ORIGINAL ARTICLE
E ec s o Penicillium ch ysogenum a . halophenolicum on k a
lignin: colo s abiliza ion and cy o oxici y e alua ion
Ma lene Reme
´dios
1
•Filomena A. Ca alho
2
•F ancisco J. Engui a
2
•
Ca los Ca doso
3
•I o C. Ma ins
2
•Nuno C. San os
2
•Ana Lu
´cia Lei a
˜o
1
Recei ed: 11 No embe 2015 / Accep ed: 21 Ma ch 2016
ÓThe Au ho (s) 2016. This a icle is published wi h open access a Sp inge link.com
Abs ac Wood indus ies and ag icul u al c ops gene -
a e an inexhaus ible supply o by-p oduc s like lignin,
which cons i u es an en i onmen al p oblem. Inc easing
e o s ha e been done o ind new applica ions o lignin.
One o hem is as a ood addi i e, bu i s chemical na u e
makes i sensi i e o b owning which cons i u es a majo
d awback o his ype o lignin applica ion. In he p esen
s udy we a e documen ing how colo s abiliza ion o a
comme cial k a lignin was achie ed a e he ea men
wi h Penicillium ch ysogenum a . halophenolicum. In
addi ion he ungal capaci y o emo e lignin is s udied
oge he wi h he e ec o i s ea men on cy o oxici y o
lignin. P. ch ysogenum a . halophenolicum was able o
ans o m lignin, ensu ing i s colo s abili y o mo e han
24 mon hs. Dynamic ligh sca e ing and a omic o ce
mic oscopy showed ha he ungus con ibu ed o
homogenize pa icle size and hyd odynamic p ope ies in
lignin suspensions wi hou inc ease he oxici y o e HeLa
cells and human p ima y ib oblas s. These indings sug-
ges new uses o k a lignin a e P. ch ysogenum a .
halophenolicum ea men p o iding an e ec i e app oach
o imp o e colo s abili y.
Keywo ds Penicillium ch ysogenum a .
halophenolicum Colo Lignin emo al Toxici y 
T ans o ma ion
In oduc ion
Lignin is he second mos abundan na u al p oduc o
ege al o igin, con e ing impe meabili y and s uc u al
suppo o plan s (Bau hoo e al. 2008;Pe
´ ez and Mo -
aleda-Mun
˜oz 2011). I s high-molecula weigh s uc u e is
gene ally achie ed by dehyd ogena i e polyme iza ion o
h ee p ima y hyd oxycinnamyl alcohols (monolignols):
coni e yl alcohol (guaiacyl p opanol, G), couma yl alcohol
(p-hyd oxyphenyl p opanol, H), and sinapyl alcohol (sy-
ingyl p opanol, S), being classi ied as a h ee-dimensional
amo phous polyme . As a majo componen o lignocel-
lulosic ma e ial, lignin is ound in was es ha a e p oduced
in la ge amoun s by many indus ies including o es y,
ag icul u e and ood. Global wood consump ion is a ound
3.5 910
9
m
3
/yea , being b oadly applied o pulp and
pape p oduc s, p oduc ion o uel and building ma e ials
(Ma inez e al. 2005). This huge p oduc ion gene a es a
la ge amoun o by-p oduc s wi h nega i e impac s o he
aqua ic, and consequen ly o he e es ial ecosys em. The
ad e se e ec is due no only o he o ganic load and
oxici y o e luen s, bu also o he es he ically unaccep -
able colo o wa e bodies la gely due o lignin and i s
de i a i es. The b ownish da k colo a ion o wa e bodies
could be he esul o enzyma ic ac i i ies o e a oma ic
compounds p esen in lignocellulose-de i ed ma e ials.
Among hem, polyphenol oxidases ca alyze he oxida ion
&Ana Lu
´cia Lei a
˜o
[email p o ec ed]
1
Depa amen o de Cie
ˆncias e Tecnologia da Biomassa,
Faculdade de Cie
ˆncias e Tecnologia, Uni e sidade NOVA de
Lisboa, Quin a da To e, Campus de Capa ica,
2829-516 Capa ica, Po ugal
2
Ins i u o de Medicina Molecula , Faculdade de Medicina,
Uni e sidade de Lisboa, A . P o . Egas Moniz,
1649-028 Lisbon, Po ugal
3
Ins i u o Nacional do Ma e A mos e a, Unidade de
Valo izac¸a
˜o dos P odu os da Pesca da Aquicul u a,
L-IPIMAR, IP-INRB, A . B ası
´lia, 1449-006 Lisbon,
Po ugal
123
3 Bio ech (2016) 6:102
DOI 10.1007/s13205-016-0414-x
o o ho-dihyd oxyl phenols in he p esence o molecula
oxygen o he co esponding o ho-quinones, which spon-
aneously polyme ize o p oduce da k o b own pigmen s
(Walke 1977).
One o cu en challenges aced by a mo e en i on-
men al- iendly science and indus y is o employ na u al
esou ces and was e p oduc s maximizing economic pe -
o mance. Indeed, new uses o lignin as uel, bioma e ials,
biocides, bios abilise s, c op cul i a ions and animal eed is
now a eali y (Lo a and Glasse 2002). Mo eo e , i was
sugges ed ha pu i ied lignins may also b ing new heal h
bene i s o animals (Bau hoo e al. 2008). Howe e , he
colo s abili y o lignin p oduc s cons i u es a majo
d awback o i s gene al use as addi i e. In his con ex , he
colo p ese a ion o ood p oduc s du ing p ocessing and
s o age is one o he main challenges o ood indus y;
since hei o ganolep ic p ope ies a e an impo an equi-
si e ha con ibu es decisi ely o ood accep ance, based
on he ini ial pe cep ion o ood condi ion and deg ee o
p ocessing, among o he cha ac e is ics (Lopez-Nicolas
and Ga cia-Ca mona 2007).
Due o i s a oma ic na u e and s uc u al complexi y,
lignin is esis an o he a ack o he majo i y o mic oo -
ganisms (Bau hoo e al. 2008;Pe
´ ez and Mo aleda-Mun
˜oz
2011). Indeed, in na u e, whi e- o ungi a e he only e i-
cien lignin deg ade s, since hey ha e he abili y o com-
ple ely mine alize his polyme , and consequen ly hey a e
qui e impo an in he global u no e o ca bon om woody
plan s (Wong 2009). The bes s udied a e Phane ochae e
ch ysospo ium,Pleo o us os ea us,Bje kande a adus a,
Pycnospo us cinnaba inus,Ce ipo iopsis sub e mispo a,
Phlebia sp., o T ame es e sicolo (Pe
´ ez and Mo aleda-
Mun
˜oz 2011). Meanwhile, he e a e o he ungi ha ha e he
abili y o pa ially deg ade lignin. Fo ins ance, a Penicillium
ch ysogenum s ain was able o using k a , o ganosol , and
syn he ic dehyd ogena i e polyme ised lignins wi h low
deg ada ion a es (Rod iguez e al. 1994). Despi e o ha ,
only a ew epo s ha e discussed lignin deg ada ion by
Penicillium s ains (Polman e al. 1994; Rod iguez e al.
1994; Hao e al. 2006; Yada and Yada 2006;Dwi edi
e al. 2011); howe e , as a as we know k a lignin emo al
and colo s abiliza ion by impe ec ungi has no ye been
desc ibed. We ha e p e iously cha ac e ized a halo ole an
P. ch ysogenum s ain, P. ch ysogenum a . halopheno-
licum, isola ed om a sal mine (Lei a
˜oe al.2012), and able
o me abolize phenolic compounds unde osmo ic s ess
(Lei a
˜oe al.2007; Guedes e al. 2011).
In he p esen wo k, we aimed o s udy he e ec o P.
ch ysogenum a . halophenolicum on k a lignin ans-
o ma ion and colo s abili y, in es iga ing i his s ain has
he po en ial o become a b owning con ol agen , o impa
lignin wi h s able colo ha may p omo e i s use as an
addi i e o bio echnological applica ions.
Ma e ials and me hods
S ain and cul u e condi ions
P. ch ysogenum a . halophenolicum was used h oughou
his s udy; his s ain was isola ed om a sal mine in
Alga e, Po ugal, and p e iously cha ac e ized (Lei a
˜o
e al. 2012).
P. ch ysogenum a . halophenolicum was main ained a
4°C on nu ien aga pla es (Di co, BD diagnos ic sys ems,
Hun Valley, MB, USA) wi h 2 % (w/ ) NaCl. P e-cul u es
o cells we e ou inely ae obically cul i a ed (160 pm in a
Ce oma
Ò
BS-T Incuba o , Sa o ius s edim bio ech,
Goe ingen, Ge many) a 25 ±1°C in 100 mL o complex
medium (MC: glucose, 30.0 g/L; NaNO
3
, 3.0 g/L; MgSO
4-
7H
2
O, 0.5 g/L; NH
4
Fe(SO
4
)
2
12H
2
O, 10.0 mg/L; K
2
HPO
4
,
1.0 g/L; yeas ex ac , 5.0 g/L; NaCl, 20.0 g/L; pH 5.6).
To in es iga e he abili y o ans o m k a lignin, he
s ain was cul i a ed in 500-mL lasks con aining 100 mL
o MC du ing 68 h. Cells we e collec ed by cen i uga ion
and washed in 0.85 % (w/ ) o NaCl. A 15 % o he p e-
inoculum was inocula ed in modi ied Jansheka medium
(Jansheka e al. 1982) con aining NaNO
3
(1.00 g) in
subs i u ion o NO
3
NH
4
(0.496 g) and wi hou i amin
solu ion and ni ilo iace a e as a componen o ace ele-
men solu ion and amended wi h 1700 mg/L k a lignin
(alkali, low sul ona e con en was ob ained om Sigma-
Ald ich, S . Louis, MO, USA). P. ch ysogenum a .
halophenolicum was ae obically incuba ed (160 pm in he
Ce oma
Ò
BS-T Incuba o ) in he da k du ing 96 h, a
25 °C. Th ee eplica es we e used. Abio ic assays we e
pe o med in pa allel wi h uninocula ed lasks (duplica es)
as nega i e con ols.
A e egula imes o cul u e, cells we e ha es ed and
12 mL o supe na an we e kep ozen a -40 °C un il
lignin de e mina ion assays, while app oxima ely 12 mL
we e main ained a oom empe a u e o lignin colo s a-
bili y expe imen s.
Mic obial d y biomass was es ima ed g a ime ically by
he me hod desc ibed by Gun he e al. (1995).
Lignin quan i ica ion and colo s abili y e alua ion
The comme cial k a lignin is an ideal subs a e o
assessing colo s abili y and biological ans o ma ion o
lignin. Fi s ly, he use o comme cial alkali lignin a oids
he conce ns abou chemical composi ion, he isola ion
me hod, o pu i y, since i is a comme cial p oduc . Sec-
ondly, comme cial alkali lignin was employed in o he
s udies o assess chemical e ec on he s uc u e o lignins
(Kadam and D ew 1986; Supa no e al. 2005; Yuan e al.
2010; DeAngelis e al. 2011; Geo ge e al. 2011; Huang
e al. 2013).
102 Page 2 o 10 3 Bio ech (2016) 6:102
123
K a lignin colo s abili y expe imen s we e conduc ed
in a lab oom i ed wi h a chambe empe a u e se a
22 °C wi hou any special s o age condi ion. A e a ime
o 7, 50 days and 7 mon hs, samples we e il e ed be o e
colo measu emen , as desc ibed in he sec ion o colo
measu emen . All assays we e pe o med in n=3 epe i-
ions. The mean alues and s anda d de ia ions we e
e alua ed using analysis o a iance (ANOVA).
Lignin concen a ions we e quan i ied by spec opho o-
me ic abso p ion a 205 nm in a Spekol
Ò
1500 UV VIS
Spek alpho ome e (Analy ic Jena AG, Ge many).
A omic o ce mic oscopy
A NanoWiza d II a omic o ce mic oscope (JPK Ins u-
men s, Be lin, Ge many), moun ed on he op o an Axio-
e 200 in e ed mic oscope (Ca l Zeiss, Jena, Ge many)
was used o imaging he samples. The AFM head is
equipped wi h a 15-lm z- ange linea ized piezoelec ic
scanne and an in a ed lase . Samples we e dilu ed o
1:200 in Milli-Q wa e and deposi ed on eshly clea ed
musco i e mica o 20 min. A e subsequen washes, he
sample was allowed o ai d y a oom condi ions. Imaging
o he samples componen s we e pe o med in ai apping
mode ( o he 1s day ungal ea ed samples) and in con-
ac mode ( o he 4 h day ungal ea ed samples). Oxi-
dized sha pened silicon ips (ACL ips om Applied
Nanos uc u es, CA) wi h a ip adius o 6 nm, esonan
equency o abou 190 kHz and sp ing cons an o 45 N/m
we e used o he measu emen s. Imaging pa ame e s we e
adjus ed o minimize he o ce applied on he scanning o
he opog aphy o he samples. Scanning speed was op i-
mized o 0.4 Hz (on he i s day o ea men sample) and
0.8 Hz (on he ou h days o ea men sample), and
acquisi ion poin s we e 512 9512 and 360 9360,
espec i ely. Imaging da a we e analyzed wi h he JPK
image p ocessing .3 (JPK Ins umen s). The wid h and
heigh o he samples we e calcula ed om he c oss sec-
ion plo s. All complex dimensions measu emen s we e
pe o med using he Gwyddion so wa e (Czech Me ology
Ins i u e, B no, Czech Republic), e sion 2.19.
Dynamic ligh sca e ing (DLS) measu emen s
Dynamic ligh sca e ing expe imen s we e ca ied ou a
25 °C on a Mal e n Ze asize Nano ZS (Mal e n, UK), wi h
a backsca e ing de ec ion a 173°, equipped wi h a He–Ne
lase (k=632.8 nm), using glass cu e es wi h ound
ape u e. K a lignin samples we e dilu ed 1/10 in MiliQ
wa e The samples we e le equilib a ing o 15 min a
25 °C be o e each measu emen s se (10 measu emen s;
each one being he a e age o 10 uns, wi h 10 s pe un).
No malized in ensi y au oco ela ion unc ions we e
analyzed using he CONTIN me hod (P o enche 1982a,b),
yielding a dis ibu ion o di usion coe icien s (D). The
measu ed D(m
2
/s) was used o he calcula ion o he
hyd odynamic diame e , D
H
(nm), h ough he S okes–
Eins ein ela ionship (Be ne and Peco a 1990):
DH¼jT
3pgD
whe e jis he Bol zmann cons an (J/K), T he absolu e
empe a u e (K), and g he medium iscosi y (Pa s). The
da a we e s a is ically analyzed wi hin each se o 10
measu emen s by obse ing he a e age and s anda d
de ia ion, and disca ding ou lie s. The a e age wi hou
ou lie s became close o he median in all he size poin s.
Colo measu emen
Samples colo was de e mined using a Macbe h eye 3000
colo ime e . The colo in he CIELAB sys em is cha ac-
e ized by h ee pa ame e s, L*,a* and b*. The ligh ness
alue (L*), aking alues om 0 % (black) o 100 %
(whi e), a* om g een (-a) o ed (?a) and b* om blue
(-b) o yellow (?b). The L*,a* and b* colo coo dina es
o each g oup o samples we e measu e a e s abili y
expe imen . These alues we e hen used o calcula e he
colo change DE* as a unc ion o he s abili y expe imen
du a ion acco ding o he ollowing equa ions:
DL¼L
L
i
Da¼a
a
i
Db¼b
b
i
DE¼ðDL2þDa2þDb2Þ1=2
whe e DL*, Da* and Db* a e he changes be ween he
ini ial (i) and he inal ( ) alues and DE* co esponds o a
colo change. The colo e olu ion assays we e pe o med
using he measu emen s a ime 7 days as s anda ds, which
co espond o he i s measu emen (i) ha was made a e
he ungal ea men , mycelia emo al and s o age a
22 °C.
Cy o oxici y e alua ion
Quan i ica ion o he oxic e ec s o k a lignin and ungal
ea ed samples a he cellula le el was pe o med by he
use o Alama Blue
Ò
es o quan i y HeLa and human
p ima y skin ib oblas s (Co iell Ins u u e o Medical
Resea ch, e . GM05565) iabili y (In i ogen) using a
modi ied e sion o an al eady desc ibed p o ocol
(Al-Nasi y e al. 2007). B ie ly, HeLa and ib oblas cells
we e pla ed in 96 well pla es a 10
5
cells pe well in RPMI
medium con aining 10 % FBS, and incuba ed a 37 °C and
3 Bio ech (2016) 6:102 Page 3 o 10 102
123
5%CO
2
du ing a leas 8 h o allow cell a achmen o he
pla e su ace. A e his incuba ion ime, RPMI was sub-
s i u ed by Op i-MEM medium o a oid se um in e e ence
wi h he assay. Cells we e hen exposed o di e en dilu-
ions o he P. ch ysogenum a . halophenolicum cul u e
supe na an s, p e iously bu e ed wi h 10 9PBS and
s e ilized by il a ion hough 0.22 lm memb anes. A e
48 h, HeLa and ib oblas cells we e washed wice wi h
PBS and incuba ed du ing 1 h a 37 °C wi h Alama Blue
Ò
ollowing he ecommenda ions om he manu ac u e .
Cell iabili y was quan i ied by de e mina ion o he luo-
escence o Alama Blue
Ò
a 590 nm a e exci a ion a
530 nm in a luo escence pla e eade (TECAM In ini e
M200). EC
50
we e de e mined by non-linea eg ession
using an equa ion o a sigmoid cu e (G aphPad P ism
5.02).
Resul s and discussion
Lignin emo al
To de e mine he abili y o P. ch ysogenum a . halophe-
nolicum o emo e k a lignin, he mic oo ganism was
cul u ed in he p esence o 1700 mg/L o comme cial k a
lignin. Since no abio ic loss o lignin was de ec ed in
con ol samples, he dec ease o lignin concen a ion in he
p esence o ungus mus be due o i s biological ac ion. The
g ow h o P. ch ysogenum a . halophenolicum and lignin
emo al is depic ed in Fig. 1. A he ini ial concen a ion o
1700 mg/L o k a lignin no lag phase was obse ed. A
lignin emo al o 68.0 % was achie ed a e 96 h o ungal
ea men , showing a clea co ela ion wi h ungal g ow h.
Indeed, da a indica e ha he e we e wo peaks in biomass,
co esponding o exponen ial g ow h phase. The i s one
should be due o he p esence o glucose in he cul u e
media composi ion besides lignin, since he ungal inocu-
lum was p epa ed in a complex medium wi h glucose, and
i s enzyma ic sys em was al eady ac i e and eady o use
his subs a e. This ac was co obo a ed by he absence o
lag phase on ungal g ow h. A e 24 h o cul u e, a sig-
ni ican inc ease on ungal biomass and a concomi an
dec ease in he lignin concen a ion we e obse ed, indi-
ca ing ha P. ch ysogenum a . halophenolicum g ow h
was due o lignin ans o ma ion. In gene al, mos o
ligninoly ic ungi epo ed equi e a high le el o con-
sump ion o easily me abolized cosubs a e o be e ec i e
in he ligninolysis. The P. ch ysogenum a . halopheno-
licum was able o emo e mo e han 1000 mg/L o k a
lignin in he i s 24 h o ba ch cul u e, which is qui e
in e es ing; whe eas he he e opolyme concen a ion did
no signi ican ly dec eased du ing he las h ee days o
ungal ea men , p obably due o he C–C bonds o alka-
line lignin ha a e highly esis an o hyd oly ic b eaking
(Yuan e al. 2010). K a lignin is en iched wi h guaiacyl
g oups, whe e he C5 posi ion in he a oma ic ing ep e-
sen s he mos abundan C–C linkage in lignin molecules
(El Mansou i e al. 2011). Fungi a e he only mic oo -
ganisms ex ensi ely s udied o he deg ada ion o lignin.
A s ain o Penicillium ch ysogenum is able o deg ade
some syn he ic and na u al lignins, and mine alize 7.9 % o
[
14
C
b
] DHP in 29 days (Rod iguez e al. 1996).
Recen ly se e al s udies we e published using com-
me cial k a lignin. Among hem, Huang e al. epo ed
ha 500 and 700 mg/L o comme cial k a lignin is
deg aded by Bacillus pumilus and Bacillus a ophacus,
espec i ely, a e 18 days o incuba ion (Huang e al.
2013). A compa a i e s udy o na u al and comme cial
k a lignin deg ada ion by Ci obac e sp. was desc ibed,
showing i s highe capaci y o use k a lignin han com-
me cial k a lignin (252 and 186 mg/L, espec i ely)
(Chand a and Bha aga a 2013). Ou esul s indica e ha P.
ch ysogenum a . halophenolicum e icien ly emo ed
k a lignin.
E ec o ungal ea men on he e olu ion o lignin
colo
To e alua e he e ec o P. ch ysogenum a . halopheno-
licum on lignin colo , scala pa ame e s (L*,a*, and b*)
we e de e mined, a e 7 days o s o age, in samples wi h
di e en imes o ungal ea men . The ini ial L* alue o
un ea ed sample was 56.45. Fungal ea men did no
induce any signi ican change in L* alues (Table 1). A e
96 h o ungal ea men he L* alue was 56.88, indica ing
ha he componen s abso bing isible ligh emain
cons an .
Fig. 1 K a lignin emo al by P. ch ysogenum a . halophenolicum
du ing 96 h unde ae obic condi ions. Squa es cell g ow h; ci cles,
k a lignin concen a ion. Da a shown ep esen s a e age o ipli-
ca es ±s anda d de ia ions
102 Page 4 o 10 3 Bio ech (2016) 6:102
123
In he samples ea ed wi h he ungus du ing 24 h he
a* alues sligh ly inc eased, possibly due o he p esence o
deg ada ion and/o oxida ion p oduc s, ollowed by a
dec ease a la e cul u e imes. Ne e heless, he di e en
imes o cul u e led o lowe alues o a*, wi h he ini ial
g een colo s ill emaining a e 96 h o ea men
(Table 1). These esul s a e compa ible wi h chemical
changes o lignin.
The b* alues o samples inc ease a e ungal ea -
men . The inc ease in yellowness could be caused by he
low-molecula -weigh phenolic compounds ob ained by he
ac ion o ungal enzymes.
AFM s udies
In na u e, lignin is an amo phous polyme ic ma e ial o en
associa ed o cellulose ibe s as main cons i uen s o plan
cell walls. Since he exac s uc u e o lignin i sel is no
known (Chaka and Ragauskas 2004; Geo ge e al. 2011),
a omic o ce mic oscopy has been applied o cha ac e ize
he opog aphy and oughness o lignocellulosic ma e ials
in hei na u al o ms (Chundawa e al. 2011). The AFM
scanning images o lignin samples wi h one (a, b) and
4 days (c, d) o ea men wi h P. ch ysogenum a .
halophenolicum a e shown on Fig. 2.
F om he igu e, i can be e iden ly no iced ha ungal
ea men on he lignin samples leads o mo phological
changes wi hin he polyme . F om ai apping mode AFM
e o (a) and heigh (b) images o he sample ea ed
h ough only 24 h, i could be seen ha i is e y he e o-
geneous in size. Opposing his, he lignin sample wi h 96 h
o ea men is much mo e homogenous and only one size
popula ion could be isualized. To quan i a i ely analyze
AFM heigh images o hese popula ions, we measu ed he
diame e and he heigh o hei su ace p o iles (Fig. 2b, d,
igh images). C oss sec ion analysis o each ea u e on he
images was done, yielding dis inc p o iles. Examples o
hese c oss sec ions a e also shown (Fig. 2b, d, le ima-
ges). The analysis indica es ha he sample wi h 96 h o
ea men had a unique popula ion, wi h an a e age diam-
e e o 321.2 ±53.7 nm and heigh o 28.8 ±8.6 nm
(n=11). We could dis inguish h ee di e en popula ions
on he lignin sample wi h only 24 h o ungal ea men ,
wi h di e en dimensions: 42.5 ±6.0 nm (diame e ) and
5.1 ±1.0 nm (heigh ) on popula ion 1 (n=9);
124.2 ±17.3 nm (diame e ) and 16.8 ±2.7 nm (heigh )
on popula ion 2 (n=16); and, 221.9 ±36.9 nm (diame-
e ) and 34.8 ±10.9 nm (heigh ) on popula ion 3 (n=23).
The e we e no signi ican di e ences in he isualized
oughness o bo h analyzed samples (Fig. 2b, d, igh
images).
F om he calcula ed dimensions o he h ee popula ions
o he 24 h ea men sample, we could hypo hesize ha
popula ions 2 and 3 we e o med by oligome s o he lignin
s uc u es o popula ion 1. A e 96 h o ea men , he
subs an ial inc ease in size o he lignin pa icles achie ed
on he en i e popula ion e eals he exis ence o polyme -
iza ion o majo agg ega ion phenomena.
DLS analysis
DLS expe imen s o un ea ed lignin and ungal ea ed
samples a e shown in Table 2. Un ea ed lignin showed a
g ea a ia ion in pa icle sizes and poo ly esol ed peaks,
wi h h ee majo ones obse ed, one co esponding,
oughly, o 6–20 nm sized species, he o he o
20–1000 nm sized species and a inal one co esponding o
species well o e 3 lm. Taking only he i s wo (which
a e be e esol ed), he maxima o he peaks co espond o
a e age hyd odynamic diame e s (D
H
) o 16.6 nm (sca -
e ing in ensi y 34.4 %) and 629.7 nm (sca e ing in ensi y
65.6 %). Upon 24 and 48 h o ea men wi h he ungus,
he sample sizes become mo e uni o m and o e all smalle .
A e 24 h o ea men h ee majo peaks we e obse ed,
bu be e esol ed han in he con ol, since he i s one
co esponds mos ly o, app oxima ely, 10–20 nm sized
species, he second one o 150–400 nm sized species and
he hi d one o species wi h mo e han 2 lm. Taking again
only he i s wo (which a e much be e esol ed), he
maxima o he peaks co esponded o a e age hyd ody-
namic diame e s (D
H
) o 15.5 nm (sca e ing in ensi y
44.9 %) and 301.3 nm (sca e ing in ensi y 42.3 %). The
Table 1 E olu ion o L* and a* and b* coo dina es o k a lignin samples a e he absence and p esence o P. ch ysogenum a . halophe-
nolicum. A e ea men , ungal micelia we e emo ed and he samples we e s o ed a 22 °C du ing 7 days be o e colo es ima ion (n=3)
Fungal ea men (h) L* a* b*
Con ol 56.45 ±0.04 a -0.81 ±0.03 a 1.87 ±0.03 a
24 56.53 ±0.22 a -0.74 ±0.02 a 2.10 ±0.15 ab
48 56.82 ±0.02 b -0.91 ±0.06 ab 2.38 ±0.01 c
72 56.64 ±0.10 a -0.98 ±0.02 b 2.18 ±0.02 b
96 56.88 ±0.05 b -0.87 ±0.04 a 2.47 ±0.03 c
Di e en le e s ( e ically) indica e signi ican di e ences (p 0.05)
3 Bio ech (2016) 6:102 Page 5 o 10 102
123

ea men wi h P. ch ysogenum a . halophenolicum
appea ed o elimina e he la ge pa icles (cen e ed a ound
629.7 nm) esul ing in much smalle s uc u es, wi h abou
hal o he hyd odynamic diame e o he con ol samples
(hyd odynamic diame e cen e ed a ound 301.3 nm). These
esul s suppo he hypo hesis ha he ungus had he
abili y o use di e en ly sized lignin molecules, as
desc ibed ecen ly by Wang e al. in he lignosul ona e
biodeg ada ion p ocess by Sphingobac e ium sp. HY-H
(Wang e al. 2013). The hi d peak did no ha e a good
esolu ion, howe e , i is clea ha he o e all sca e ing
pa icles size dec eased, and ha also in pa allel, he
amoun o pa icles wi h sizes abo e 1 lm is diminished,
esul ing on an o e all ex ensi e educ ion and uni-
o miza ion in pa icles size.
The same p o ile was obse ed a e 96 h o ea men ,
bu in such an ex en ha he sample can be said o be
essen ially homogeneous, wi h one peak a 245.7 nm
(sca e ing in ensi y 92.4 %), wi h obse ed easonable
ag eemen wi h he AFM da a (321.2 ±53.7 nm). A small
un esol ed peak o pa icles wi h mo e han 1 lm was also
isible, bu wi h a limi ed ac ion o he in ensi y. Mean-
while, in he un ea ed samples h ee peaks clea ly emains
a e 96 h. These esul s we e consis en wi h he
Fig. 2 AFM scanning images o k a lignin wi h 1 and 4 days o
ea men wi h P. ch ysogenum a . halophenolicum. Ai apping
mode AFM e o (a) and heigh (b) images o k a lignin wi h 1 day
o ungal ea men (ho izon al scale 1lm91lm; heigh scale up
o 27.6 nm). Ai con ac mode AFM e o (c) and heigh (d) images o
k a lignin wi h 4 days o ungal ea men (ho izon al scale:
700 nm 9700 nm; heigh scale up o 66.5 nm). C oss sec ion
analysis o he AFM heigh images could also be pe o med. Th ee
examples o c oss sec ions o he di e en isualized popula ions
(b igh image) and one example o a homogeneous popula ion (d,
igh image) o he k a lignin p o iles a e shown. Wi h his ype o
analysis, lignin size, heigh , shape and oughness can be de e mined
Table 2 A e age pa icles size o ungal ea men samples (hyd odynamic diame e ) es ima ed by dynamic ligh sca e ing (DLS)
Incuba ion ime (h) Samples Peak 1 Peak 2 Peak 3
24 Fungal ea men 15.5 nm (44.9 %) 301.3 nm (42.3 %) 3590.0 nm (12.8 %)
Con ol 16.6 nm (34.4 %) 629.7 nm (65.6 %) No majo peak
48 Fungal ea men 15.2 nm (5.0 %) 191.5 nm (80.6 %) 3551.0 nm (12.8 %)
Con ol 17.0 nm (29.5 %) 718.4 nm (64.0 %) 4202.0 nm (6.5 %)
96 Fungal ea men No peak 245.7 nm (92.4 %) 4397.0 nm (7.6 %)
Con ol 13.0 nm (35.3 %) 250.5 nm (43.0 %) 2053.0 nm (21.7 %)
Size ep esen s he peak a e age based on size dis ibu ion by olume; he pe cen ages ep esen he peak a ea, da k shadowed a ea highligh s
homogeneously sized samples (e.g., samples wi h o e 80 % o he pa icles included wi hin a single size dis ibu ion peak)
102 Page 6 o 10 3 Bio ech (2016) 6:102
123
hypo hesis ha he p esence o P. ch ysogenum a .
halophenolicum could p omo e he polyme iza ion o lig-
nin in o well-de ined and homogenously sized samples.
O e all, and in spi e o he small di e ences be ween
obse ed AFM and DLS pa icles size due o he di e en
p ecision le el o he wo echniques (DLS size es ima ion
may be dis o ed by la ge pa icles, while he la ening o
he sample o a discoidal shape on he AFM subs a e
and/o con olu ion wi h he AFM ip dimensions also
a ec s size de e mina ions), he esul s ob ained using
AFM opog aphic images we e in acco dance wi h hose
achie ed ia DLS, showing ha no only he low-molec-
ula -weigh o ganic compounds, bu also he polycyclic
a oma ic compounds can be deg aded by P. ch ysogenum
a . halophenolicum wi h he concomi an uni o miza ion
o he lignin samples in o a single sized species a e 96 h
o ea men wi h he ungus.
Lignin colo s abili y
As desc ibed abo e, DLS and AFM show ha , a e 96 h o
ungal ea men , he lignin samples become highly
homogenized in e ms o size, es ima ed o be below
390 nm, he app oxima e lowe limi o ligh de ec ion by
he human eye (S a 2005). This ac p e en s, among
o he phenomena ha may in e e e wi h colo s abili y, he
occu ence o ligh sca e ing phenomena. This can occu
in ela i ely dense solu ions, especially, i hey o m di -
e en ly sized pa icles ha migh e en ually modi y he
colo pe cep ion. Howe e , in his pa icula case, ligh
sca e ing by hese uni o mly sized lignin pa icles
( 390 nm) would no be a p oblem since i would be
al eady in he ul a iole ligh spec um, in isible o he
human eye, which helps s eng hen he po en ial o lignin
ea ed samples as colo s abilize . To u he e alua e his
po en ial, he ligh ness alues o samples ea ed wi h he
P. ch ysogenum a . halophenolicum a e 50 days and
7 mon hs unde s abili y assay condi ions, we e compa a-
ble o sligh ly highe han he ini ial L* alues (Table 3).
Mo eo e , colo s abili y inc eased a e 50 days o
s o age, since he o e all colo change (DE*) alues o he
samples ea ed wi h he ungus we e smalle han hose o
he con ol samples (Table 3).
Fo ea ed ungal samples, he colo change o 24 h
was 0.55. The colo change alue was 0.17 o 96 h in he
ea ed ungal samples. These esul s showed a ace
isual change in samples ea ed mo e han 48 h wi h P.
ch ysogenum a . halophenolicum, acco ding o he scale
p oposed by Di ckx e al. (Di ckx e al. 1992). Fu he -
mo e, he o e all colo change dec eased as ungal con-
ac ime inc eased, sugges ing heigh ened colo s abili y.
A he end o 7 mon hs s o age, a sligh inc ease o DE*
in he ea ed samples was obse ed, when compa ed o
he alues ob ained a 50 days o s o age. Ne e heless, in
all samples ea ed wi h ungus a e 7 mon hs o s o age,
he o e all colo change was lowe han ha o he con-
ol wi h 50 days. Fu he mo e, in he cul u e ea ed
du ing 96 h no signi ican colo change was obse ed
be ween 7 and 26 mon hs o s o age, which is in excellen
ag eemen wi h he DLS and AFM size es ima ion
showing s able uni o m molecules (a e ungal ea -
men ). O e all, his s ongly suppo s he hypo hesis ha
he ea men wi h P. ch ysogenum a . halophenolicum
s abilizes he lignin molecules p ope ies, especially in
wha ega ds o colo and o i s possible use as a colo
modi ies/s abilize .
Cy o oxici y o k a lignin be o e and a e ungal
ea men
The e alua ion o he po en ial oxic e ec s o lignin
me aboli es was conduc ed by he de e mina ion o he
ela i e p opo ion o iable cells, by he Alama Blue
Ò
assay. This es is based on he con inuous con e sion by
iable cells o esazu in, a non- luo escen cell pe meable
pigmen , o eso u in, a luo escen compound ha can be
de e mined by luo ime ic assay, yielding a quan i a i e
measu emen o cell su i al. We used HeLa cells and
human p ima y skin ib oblas s iabili y as an indica o o
he po en ial oxic e ec s o lignin and i s me aboli es. The
EC
50
alues o k a lignin showed ha i had cy o oxic
e ec s, bu only a e y high concen a ions (Fig. 3). In
incuba ions wi h ib oblas s, he EC
50
o cy o oxici y was
highe han HeLa cells (EC
50
o 5177 ±1726 and
1223 ±328 mg/L, espec i ely in ib oblas s and HeLa
cells). A he concen a ions o 750, 1000, 2000 mg/L, we
obse ed a signi ican dose-dependen inc emen in he
HeLa cells iabili y. A good co ela ion be ween he
cy o oxic e ec s on HeLa cells and on ib oblas s cells
a e 48 h was obse ed ( =0.9775 and =0.9509,
espec i ely). As i is p e iously epo ed he di e ences
be ween a cance cell line and p ima y ib oblas s can be
a ibu ed o di e ences in cell sensi i i y o he compound
Table 3 E olu ion o colo di e ence (DE*) a e 50 days, 7 and
26 mon hs o s o age
Fungal ea men (h) DE*
50 days/
7 days
7 mon hs/
7 days
26 mon hs/
7 days
Con ol 0.59 ±0.52 1.20 ±0.62 n.d.
24 0.55 ±0.19 0.90 ±0.67 1.06 ±0.19
48 0.26 ±0.01 0.60 ±0.04 n.d.
96 0.17 ±0.04 0.61 ±0.05 0.68 ±0.05
n.d. no de e mined
3 Bio ech (2016) 6:102 Page 7 o 10 102
123
ha is assayed and would be mainly ela ed wi h he cell
di ision a e (Uga ondo e al. 2008).
To assess i lignin me aboli es gene a ed om P.
ch ysogenum a . halophenolicum biological ac i i y a e
non oxic o HeLa and ib oblas cells, new expe imen s
we e done. The o iginal samples we e dilu ed in cell cul-
u e medium be o e o cy o oxici y es ing o ob ain a
concen a ion ange simila o hose whe e s anda d lignin
concen a ion begins o be oxic o he es ed cells. Resul s
shown in Fig. 4clea ly indica e ha bo h HeLa and
ib oblas cells iabili y we e no signi ican ly modi ied by
he exposu e o P. ch ysogenum a . halophenolicum, in
compa ison wi h he s anda d lignin a he same concen-
a ions, indica ing ha he lignin me aboli es gene a ed by
P. ch ysogenum a . halophenolicum we e no cy o oxic.
These esul s we e qui e impo an since i is epo ed
ha many dyes a e belie ed o be oxic, some o hem as a
esul o mic obial me abolism (Cou o 2009). Addi ionally,
Penicillium species a e known o p oduce a high abun-
dance o seconda y me aboli es including myco oxins such
as oc a oxin A, ci inin, e ucosidin, pa ulin, among
o he s, which ha e di e en deg ees o oxici y on a ge
o gans o a e ca cinogenic (Cha
´ ez e al. 2011). The e o e,
i is impo an o assess he cy o oxic e ec s o lignin
samples a e ea ed wi h P. ch ysogenum a . halophe-
nolicum o disca d any possible cy o oxic p ope ies.
Despi e he po en ial o biological haza d is low o
ungi con e ed eed as so a , assess cell iabili y is o
majo impo ance o ensu e consume heal h and sa e y.
The p esen da a indica ed ha P. ch ysogenum a .
halophenolicum, in his es condi ions, did no inc ease
lignin oxici y, showing ha k a lignin can be used o e
an e ec i e concen a ion ange ha is sa e o no mal and
cance cell lines s udied.
The en i onmen al p o ec ion agencies a e becoming
mo e es ic i e ega ding wa e discha ge om indus ial
e luen s. Se e al indus ies, besides he classical p ima y
and seconda y ea men s, use physical and chemical e -
ia y ea men s o emo e he colo cause by lignin and
lignin de i a i es, which a e expensi e and no always lead
o high pe o mance. Biological echnology is a easible
and p omising al e na i e. Fungi ha e a ac ed a g ea deal
Fig. 3 K a lignin dose
esponse cu es using he
Alama Blue assay. Cu es a e
examples ob ained by a e age
o i e expe imen al eplica es
and illus a e he esponse o he
human p ima y skin ib oblas s
(a) and HeLa cells (b)
Mock
Lignin s anda d
24 h
48 h
96h
0
20
40
60
80
100
Cell su i al (%)
BA
Mock
Lignin s anda d
24h
48h
96 h
0
20
40
60
80
100
Cell su i al (%)
Fig. 4 HeLa and ib oblas cells iabili y quan i ied by Alama Blue
assay a e incuba ion wi h ungal ea ed and un ea ed k a lignin
p epa a ions. aHeLa cells, bhuman p ima y skin ib oblas s. Mock,
un ea ed cells; K a lignin s anda d, cells incuba ed wi h soluble
lignin a a concen a ion o 50 mg/L; 24, 48 and 96 h ep esen he
ime o ungal ea men . Cell su i al was calcula ed as he a io o
luo escence be ween sample o s anda d and mock
102 Page 8 o 10 3 Bio ech (2016) 6:102
123
o in e es as po en ial biomass and ecalci an compounds
deg ade s such as lignin due o hei abili y o p oduce a
b oad di e si y o ex acellula ligninoly ic enzymes some
o hem wi h lack speci ici y o a pa icula subs a e. I
whi e- o ungi comple ely mine alize lignin, o he ungi
and bac e ia lead o he p oduc ion o b own pigmen s
which limi s he p ac ical use o such ag o esidues.
Meanwhile, al hough lignin was gene ally conside ed o be
nu i ionally ine , new scien i ic da a on heal h-p o ec i e
mechanisms o ce eals endo se he opposi e idea (Fa de
2010). Meis e ad anced a new possible use o modi ied
lignin o pe and human ood as oughage, a ibe sou ce, o
a cance p o ec ion agen (Meis e 2002). Recen ly, To o a
e al. desc ibed he use o k a lignin as he aw ma e ial
o mic ocapsula ion p ocesses assembled by ul asound
in o lignin mic ocapsules o s o age and elease Couma in-
6 (To o a e al. 2014). Se e al esea che s poin ed ou ha
a lignin wi h a mo e cons an s uc u e and size will
enhance i s po en ial u iliza ion in high- alue p oduc s
(No g en and Edlund 2014; Qu e al. 2015).
In summa y, his wo k showed ha P. ch ysogenum a .
halophenolicum was able o ans o m k a lignin, dis-
playing a highe capaci y o g ow on lignin subs a es.
AFM and DLS assays indica ed ha k a lignin bio ans-
o ma ion p oceeds ei he ia low-molecula phenolic
compounds o oligome s. P. ch ysogenum a . halophe-
nolicum ea men esul ed in a s abiliza ion o comme cial
soluble k a lignin colo . I is also ema kable ha lignin
colo s abili y was eco ded o mo e han 24 mon hs a
22 °C unde ligh condi ions o s o age. These esul s we e
compa ible wi h he hypo hesis ha P. ch ysogenum a .
halophenolicum could be a po en ial ool o s abilizing
lignin agains colo change by ans o ming he he e oge-
neous lignin composi ion in a mo e homogenous s uc u e.
The inc eased lignin s abili y was achie ed wi hou
inc easing oxici y o e HeLa and ib oblas cells. These
indings could p omo e he applica ion o P. ch ysogenum
a . halophenolicum o he de elopmen o lignin- o i ied
o mula ha would imp o e ood ibe con en .
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