS analysis om he depolyme iza ion eac ions ca ied in SeolA, B and O gB
p esen ed p oduc s whose o igin can be associa ed o side eac ions om e hanol ( iz. hexanol, 2-
e hyl-bu an-1-ol, 4-me hyl e ahyd o-2H-py an-2-one, e c.). Bo h Ru/C and Cu-con aining HTC
Figu e 4.
DOSY spec um o SeolA523-Ru. Figu es on he le co espond o PS calib a ion and igu es
on he igh o PEG calib a ion. Red do ed squa es co espond o he in eg a ion egions in he a oma ic
and in he alipha ic egions.
I is impo an o no e ha in all depolyme iza ion eac ions o O gB, ca ied ou in E OH in
he p esence o ei he NaOH o HTCs wi h di e en Ni-Cu loadings, no signi ican di e ences in M
n
(ca. 700 Da) could be ound among he di e en Ni-Cu loadings o in he blank eac ion. Howe e ,
when BCD was ca ied ou in H
2
O in he p esence o NaOH, M
n
was much highe , ca. 1200 Da,
which sugges s ha ac iona ion o lignin is caused by he e hanolic sol en a high empe a u es ( iz.
he mos-sol olysis). Simila ly, di ec hyd ogenolysis in he p esence o Ru/C in e hanol and BCD in
e hanol/H2O a 523 K o SeolA (see Figu e 3), B and O gB, M
n
was in he ange o 600–700 Da, ha also
sugges he essen ial ole o he mos-sol olysis in lignin depolyme iza ion.
Di usion aces in he ange o 300–330 Da using he PS calib a ion cu e o in he ange o
185–300 Da using PEG cu es, could be de ec ed in all he DOSY spec a. This ag ees wi h he peak
ha can be obse ed in mos SEC ch oma og ams in he ange o 275–350 Da. These di usion aces in
he a oma ic egion can be a ibu ed o he p esence o dime s (e.g., guaiacylglyce ol-
β
-guaiacyl e he ,
gigan ol, e c.). I mus be highligh ed ha in all he s udied samples de i ed om echnical lignins,
SeolA, B and O gB, di usion aces in his appa en mass ange could be de ec ed in he a oma ic
egion, which sugges s ha mos o his peak may come om he p esence o wo-uni a oma ic
compounds. Howe e (see Figu e 4and below), di usion aces co esponding o hese ela i ely low
appa en masses we e much mo e in ense in he alipha ic egions.
DOSY spec a p esen ed in mos cases a ema kable di e ence be ween he di usion coe icien s
associa ed o he a oma ic and he alipha ic egions, being he la e signi ican ly highe . Di usion
aces in he DOSY spec a (see Figu e 4) associa ed o he a oma ic egion a e ela ed o appa en
masses abo e 400 Da, and cen e ed in he ange o 486–630 Da, whe eas he alipha ic egions a e below
500 Da, acco ding o PS calib a ion, and below 300 Da acco ding o PEG calib a ion ( o he compa ison
o a e age appa en masses in bo h egions see Table 6). In a p e ious wo k [
6
], hese aces in he
alipha ic egion we e associa ed o he peeling eac ion o saccha ide impu i ies in he s a ing lignins,
as is he case o BioA and B. Howe e , di usion aces a low appa en masses we e also obse ed
in he depolyme iza ion o echnical lignins, as i can be clea ly seen o SeolA
523-Ru (see Figu e 4)
.
GC-MS analysis om he depolyme iza ion eac ions ca ied in SeolA, B and O gB p esen ed p oduc s
whose o igin can be associa ed o side eac ions om e hanol ( iz. hexanol, 2-e hyl-bu an-1-ol,

Biomolecules 2020,10, 1338 22 o 30
4-me hyl e ahyd o-2H-py an-2-one, e c.). Bo h Ru/C and Cu-con aining HTC ca alys s a e able
o p omo e hyd ogen ans e eac ions om e hanol ha p omp he Gue be eac ion [
47
,
59
] ha
p oduces long chain alcohols. Gi en he eac ion condi ions, i can be expec ed ha highe mass
e hanol de i a i es, ha we e no de ec ed by GC-MS we e p oduced.
Semi-q-DOSY was used in Sec ion 3.1 o es ima e he pu i y o isola ed lignin. A simila app oach
was used o es ima e he pe cen o C a oms ha co espond o a oma ic ings in he eac ion mix u es
(see Table 6). The pe cen age o a oma ic C is ela i ely low in he depolyme iza ion o BioA and B
(54% and 58%) because o he peeling eac ion o pa ially deg aded saccha ides in he s a ing lignins.
O gB
463
(73%) p esen s highe a oma ic C con en han BioA
473
and B
473
because no deg ada ion o
saccha ides occu ed upon BCD.
SeolA
Ru
, B
Ru
and O gB
Ru
also p esen ed ela i ely low con en s in a oma ic C (65%, 71% and
68% espec i ely) acco ding o DOSY in eg a ion. Howe e , in his case he dec ease in he a oma ic
pe cen age o C a oms accoun s o he o ma ion o alipha ic compounds upon eac ion o e hanol
a high empe a u es, ollowing he hyd ogen ans e mechanism in a simila ashion han in he
Gue be eac ion. The es ima ed a oma ic C is highe in SeolA
493
and A
523
(83% and 66%) han in
SeolA
493-Ru
and A
523-Ru
(61% and 56%) espec i ely ha is due o mo e ex ensi e e hanol side eac ion
upon eac ion a 523 K. A simila end was obse ed in SeolA
523
and A
523-Ru
(66% and 56%) and
O gB523 and B523-Ru (61% and 44%).
This e ec was mo e p onounced when O gB eac ed wi h NaOH. 73% and 62% o a oma ic C
we e es ima ed when he eac ion was un a 463 K in H
2
O and e hanol espec i ely, whe eas only
16% was es ima ed when he eac ion was un in e hanol a 543 K. E en mo e in e es ing, when he
BCD was ca ied ou using HTCs a 543 K, he es ima ed a oma ic ca bon inc eased wi h inc easing
nickel con en s. Thus, 38% o a oma ic C was es ima ed o ea men s wi h HTC-0 and HTC-1, 44% o
HTC-2.5, 49% o HTC-4 and 57% o HTC-5. This pe cen age o a oma ic ca bons was low, sugges ing
ha he Gue be eac ion ook place o a g ea ex en , which is consis en wi h p e ious epo s ha
desc ibed ha he Gue be eac ion is ca alyzed by HTC-Cu ca alys s [47].
31
P NMR analyses a e de i a iza ion o some eac ion mix u es p o ided he numbe o a oma ic
hyd oxy g oups pe g am o sample (see Table 6). This can be also a good pa ame e o e alua e he
pu i y o he mix u e in e ms o poly-(hyd oxy)-a oma ic con en . Indeed, his pa ame e ollowed
he same end ha he a oma ic C con en in compa able eac ion mix u es. In he case o he
depolyme iza ion o O gB i can be obse ed ha O gB
463
ca ied ou in H
2
O p esen ed 5.09 mmol
a oma ic OH/g while he same eac ion in e hanolic medium p o ided a eac ion mix u e wi h 2.20 mmol
a oma ic OH/g. Simila ly, O gB
HTC2.5
con ained 2.61 mmol a oma ic OH/g. In he eac ion mix u es o
he SeolA se ies i can be also obse ed ha he con en o a oma ic OH/g dec eased om SeolA
493
o A
493-Ru
and om SeolA
523
o A
523-Ru
, which can be a ibu ed o he inc ease in alipha ic ca bon
de i ed om side eac ions p oduced by he p esence o e hanol.
Some o he di usion aces de ec ed in he alipha ic egion may also come om C-and O-alkyla ion
unde eac ion condi ions wi h scE OH, as i has been p e iously desc ibed [
39
,
47
,
60
]. HSQC o
SeolA
523
and A
523-Ru
in he alipha ic egion (see Figu e S14 in Suppo ing In o ma ion) p esen ed
c oss-peaks ha a e compa ible wi h alkyla ed a oma ic compounds. A simila obse a ion can be
made o O gB
HTC2.5
(see Figu e S15 in Suppo ing In o ma ion), bu no in hose eac ions ca ied in
H
2
O. Howe e , he in ensi y o hese signals was ela i ely low and was only de ec ed a e selec i e
peak-picking ea men o he aw spec um, and hei di usion coe icien was sligh ly highe han
ha o he co esponding a oma ic egion. Finally, O-alkyla ed and C-alkyla ed (o he s han ga he ed
in Tables 4and 5) p oduc s we e no de ec ed by GC-MS.
4. Discussion
The esul s shown abo e e eal ha BCD in he aqueous medium is s ill a aluable op ion o
he depolyme iza ion o highly ecalci an lignins, because he basic media p omo es he o ma ion
o phenola e anions ha inc ease he solubili y o he lignin, which e ol e o quinone me hides
Biomolecules 2020,10, 1338 23 o 30
ini ia ing he p ocess. I mus be no ed ha BioA and B we e ob ained a e acidic he mochemical
ea men o popla and pine chips. This acidic p e ea men p omo es he o ma ion o C–C bonds
be ween he a oma ic uni s ha inc eased he ecalci ance o he lignin, which explains hei low
solubili y in o ganic sol en s [
42
]. O ganosol -de i ed lignin om popla sawdus , O gB, was poo ly
solubilized in aqueous medium, hence yielding low amoun s o depolyme iza ion p oduc s in NaOH
BCD, whe eas he opposi e si ua ion ook place in e hanolic medium, which no only solubilized O gB
lignin e ec i ely bu also ac ed as eac an causing he mo-sol olysis. BCD eac ion condi ions o
O gB in e hanol we e ha sh ha p o oked epolyme iza ion and, he e o e, low monome yields.
As conce ns he e ogeneously ca alyzed depolyme iza ion o lignins using Ni-, Cu- and NiCu-HTC
ca alys s, he esul s show ha he yield o a oma ics only inc eased sligh ly in compa ison o
homogeneous BCD. The ca alys s p ima ily exe ed a ole in modi ying he selec i i y o he di e en
p oduc s. Mo eo e , imp egna ing he HTC suppo s wi h Ni and/o Cu also caused ac i i y and
selec i i y o a y, and syne gis ic e ec s we e obse ed in he simul aneous use o Ni and Cu as
ac i e me als. None heless, he o mula ion o bime allic NiCu-HTC-s mus be ca e ully done. In ac ,
he inc ease in monome s con aining ca bonyl g oups (15 and 26) aking place upon depolyme iza ion
using he di e en HTC-M ca alys s p epa ed can be associa ed wi h he dec ease in coppe con en .
These compounds a e mo e p one o epolyme iza ion eac ion unde basic condi ions ha can be in
he o igin o he dec ease in he o e all monome yield a he lowes coppe loadings.
One impo an ea u e in HTC/Ni-Cu ca alys s is ha , in he opposi e way o BCD using NaOH,
monome s wi h side alkyl chains a e p oduced wi h no iceable amoun s o alkyl-guaiacols, 12–13 and
15 and alkyl-sy ingols, 26, which a e ha dly de ec ed in NaOH depolyme iza ion o BioB and O gB
in aqueous medium. This di e ence can be due ei he o he depolyme iza ion mechanism i sel o
o he C-alkyla ion ha may occu a e depolyme iza ion, as i has been al eady desc ibed in he
p esence o Cu-Mg-Al mixed oxides as epo ed by Huang e al. [
47
]. Conce ning he depolyme iza ion
mechanism, in he case o Ni-HTC, K uge e al. [
44
] sugges ed ha he ni a e ion has an impo an
ole. Unde he eac ion empe a u e used in his s udy, ni a es can be decomposed o NO and NO
2
ha would p omo e lignin ni a ion ha may accele a e he
β
-O-4 clea age. In he same s udy, i was
ound inylphenol, om deca boxyla ion o p-couma ic acid, as he majo monome in he Ni-HTC
depolyme iza ion o lignin. In addi ion, as men ioned be o e, Ni-modi ied ca alys s p esen s ong
binding si es o e he linkages and speci ically clea e he C–O linkages, yielding side alkyl chains in he
p esence o hyd ogen [
45
], which in his case would be p o ided by he dehyd ogena ion o e hanol ha
is in he o igin o he Gue be eac ion (see below) [
59
]. Conce ning C- and O- alkyla ion, he Gue be
eac ion and alkyla ion using HTC-Cu ca alys s ha e also been desc ibed by
Huang e al. [47]
. In some
cases, me hyla ion o he a oma ic ing was also obse ed when using me hanol as sol en medium
and HTC-Cu ca alys s [
61
]. In any case, nei he O-alkyla ed phenols no o he a oma ics han hose
ga he ed in Tables 4and 5we e de ec ed by GC-MS in he p esen s udy. Al hough he as-used eac ion
condi ions a e mo e simila o hose desc ibed by K uge e al. han hose desc ibed by Huang e al.
(543 K s. 613 K and 653 K), he ela i ely high con en s o homosy ingaldehyde and e hylguaiacol in
he HTC-ca alyzed eac ion do no allow us o o ally disca d C-alkyla ion.
The Gue be eac ion consis s o a sequence o eac ions ha s a s wi h me al-ca alyzed
dehyd ogena ion ollowed by aldol-condensa ion, dehyd a ion and me al-ca alyzed hyd ogena ion,
which can lead o long chain alcohols unde ou eac ion condi ions. Indeed, as can be in e ed om
he DOSY and HSQC analyses p esen ed abo e, he ex en o he Gue be eac ion when using he Ni-,
Cu- and NiCu-HTC ca alys s is e y la ge, which is in ag eemen wi h p e ious wo ks [59].
As s a ed in he in oduc ion sec ion, o he au ho s s udied depolyme iza ion o lignin- ich
bio e ine y s eams de i ed om enzyma ic hyd olysis o co n s o e using Ni-HTC ca alys s [
44
].
Despi e selec ing milde ope a ing condi ions in he p esen s udy, simila M
n
alues we e a ained,
and monome yields we e simila han hose p esen ed by K uge e al. [
44
]. Howe e , i seems likely
ha in ha s udy nei he saccha ide deg ada ion eac ions no sel -condensa ion eac ions o he
alcohol used as eac ion medium we e conside ed, which mean ha monome yields we e lowe
Biomolecules 2020,10, 1338 24 o 30
han expec ed om SEC. Mos o he ac iona ion a ained is likely caused by he syne gis ic e ec
o he sol en and he HTC solids. I is in e es ing ha he ole o Ni in he depolyme iza ion is
somewha less e ec i e han he ca aly ic ole o ni a es, which di e s wi h he obse a ions ound
in
S u geon e al. [43].
Howe e , i mus be no ed ha he yields o monome s we e no epo ed
in ha wo k. I could be possible ha he dec ease in M
n
no iced in hei SEC analyses would be
mainly caused by he mal c acking eac ions. In ac , he p e ailing monome was guaiacylke one,
which seems logical i he mal c acking is he p e ailing depolyme iza ion ou e.
The esul s ob ained using he Ru/C comme cial ca alys showed ha depolyme iza ion o
echnical lignins ob ained om pine o popla wood was mode a e in e ms o ac i i y, and ha he
noble me al ca alys could no ou pe o m he HTC-M ca alys s in O gB. I is wo h no ing, howe e ,
ha bio-a oma ic based compound yield o O gB was sligh ly highe han in SeolA and B, which can
be a ibu ed o a milde p ocess upon i s isola ion (i.e., au ohyd olysis s ep was no ca ied o O gB).
No wi hs anding ha clea age o alkyl side-chain bonds eadily ook place, C–C double bonds and
ca bonyl g oups a e mo e ex ensi ely educed in he p esence o his ca alys . Mo eo e , a numbe o
alipha ic compounds as oc anol, hexanol o 4-me hyl e ahyd o-2H-py an-2-one we e also de ec ed by
GC-MS, which sugges s ha hyd ogenolysis p oceeded ia hyd ogen ans e om e hanol, as i has
been p e iously desc ibed o lignin depolyme iza ion wi h Ru/C [
62
,
63
] o molybdenum ca bide [
64
].
The o ma ion o hese byp oduc s may ollow a simila mechanism o ha desc ibed o HTC-ca alyzed
eac ions (see abo e). Indeed, he p oposed mechanism o Ru ca alyzed hyd ogenolysis in MeOH a
493 K indica es ha he plausible pa hway o Ru/C-ca alyzed hyd ogenolysis o lignin leading o C3
and C2- agmen ed monophenols s a s om a nucleophilic a ack o MeOH on he benzylic posi ion
o
β
-O-4 uni s o lignin o gi e
α
-OMe
β
-O-4 s uc u es and o maldehyde. Ru/C ca alys s play an
essen ial ole on he egene a ion o MeOH om o maldehyde and on he hyd ogena ion o he
alkenyl side chains [
17
]. In ac , when he eac ion was un unde ni ogen a mosphe e, no signi ican
di e ences we e ound in compa ison o he homologous un ca ied ou unde hyd ogen a mosphe e,
which con i med ha he main eac ion mechanism is hyd ogen ans e om e hanol ca alyzed
by u henium.
Fu he mo e, i has been desc ibed ha using Ru/C in he p esence o a base causes a dec ease
in he con en o monome a e lignin depolyme iza ion [
17
]. This jus i ied he decision adop ed
in his s udy o spli ing hyd ogenolysis in basic media in o wo sepa a e s eps o echnical lignins:
BCD in E OH/H
2
O and Ru/C ca alyzed hyd ogenolysis. The di e en na u e o he lignins is in he
o igin o he highe a oma ic monome yields o O gB and SeolB (7–10%) compa ed o SeolA (1.1%)
upon ea men wi h NaOH in E OH/H
2
O. Fu he ea men wi h Ru/C, howe e , caused a no iceable
inc ease in phenolic monome s yields in SeolA
493
andA
523
han in SeolB
523
and O gB
523
. Thus, as a
humbnail ule, monome yields we e lowe in pine-de i ed lignins han in popla de i ed lignins.
So wood lignin has a mo e condensed s uc u e han ha dwood lignin, because G subuni s ha e one
less me hoxy g oup (in compa ison wi h S subuni s) which acili a es he o ma ion o C–C bonds
(such as 5-5 and β-5) and hinde s depolyme iza ion.
In addi ion, as al eady e idenced in ou p e ious wo k [
6
], DOSY NMR, has p o ed o be a e y
aluable ool o es ima e he ac ual appa en mass o he poly-(hyd oxy)-a oma ic ac ion o lignins
and hei s uc u al composi ion. Reac ion mix u es a e lignin depolyme iza ion consis o a mix u e
o monome ic phenols and poly-(hyd oxy)-a oma ic e he s. The applica ion o his la e ac ion o
poly-(hyd oxy)-a oma ic e he s is s ongly dependen on hei a e age o numbe molecula weigh ,
M
w
o M
n
espec i ely. Howe e , he es ima ion o he appa en masses using he di usion coe icien ,
D, is no i ial because D is s ongly dependen , among o he ac o s, on he iscosi y o he medium
and he sol en -di usa e in e ac ions. In e es ingly, i could be obse ed ha , in he es ima ion o
poly-(hyd oxy)-a oma ic e he s wi h ela i ely high appa en masses, co ela ion wi h he alues
es ima ed by SEC we e be e using PS cu es whe eas in he case o low appa en masses his co ela ion
was be e using PEG cu e. The eason lies in he di e en di usa e-di usa e and di usa e-sol en
in e ac ions ha a ise om he inc easing hyd ophobici y o he highes mass ac ions ha a ise
Biomolecules 2020,10, 1338 25 o 30
om and inc easing impo ance o dispe sion o ces as he mass inc eases. Al hough some small
disc epancies can be ound be ween he appa en masses o he a oma ic egion, which co esponds
o he appa en mass o poly-(hyd oxy)-a oma ic ac ion, and Mn, alues a e in good ag eemen .
Finally, semi-q-DOSY combined wi h
31
P has been used o es ima ing he pe cen o a oma ic
ca bon a oms in he eac ion mix u es. Al hough his is no a quan i a i e measu e, his has allowed
o app oxima ely de e mining he con en o poly-(hyd oxy)-a oma ics in he eac ion mix u es and
e alua ing he ex en o e hanol side eac ions ( iz. ans e hyd ogena ion o he Gue be eac ion).
In his case, his con en dec eases in he eac ions ha we e ca ied ou in e hanol and, amongs hem,
in hose conduc ed wi h he ha shes condi ions because o he side eac ions o he sol en .
5. Conclusions
BioA and B downs eam bio e ine y lignins we e ob ained om P. adia a and Populus Sp.,
espec i ely. SeolA and B we e ob ained a e au ohyd olysis and soda e hanosol o P. pinas e and
was e popla sawdus , while O gB was ob ained om he la e ia o ganosol in isop opanol.
I has been shown ha he e is no a single solu ion o lignin depolyme iza ion. The e o e,
ca alys and eac ion condi ions mus be ca e ully selec ed depending on he eeds ock and he lignin
isola ion me hod.
BioA and B could only be depolyme ized by NaOH BCD in aqueous medium. Howe e , he bes
a oma ic monome yields in he depolyme iza ion o echnical lignins we e achie ed by BCD in
e hanol/H
2
O ollowed by Ru/C ca alyzed hyd ogenolysis in e hanol. The ca alys plays an essen ial ole
in he selec i i y. Ni-, Cu- and bime allic NiCu-ca alys s suppo ed on calcined hyd o alci e p oduced
sligh ly lowe a oma ic monome yields in he he e ogeneous BCD o O gB in scE OH, al hough,
he syne gis ic e ec o Ni and Cu caused a change in he selec i i y yielding monome s wi h alkyl
side chains bea ing ca bonyl g oups.
The sol en and he eac ion empe a u e a e also key in he ou come o he depolyme iza ion
eac ion. On one hand, he esul ing poly-(hyd oxy)-a oma ic e he ac ions p esen ed simila appa en
masses and SEC p o iles when he depolyme iza ion eac ions we e ca ied in a gi en sol en a a
gi en empe a u e ega dless o he ca alys used, sugges ing ha he mo-sol olysis is an essen ial
pa in lignin depolyme iza ion. On he o he hand, when he eac ion was ca ied ou in e hanol a
high empe a u es, a se ies o compounds de i ed om he hyd ogen ans e mechanism in Ru/C
ca alyzed eac ions and he Gue be eac ion in HTC-M ca alyzed eac ion we e de ec ed.
DOSY-NMR has p o en o be a use ul and eliable ool in selec i ely de e mining he appa en mass
o he di e en ac ions in he eac ion mix u es. Semi-q-DOSY combined wi h
31
P has been used o
he i s ime in he es ima ion o he pu i y, no only o lignins, bu also o he poly-(hyd oxy)-a oma ic
ac ions a ising om lignin depolyme iza ion and i has been essen ial o es ima e he ex en o he
sol en side eac ions.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h p://www.mdpi.com/2218-273X/10/9/1338/s1,
SI. 1 XRD analyses; Figu e S1. Rep esen a i e XRD di ac ion pa e ns o he esh and calcined NiCu-HTC
samples; SI. 2 Chemical cha ac e iza ion lignin and eac ion mix u es analysis; Table S1. A oma ic monome s
and e en ion imes; SI. 3. Expe imen al de ails o NMR cha ac e iza ion; Table S2 In eg a ion egions used in
he de e mina ion o a e aged di usion coe icien s. Table S3. In eg a ion egions used in he de e mina ion o
a oma ic –OH con en s and G/S a io; SI. 4. SEC and DOSY spec a; Figu e S2. No malized SEC ch oma og ams
o isola ed lignins. BioB, SeolB, O gB; BioA and SeolA; Figu e S3. DOSY spec a o BioB and BioA;
Figu e S4
.
Raw DOSY spec a o SeolA, SeolB, and O gB; Figu e S5 DOSY spec a o BioB
473
in he alipha ic egion;
Figu e S6
. Alipha ic egion o he DOSY spec a o O gB, O gB
463
and O gB
HTC2.5
; Figu e S7. DOSY in he
alipha ic egion o SeolA, SeolA
493
and SeolA
493-Ru
. DOSY in he a oma ic egion o SeolA, SeolA
523
and
SeolA
523-Ru
; Figu e S8. DOSY spec a and SEC o O gB ob ained using e hanol and isop opyl alcohol; Figu e S9.
SEC ch oma og ams o he depolyme iza ion o O gB wi h NaOH in e hanol a 463 K and HTC-2.5 in e hanol a
543 K; SI. 5. HSQC and HSQC-TOCSY spec a; HSQC spec a o SeolA; Figu e S11. HSQC spec a o O gB;
Figu e S12. HSQC spec a o SeolB; Figu e S13. HSQC spec a o ace yla ed BioB, BioA and HSQC-TOCSY o
BioA; Figu e S14. Alipha ic egion in he HSQC spec a o SeolA523 and SeolA523-Ru; Figu e S15. HSQC spec a
o in he alipha ic egion o O gBHTC2.5.