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1
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Simul aneous alo iza ion and de oxi ica ion o he hemicellulose ich 5
liquo om he o ganosol ac iona ion 6
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Ma ía Ga cía-To ei o, José Ca los Ma ínez-Pa iñoa, Bea iz Gullón, Thelmo A. Lú-8
Chau, Ma ía Te esa Mo ei a, Juan M. Lema, Gemma Eibes*
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Dep . o Chemical Enginee ing, Ins i u e o Technology, Uni e sidade de San iago de 11
Compos ela, 15782 San iago de Compos ela, Spain 12
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aP esen add ess: Depa men o Chemical, En i onmen al and Ma e ials Enginee ing, 14
Ag i ood Campus o In e na ional Excellence (ceiA3), Uni e sidad de Jaén, Jaén, Spain 15
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*Co esponding au ho : Gemma Eibes 17
e-mail add ess: [email protected] 18
Tel: +34-881816020, Fax: +34-881816702 19
Pos al add ess: Rúa Cons an ino Candei a s/n, 15782, San iago de Compos ela, Spain 20
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2
Abs ac 23
F ac iona ion o lignocellulosic biomass wi h sol en s (o ganosol p ocess) gene a es a 24
hemicellulose- ich liquo wi h a high con en o phenolics which is pa icula ly oxic. 25
This wo k add esses he u iliza ion o his s eam as a po en ial ca bon sou ce o he 26
p oduc ion o ligninoly ic enzymes (LE). Among six basidiomyce es species, I pex 27
lac eus and Ganode ma lucidum p esen ed he highes ac i i ies o manganese pe oxidase 28
(646±122 U L-1) and laccase (1,497±161 U L-1), espec i ely, g owing on a medium 29
composed mainly o cellulose ibe s, lignin and hemicellulose. The in luence o each 30
lignocellulosic ac ion on he LE p oduc ion mechanisms was s udied in mo e de ail. The 31
high concen a ion o phenolic compounds in he hemicellulose- ich s eam ac ed as 32
induce o LE p oduc ion, wi h le els e en g ea e han hose o xylose. Acu e oxici y 33
es s on Vib io ische i e ealed a subs an ial educ ion o he oxici y a e he ungal 34
ea men (by ca. 12–21 imes). The p oposed alo iza ion and de oxi ica ion o his 35
cu en ly non-exploi ed and abundan by-p oduc s eam is a p omising s a egy o 36
enhance he indus ial easibili y o he o ganosol ac iona ion p ocess. 37
38
Keywo ds: manganese pe oxidase, laccase, lignocellulose, whi e- o ungi, o ganosol 39
ac iona ion 40
41
42
3
1. In oduc ion 43
Whi e- o ungi a e he only g oup o mic oo ganisms cu en ly known ha deg ade all 44
basic wood polyme s, i.e., cellulose, hemicellulose and lignin in o low-molecula weigh 45
compounds ha can be assimila ed o g ow h (Cama e o e al., 2014). This is achie ed 46
by hei abili y o p oduce se e al hyd oly ic enzymes (cellulases and hemicellulases) and 47
hei unique ne wo k o oxida i e (ligninoly ic) enzymes. The s udy o he ex acellula 48
ligninoly ic enzyme (LE) sys em o Phane ochae e ch ysospo ium demons a ed ha 49
lignin pe oxidase (LiP, EC 1.11.1.14), manganese pe oxidase (MnP, EC 1.11.1.13) and 50
laccase (EC 1.10.3.2) a e he p ima y enzymes associa ed wi h he deg ada ion o lignin 51
(Kuwaha a e al., 1984). 52
The LE sys em plays a undamen al ole in he biocon e sion o lignocellulose. 53
Fu he mo e, LE can also be applied o o he pu poses, such as he p oduc ion o second-54
gene a ion bio uels, o ganic syn hesis (an ibio ics, polyme s, building blocks), cosme ics 55
(skin-ligh ening agen s), nanobio echnology (bio uel cells and biosenso s o biomedical 56
applica ions), bio emedia ion, biopulping and biobleaching in pape indus y as well as 57
he ood and ex ile indus y (Alcalde, 2015; Yada and Yada , 2015). 58
Howe e , o mee ma ke demands, he la ge-scale p oduc ion o hese enzymes a 59
low cos is manda o y. Al hough signi ican e o s ha e been de o ed o enhance LE 60
p oduc ion by he e ologous p o ein exp ession (Alcalde, 2015), he le els o enzyma ic 61
p oduc ion a e s ill a he limi ed (Eibes e al., 2009) and s ill ha e o be ob ained om 62
wild s ains (Elisash ili and Kachlish ili, 2009). 63
LE p oduc ion highly depends on he ungal species, sou ce o lignocellulosic 64
subs a e and cul i a ion me hod (Elisash ili e al., 2008). The p esence o lignocellulose 65
was ound o be a p e equisi e o LE p oduc ion by whi e- o ungi in subme ged cul u es 66
(Kapich e al., 2004; Gassa a e al., 2012) and he a ious lignocellulosic ac ions ha e 67
4
been epo ed o in luence LE p oduc ion in a di e en manne . Fo example, P. 68
ch ysospo ium was ound o sec e e laccase in he p esence o cellulose, bu no glucose 69
(S ini asan e al., 1995). T. e sicolo p oduced highe laccase yields in he p esence o 70
na u al lignocellulose-con aining subs a es such as whea s aw o wood, a he han wi h 71
glucose (Schlosse e al., 1997). Toxic a oma ic compounds and lignin also ha e a 72
ema kable in luence on LE p oduc ion wi h T. e sicolo o Phlebia adia a (Rogalski 73
e al., 1991a; Rogalski e al. 1991b). Unde s anding he physiological mechanisms 74
egula ing enzyme syn hesis by whi e o ungi could he e o e be use ul o imp o ing 75
he e icien p oduc ion o LE. 76
The o ganosol p ocess, i.e. pulping wi h e hanol-wa e , allows he ac iona ion o 77
he lignocellulosic ma e ials on i s main cons i uen s, i.e. cellulose ibe s, lignin and 78
hemicellulose ac ion (Lau e e al., 2014). Howe e , only a ew wo ks ha e s udied 79
po en ial applica ions o he hemicellulose liquo a e lignin eco e y (Kau o e al., 80
2013; Hallbe g e al., 2011). The p esence o deg ada ion p oduc s om he 81
ca bohyd a es, soluble low molecula weigh lignin and o he possible inhibi o s may 82
limi i s con e sion o bio uel (Kau o e al., 2013). On he o he hand, conside ing his 83
ac ion as was e s eam, he la ge cos s associa ed wi h was ewa e ea men would limi 84
he economic iabili y o he o ganosol bio e ine y (Lau e e al., 2014). 85
Recen ly, he in e es on he alo iza ion o ag o-indus ial esidues o p oduce LE a 86
lowe cos has inc eased (Palma e al., 2016). Se e al esidual s eams, such as apple 87
pomace sludge (Gassa a e al., 2012), ishe y esidue, b ewe y was e, pulp and pape 88
indus y sludge (Gassa a e al., 2010), suga cane esidue (Maza e al., 2015), oli e oil 89
was ewa e (Mann e al., 2015), oa husks, was e om pape p ocess indus y (Winquis 90
e al., 2008) ha e been e alua ed as ca bon sou ce. Howe e , o ou knowledge, he 91
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applica ion o he hemicelluloses ac ion om o ganosol p ocess o ligninoly ic 92
enzyme p oduc ion has no been epo ed ye . 93
This s udy e alua es he p oduc ion o LEs in subme ged e men a ions by di e en 94
ungal s ains using he hemicellulose liquo o an o ganosol p ocess as ca bon sou ce. 95
Wi h he aim o be e analyzing he speci ic equi emen s o he ungal s ains o LE 96
p oduc ion, he indi idual o combined addi ion o he o he ac iona ion p oduc s 97
(cellulose ibe s and lignin) was e alua ed be o e using he hemicellulose liquo as he 98
only ca bon sou ce. Mo eo e , he acu e oxici y o he ea ed liquo was assessed wi h 99
he bioluminescen pho obac e ium Vib io ische i. 100
101
2. Ma e ials and me hods 102
2.1. Lignocellulosic subs a es 103
The lignocellulosic subs a es used as ca bon sou ce in his s udy we e cellulose 104
ibe s (C6), o ganosol lignin (L) and a concen a ed C5-suga s liquid ac ion (C5), all 105
p o ided by he F aunho e Cen e o Chemical-Bio echnological P ocesses (CBP, 106
Leuna, Ge many). The physico-chemical cha ac e isa ion o hese subs a es is gi en in 107
Table 1. The ac iona ion o beech wood in he o ganosol p ocess was pe o med a he 108
F aunho e -CBP. Deba ked beech wood chips we e pulped wi h a 1:1 e hanol/wa e 109
mix u e a 170°C o 100 minu es using 0.5% o sulphu ic acid (based on d y wood) a a 110
liquo - o-wood a io o 3.2:1 and he p essu e was main ained a 20 ba using ni ogen 111
gas (Lau e e al., 2014). The solid ac ion ob ained a e he pulping o he beech wood 112
was washed; disin eg a ed and dewa e ed, ob aining he cellulose ibe s (C6). The liquid 113
phase was composed mainly o lignin, C5-suga s om hemicellulose ac ion and 114
deg ada ion p oduc s. Lignin (L) was p ecipi a ed om his s eam by he addi ion o 115
wa e and il e ed o i s sepa a ion (Schulze e al., 2016). By he eco e y o he e hanol 116
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om he il a es o p ecipi a ed lignin, a aw hemicellulose ac ion was ob ained (C5). 117
This aw hemicellulose s eam was subsequen ly concen a ed. 118
2.2. Mic oo ganisms and inocula p epa a ion 119
I pex lac eus (F . 238 617/93), Len inus ig inus (PW94-2), S e eum hi su um 120
(PW93-4) and Phane ochae e ch ysospo ium (BKM-F-1767) we e ob ained om he 121
cul u e collec ion o he Depa men o Chemical Enginee ing o he Uni e si y o 122
San iago de Compos ela (Spain). Bje kande a sp. R1 was isola ed om a Chilean o es 123
in Temuco and iden i ied as a new anamo ph o Bje kande a sp. (Taboada-Puig e al., 124
2011) Ganode ma lucidum was isola ed om mush oom spen subs a e, kindly p o ided 125
by Hi as da Te a S.L. (Pon e ed a, Spain). All ungal s ains we e main ained on MEA 126
aga a 4ºC. 127
Mycelia we e p oduced in s a ic cul u es o ungal inocula p epa a ion. Fi e plugs 128
o ungal mycelium om esh aga pla es we e inocula ed wi h 200 mL o glucose-129
pep one medium (Kimu a e al., 1990) in Fe nsbach lasks and incuba ed a 30ºC. A e 130
7 days, ungal cul u es we e homogenized in a s e ilized blende o 30 s and used as 131
inoculum. 132
2.3. Cul u e condi ions 133
Subme ged e men a ions we e pe o med in 250 mL E lenmeye lasks con aining 90 134
mL o cul u e medium and 10 mL o inoculum. The ungal cul u es we e incuba ed on a 135
o a y shake a 150 pm, ini ial pH 4.5 and 27ºC. All cul u e media we e p epa ed wi h 136
dis illed wa e and con ained 1 g L-1 pep one (as ni ogen sou ce), 0.5 mM MnSO4 and 137
0.15 mM CuSO4 (as induce s o enzyme p oduc ion). Six di e en combina ions o 138
cellulose ibe (C6), o ganosol lignin (L), concen a ed hemicellulose ac ion (C5), 139
and/o xylose (X) we e e alua ed as ca bon sou ce. The concen a ion o hese 140
componen s in each cul u e medium is shown in Table 2. Cellulose and lignin 141
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concen a ion was calcula ed conside ing he humidi y and concen a ion o each o he 142
componen s in bo h s eams (Table 1). Hemicellulose concen a ion was exp essed in 143
equi alen s o TRS, conside ing ha he concen a ed hemicellulose ac ion had a TRS 144
concen a ion o 225 ± 5.6 g L-1. 145
2.4. Analy ical me hods 146
An adap ed p o ocol om NREL (Na ional Renewable Ene gy Labo a o y, Golden, 147
USA) was used o he de e mina ion o he biomass composi ion in glucan, xylan and 148
lignin (Slui e e al., 2007). Acid hyd olysis allows lignin sepa a ion om he suga 149
ac ion, and he amoun o lignin eco e ed is de e mined by weigh . The concen a ions 150
o xylan and glucan we e calcula ed om he concen a ion o he co esponding 151
monome ic suga s using a co ec ion o 0.88 and 0.90 o C5 and C6 suga s, espec i ely. 152
The Folin-Ciocal eu’s me hod was used o he quan i ica ion o he o al phenolic 153
compounds concen a ion (Single on and Rossi, 1965). 154
To al educing suga s (TRS) we e de e mined by he dini osalicylic acid (DNS) 155
me hod (Mille , 1959). Glucose, xylose, hamnose and ace ic acid concen a ions in he 156
C5-suga s ac ion we e de e mined by HPLC (Hewle Packa d ch oma og aph), 157
equipped wi h an ION-300 column (López e al., 2011). The concen a ion o he xylose 158
oligome s was de e mined by measu ing he eleased xylose a e pos -hyd olysis 159
ca alyzed wi h sulphu ic acid. The liquid sample om he C5-suga ac ion o he ungal 160
cul u e supe na an was dilu ed and H2SO4 was added o each a inal concen a ion o 161
4% ( / ). The inal pH o he sample was 0.7. I was au ocla ed a 121ºC o 20 min in a 162
sealed lask. Flasks we e weighed be o e and a e he he mal ea men o allow 163
co ec ion o any possible e apo a ion losses. Finally, samples we e il e ed h ough 0.45 164
μm cellulose ace a e memb anes and analysed by HPLC o glucose, xylose, and 165
a abinose using a 1100 se ies Hewle -Packa d ch oma og aph i ed wi h a e ac i e 166
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index de ec o ope a ed a 50°C. O he analysis condi ions we e as ollows: Aminex 167
HPX-87H column (BioRad, He cules, CA); mobile phase, 0.003 mol/L H2SO4; low, 0.6 168
mL/min. 169
2.5. Enzyma ic ac i i y assays 170
Laccase ac i i y was de e mined by measu ing he oxida ion o 5 mM 2,2’-azino-171
bis(3-e hylbenzo hiazoline-6-sul onic acid) (ABTS) in 100 mM sodium ace a e bu e , 172
pH 5 (ε436=29.3 mM-1 cm-1) (Taboada-Puig e al., 2011). MnP ac i i y was measu ed by 173
ollowing he oxida ion o 1 mM 2,6-dime hoxyphenol (DMP) a 468 nm in a 50 mM 174
sodium malona e bu e (pH 4.5) con aining 1 mM MnSO4 a e s a ing he eac ion wi h 175
0.4 mM H2O2 (ε468=49.6 mM-1 cm-1) (Taboada-Puig e al., 2011). LiP ac i i y was 176
de e mined by measu ing he oxida ion o 2 mM e a yl alcohol o e a aldehyde a 310 177
nm in 50 mM o a a ic acid (pH 3) and 0.4 mM o H2O2 (ε310=9.3 mM-1 cm-1) (Taboada-178
Puig e al., 2011). All enzyma ic ac i i ies we e exp essed as in e na ional uni s (U), 179
de ined as he amoun o enzyme ha ans o ms 1 µmol subs a e/min o he amoun o 180
enzyme ha eleases 1 µmol p oduc /min a 30ºC. 181
2.6. Mic o ox® oxici y assays 182
Mic o ox® oxici y assays we e pe o med by using a Mic o ox® model 500 183
Analyze . The luminescen ma ine bac e ium Vib io ische i was he bioassay 184
mic oo ganism used o hese expe imen s. The esul s we e exp essed as EC50 a 5, 15 185
and 30 min, which co esponds o he olume pe cen age ha causes a educ ion in he 186
ligh ou pu o he Mic o ox® es o ganism by 50% in 5, 15 and 30 min o con ac , 187
es ima ed acco ding o he “Basic Tes ” p o ocol o he so wa e (Mic obics, 1992). 188
2.7. Da a analysis 189
A s a is ical analysis was conduc ed o a co ec compa ison o esul s using he 190
so wa e R .2.12.0 (The R Founda ion o S a is ical Compu ing). Fi s , a one-way 191
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analysis o a iance (ANOVA) was ca ied ou o de e mine i he esul s ob ained a 192
di e en condi ions we e signi ican ly di e en . Then, i he ANOVA con i ms he 193
exis ence o a signi ican di e ence (p < 0.05), a pos -hoc analysis (Tukey’s HSD) was 194
pe o med o a le el o signi icance (α) o 0.05. 195
196
3. Resul s and discussion 197
3.1. Fungal sc eening 198
Six di e en whi e- o ungi: I. lac eus, Bje kande a sp. R1, S. hi su um, L. ig inus, G. 199
lucidum and P. ch ysospo ium, we e sc eened o ligninoly ic enzyme p oduc ion in a 200
cul u e medium composed o cellulose ibe s, o ganosol lignin and hemicellulose 201
ac ion (C6+L+C5) (Table 2). This medium p omo ed he g ow h o all es ed ungi in he 202
o m o pelle s, excep P. ch ysospo ium, which did no p oduce any ligninoly ic enzyme. 203
Inhibi ion o LE p oduc ion by P. ch ysospo ium was p obably caused by a non-iden i ied 204
componen om he hemicellulose s eam. Componen s such as o al phenolics, ace ic 205
acid, Na+ and SO42- we e no expec ed o inhibi LE p oduc ion (Kou o sios and Ze akis, 206
2014). 207
Di e en suga consump ion p o iles we e obse ed (Fig. 1A and 1B), bu he 208
common end among all he ungal s ains was he unconsumed suga ac ion ha 209
emained a he end o he e men a ions (app oxima ely 1 g L-1 o TRS). L. ig inus and 210
G. lucidum we e he s ains wi h he as es suga s consump ion a es; in bo h cases, he 211
TRS concen a ion was educed o 1 g L-1 a day 4. All ungal cul u es p esen ed a lag 212
phase o wo days, wi h he excep ion o S. hi su um, which had he longes lag phase; i 213
ex ended up o ou days, bu he ea e , suga s we e deple ed in 3d. An inc ease o TRS 214
a day 5 was de ec ed in he case o I. lac eus, p obably due o he hyd olysis o he 215
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compounds and long-chain a y acids ha a e oxic o mic oo ganisms and plan s (Mann 365
e al., 2015). 366
A e he ungal ea men and he eco e y o he enzyme by ul a il a ion (10 367
kDa), he oxici y o he s eam dec eased nea ly 21 and 12 imes o I. lac eus and G. 368
lucidum s ains, espec i ely. The de oxi ica ion p omo ed by I. lac eus seemed mo e 369
e ec i e han ha o G. lucidum, al hough he emo al o phenolic compounds was e y 370
simila in bo h eac o s (≈85%). The analysis o he a oma ic composi ion o bo h ea ed 371
liquo s could help o cla i y he di e en beha iou obse ed in he de oxi ica ion (Asses 372
e al., 2009). N ougias e al. (2012) e alua ed he de oxi ica ion o oli e mill was ewa e 373
by di e en WRF, and hey obse ed ha Ganode ma s ains we e less e icien han 374
Pleu o us sp. in he educ ion o oxici y (by ca. 5–8 and 15 imes, espec i ely). 375
376
4. Conclusions 377
The di e en lignocellulosic ac ions de i ed om he o ganosol ac iona ion o beech 378
wood in luenced he mechanisms associa ed wi h LE p oduc ion be ween ungal species. 379
Addi ionally, he esidual liquid s eam, con aining mainly hemicellulosic suga s and 380
phenolic compounds, was obse ed o be an excellen LE induce . The u iliza ion o his 381
esidue as he sole ca bon sou ce p o ides an oppo uni y o p oduce LE in an economical 382
and simple medium and, impo an ly, he ungal ea men esul s in a subs an ial dec ease 383
in he liquo oxici y. Fu he mo e, he alo iza ion and de oxi ica ion o his byp oduc 384
s eam is a p omising s a egy ha may enhance he indus ial easibili y o he o ganosol 385
p ocess. 386
387
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Acknowledgmen s 388
This esea ch was suppo ed by he Spanish Go e nmen (Minis y o Economy and 389
Compe i i eness) h ough he ERA-IB2 p ojec 2G-Enzymes (PCIN-2015-031) and 390
ENTIRE and MODENA p ojec s (CTQ2013-44762-R and CTQ2016-79461-R). The 391
au ho s belong o he Galician Compe i i e Resea ch G oup GRC2013-032 and o he 392
CRETUS S a egic Pa ne ship (AGRUP2015/02). All hese p og ammes a e co- unded 393
by FEDER (EU). Au ho s a e g a e ul o F aunho e CBP o p o ision o he o ganosol 394
ac ions. 395
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528
23
Tables 529
530
Table 1: Physico-chemical cha ac e is ics o he cellulose ibe s, o ganosol lignin and 531
concen a ed hemicellulose ac ions ob ained om he o ganosol p ocess. Mean alue 532
± s anda d e o we e calcula ed using iplica e se s 533
534
Pa ame e s
Cellulose
ibe s
(C6)
O ganosol
lignin
(L)
Pa ame e s Hemicellulose
(C5)
pH 3.4 2.6
pH 5.5
Humidi y (%, w/w) 69.8 ± 0.4 12.7 ± 0.6
TN (mg L-1) 748 ± 55
Ashes (%, w/w)
0.16 ±
0.02
0.14 ± 0.02
Glucose (g L-1) 10.8
Glucan (%, w/w d y
basis)
73.5 ± 7.4 0
Xylose (g L-1) 144.3
Xylan (%, w/w d y
basis) 11.3 ± 3.9 2.4 ± 0.09
Rhamnose (g L-1) 44.1
Xylose oligome (g
L
-1
)
75.7
Acid soluble lignin
(%, w/w d y basis) 2.1 ± 0.2 2.0 ± 0.1
Ace ic acid (g L-1) 17.1
To al phenols (g L-
1
)
40.8 ± 0.8
To al lignin (%, w/w
d y basis) 10.4 ± 1.0 92.7 ± 0.9
Na+ (g L-1) 20.7
SO42- (g L-1) 18.8
TN: o al ni ogen 535
536
24
537
Table 2: Ca bon composi ion o he di e en media es ed 538
Medium componen (g L-1)
C
5
+C
6
+L
C
5
C
5
+C
6
C
5
+L
X
X+C
6
+L
Cellulose ibe s (C
6
)
10
--
10
--
--
10
O ganosol lignin (L)
10
--
--
10
--
10
Hemicellulose (C
5
)b
5
5c
5
5
--
--
Xylose (X)
--
--
--
--
5
5
c Unless o he wise s a ed 539
540
25
541
Table 3: Concen a ion o suga s and phenols in he C5+C6+L medium 542
Fungi Day Glucose
(g L-1)
Xylose
(g L-1)
Rhamnose
(g L-1)
Xylose
oligome s
(g L
-1
)
Phenolic compounds
(g L-1)
I. lac eus 3 0.14 ± 0.04 1.53 ± 0.23 0.62 ± 0.07 1.70 0.450 ± 0.010
7 1.59 ± 0.21 0.70 ± 0.13 0.54 ± 0.08 NA NA
9 0.22 ± 0.07 0.55 ± 0.26 0.45 ± 0.21 1.50 0.101 ± 0.002
G. lucidum 3 0.21 ± 0.09 1.37 ± 0.14 0.68 ± 0.02 1.70 0.380 ± 0.010
7 0.07 ± 0.02 0.09 ± 0.02 0.24 ± 0.03 NA NA
9 0.09 ± 0.01 0.09 ± 0.03 0.21 ± 0.02 1.61 0.124 ± 0.002
NA: no a ailable 543
544
545
