179
B azilian A chi es o Biology and Technology
Vol.47, n. 2 : pp. 179-183, June 2004
ISSN 1516-8913 P in ed in B azil BRAZILIAN ARCHIVES OF
BIOLOGY AND TECHNOLOGY
A N I N T E R N A T I O N A L J O U R N A L
Fed-ba ch Decolo iza ion o Poly R-478 by T ame es
e sicolo
Ma ía Te esa Mo ei a1, Césa Viaca a2 and Gladys Vidal3∗
1 Dep . o Chemical Enginee ing; Ins i u e o Technology; Uni e si y o San iago de Compos ela; E-15706 San iago
de Compos ela - Spain. 2 Dep . o Chemical Enginee ing; Uni e si y o La F on e a; P. O. Box 54-D; Temuco -
Chile. 3 En i onmen al Science Cen e EULA-Chile; Uni e si y o Concepción; [email p o ec ed]; P. O. Box 160-C;
Concepción - Chile
ABSTRACT
Physiological aspec s we e e alua ed o de e mine op imal condi ions o he decolo iza ion o a syn he ic dye, Poly
R-478, by whi e- o ungus T ame es e sicolo # 52J. The decolo iza ion expe imen s we e ca ied ou in
semicon inuous ope a ion du ing h ee cycles o imp o e he p ocess e iciency. The bes decolo iza ion e iciencies
(65% o 80%) we e ob ained in ungal cul u es pe o med in ni ogen limi ed condi ions unde ae obic condi ions.
Key wo ds: Decolo iza ion, T ame es e sicolo , Poly R-478, ligninoly ic enzymes
∗ Au ho o co espondence
INTRODUCTION
The e is cu en ly conside able en i onmen al
in e es in he p oblem o colo emo al o a wide
ange o was ewa e s. In he ex ile manu ac u ing
indus y, up o 50% o he dyes a e los a e he
dyeing p ocess and he e o e, hey a e disposed
ou in la ge quan i ies in he e luen s. Mo eo e ,
mos o he dyes a e o en i onmen al conce n
because o hei oxic p ope ies on li ing
ecosys ems (Dawson, 1981). These e luen s cause
a signi ican loss in luminosi y and inc ease in
empe a u e, which g ea ly diminishes he
dissol ed oxygen concen a ion in was ewa e s,
wi h he subsequen al e a ion o he aqua ic li e
(Young and Yu, 1997). Be o e hei disposal, hey
a e o be ea ed o educe hei le els o oxici y
and hus, o minimize pollu ion impac . Physic
chemical ea men s ha e high ope a ional cos s
associa ed and limi ed applicabili y, which ende
hese echniques una ac i e. O he con en ional
p ocesses based on biological ea men (ae obic-
anae obic) a e ela i ely ine ec i e in e luen
decolo iza ion, because high molecula weigh
compounds a e no easily deg aded by bac e ia,
and colo ed compounds pass h ough he ea men
sys em la gely undeg aded (Lema e al., 2002).
Ligninoly ic pe oxidases sec e ed by whi e- o
ungi may comp ise a biological ea men
p oposal since hey possess an ex acellula
deg ada ion sys em, which enables he deg ada ion
o lignin (Bumpus e al., 1985). This deg ading
abili y has opened new p ospec s o he
de elopmen o bio echnological p ocesses aimed
a he deg ada ion o complex polyme s, such as
xenobio ic (polya oma ics, polyphenolics, e c.)
compounds (Field e al., 1993), e luen
decolo iza ion (Lema e al., 2002) and
biobleaching o k a pulp (Mo ei a e al., 1997).
Se e al epo s ha e desc ibed bio eac o designs
o indus ial e luen decolo iza ion om whi e-
o ungi in subme ged and immobilized liquid
Mo ei a, M. T. e al.
B azilian A chi es o Biology and Technology
180
cul u es (Palle la and Chambe s, 1997; Roye e
al., 1991; Mielgo e al., 2002). The aim o he
wo k was ocused on he knowledge o he
deg ada i e capabili y o a complex compound
wi h he inal objec i e o acili a ing he
de elopmen o decolo iza ion sys ems. The
decolo iza ion o a polyme ic dye Poly R-478,
wi h s uc u al simila i y o lignin, has been
selec ed as a model compound.
MATERIALS AND METHODS
Mic oo ganism and cul u e condi ions
T ame es e sicolo #52J was kindly p o ided by
M. Paice, Pap ican (Pulp and Pape Ins i u e o
Canada). The ungus was main ained a 4ºC on
pep one yeas ex ac slan s om which i was
ans e ed o glucose mal ex ac (ME) pla es
(Mes e e al., 1996). The ME pla es we e
incuba ed a 30ºC o 7 days. Fi e aga plugs (5-
mm diame e ) we e punched om he leading edge
and cul u ed in Fe nsbach lasks wi h 100 mL o
N-limi ed cul u e medium (C/N a io 62.5/1)
con aining 10 g/L glucose, 2.2 mM NH4+-N as
ammonium a a e and BIII mine al medium
(Tien and Ki k, 1988) o 6 days a 30ºC. A e
his ime, he mycelium was c ushed in a Blende
o 1 min be o e i s use as he inoculum.
E lenmeye lasks (250 mL) con aining 0.1 g/L
dye, 90 mL o he N-limi ed medium was
inocula ed wi h 10% ( / ) homogenised mycelium
in iplica e assays. Abio ic con ols we e un in
pa allel. Decolo iza ion expe imen s we e
pe o med in an o bi al shake (150 pm) a 30°C
unde an ai o oxygen a mosphe e by he
pe iodical lushing o O2 gas (0.8 ba manome ic
p essu e). The cul u e b o h was pe iodically
moni o ed o MnP ac i i y and glucose. A e
glucose deple ion condi ions and pe oxidase
peaks, wo ed-ba ch addi ions o he dye (0.1 g/L)
and glucose (5 g/L) was in oduced o pe o m
decolo iza ion.
Dye decolo iza ion a e
The decolo iza ion o Poly R-478 dye
(polyan h aquinone) was moni o ed by he
pe cen age educ ion in he abso bance a io a 520
and 350 nm. Be o e he abso bance eadings, 0.2
mL o cen i uged sample (8,000 pm, 10 min)
was dilu ed wi h 0.8 mL o dis illed wa e and
measu ed a 30ºC wi h a 1-cm cu e e in a
spec opho ome e (Hi achi U-2000, USA).
Enzyme and analy ical assays
Enzyme ac i i ies we e spec opho ome ically
de e mined a 30oC. MnP and Laccase ac i i ies
we e measu ed by he oxida ion o 2,6-
dime hoxyphenol (DMP) a 468 nm (Field e al.,
1993). The ex inc ion coe icien s used o he
dime ic p oduc o dime hoxyphenol (DMP)
oxida ion was 49,600 M-1cm-1. The eac ion was
ini ia ed by addi ion o 0.4 mM H2O2. One uni o
enzyme ac i i y (U) is equi alen o 1µmol o
p oduc o med pe min. Reducing suga s we e
de e mined by he dini osalicilyc acid me hod
using D-glucose as s anda d acco ding o Ghose
(1987). Residual hyd ogen pe oxide was measu ed
by analy ical indica o s (Me ckoquan pe oxid- es )
(Mielgo e al., 2002).
RESULTS AND DISCUSSION
A emp s o induce decolo izing ac i i y by he
ex acellula ligninoly ic sys em o T ame e
e sicolo we e ca ied by conside ing di e en
nu i ional egimes (N-limi a ion o N-su iciency
exp essed as C/N a io 62.5/1 o 12.5/1,
espec i ely) wi h passi e ae a ion o pe iodical
lushing o oxygen. Decolo iza ion in epea ed
cycles was planned o imp o e he p ocess
e iciency.
Acco ding o he esul s ob ained by he
combina ion o he di e en ni ogen egimes and
ae a ion supply, he bes decolo iza ion
pe cen ages co esponded o he assays ca ied ou
in ni ogen limi a ion and unde an ai a mosphe e
(Table 1). These cul u e condi ions led o
seconda y me abolism, which a ou ed he
decolo iza ion o he dye. In con as o esul s
epo ed o Phane ochae e ch ysospo ium (Glenn
and Gold, 1983), an oxygen a mosphe e did no
ha e a bene icial in luence on decolo iza ion, as i
is e iden o he compa ison o decolo iza ion
pe cen ages unde ai o oxygen.
Table 1 - Maximum decolo iza ion pe cen ages o he
dye Poly R-478. Assay
Assay Fi s
Second Thi d
Oxygen/N-su iciency 38.7 39.4 38.6
Ai /N-su iciency 53.1 51.5 53.3
Oxygen/N-limi a ion 59.5 61.3 61.5
Ai /N-limi a ion 72.3 82.0 66.3
Fed-ba ch Decolo iza ion o Poly R-478 by T ame es e sicolo
B azilian A chi es o Biology and Technology
181
A highe concen a ion o ni ogen (C/N a io
12.5/1) g ea ly diminished he inal decolo iza ion
pe cen age wi h a e age educ ions o 30%. On
he o he hand, he supply o oxygen also
nega i ely a ec ed he decolo iza ion e iciency o
alues a ound 60% in compa ison wi h he bes
esul s o 82%, ob ained in he second cycle o he
assay pe o med in N-limi a ion and ai supply.
The decolo iza ion o he dye could be a ibu ed
o he p esence o pe oxidases and oxidases as
MnP and Laccase. The e o e, we in es iga ed he
physiological egula ions o he enzyma ic ac i i y
o hese wo enzymes unde he men ioned cul u e
condi ions. Figs. 1 and 2 show he enzyma ic
ac i i ies ob ained in he p esence o di e en
nu i ional egimes and passi e ae a ion o
pe iodical lushing o oxygen.
Conside ing he i s cycle, he le el o enzyma ic
ac i i y as MnP and Laccase did no signi ican ly
a y on he ni ogen egime. Howe e , his
endency was di e en in he ollowing cycles.
Figu e 1 - T ends o glucose and ligninoly ic ac i i y du ing Poly R-478
decolo iza ion in expe imen s wi h su iciency o ni ogen and passi e
ae a ion (a) o oxygen pe iodical lushing (b) Enzymes ac i i ies: MnP
( ), Laccase (), H2O2 (∗) and Subs a e: Glucose (•).
0 100 200 300 400
0
1
2
3
4
5
6
0
50
100
150
200
250
0 100 200 300 400
0
1
2
3
4
5
6
0
20
40
60
80
100
120
Time (h)
b)
a)
Enzyme ac i i ies (U/L )Enzyme ac i i ies (U/L)
Glucose (g/L), H
2
O
2
(mg/L)Glucose (g/L), H
2
O
2
(mg/L)
Mo ei a, M. T. e al.
B azilian A chi es o Biology and Technology
182
Figu e 2 - T ends o glucose and ligninoly ic ac i i y du ing Poly R-478
decolo iza ion in expe imen s wi h limi a ion o ni ogen and passi e
ae a ion (a) o oxygen pe iodical lushing (b) Enzymes ac i i ies: MnP
( ), Laccase (), H2O2 (∗) and Subs a e: Glucose (•).
Acco ding o esul s co esponding o
decolo iza ion and enzyma ic i e s, bo h p ocesses
a e no necessa ily closely in e connec ed as
would be expec ed in p inciple. The mo e
a ou able ni ogen egime o decolo iza ion
co esponded o ni ogen limi a ion, in con as o
he enhanced ligninoly ic ac i i y ound in
ni ogen su icien condi ions, which was
especially e idenced o Laccase in he subsequen
cycles o ope a ion. Based on he a ibu ed
decolo iza ion ole o MnP and Laccase, his
appa en con adic ion can be explained by wo
ac s: i) decolo iza ion should be a ibu ed o an
e en occu ing in subs a e limi ed cul u es,
which is ypically associa ed o he sec e ion o
ligninoly ic enzymes and ii) he enhanced
ligninoly ic ac i i y is possibly due o he highe
biomass concen a ion unde en iched ni ogen
condi ions; he supplemen a ion o glucose pulses
wi h esidual ni ogen s ill pe mi ed he g ow h o
mic oo ganism and new p oduc ion o enzyma ic
ac i i y, especially Lacasse. The mycelium
emained uncolo ed a e he ea men wi h no
appa en adso p ion o he Poly R-478 on i s
su ace.
A e decolo iza ion assays, he abso bance
p o iles o he dye be o e and a e ea men we e
compa ed. As i can be deduced om, a signi ican
b eakdown o he molecule associa ed o he
ch omopho e g oup was ob ained (Fig. 3); he
majo emo al o he abso bance peak a 520 nm
was ob ained as a esul o he enzyma ic
ea men .
0 100 200 300 400
0
1
2
3
4
5
6
0
50
100
150
200
250
0 100 200 300 400
0
1
2
3
4
5
6
0
20
40
60
80
100
120
140
160
Time (h)
a)
b)
Enzyme ac i i ies (U/L)
Glucose (g/L), H
2
O
2
(mg/L)
Enzyme ac i i ies (U/L)
Glucose (g/L), H
2
O
2
(mg/L)
Fed-ba ch Decolo iza ion o Poly R-478 by T ame es e sicolo
B azilian A chi es o Biology and Technology
183
Figu e 3 - Scan Poly R-478 dye be o e and a e
T ame es e sicolo ea men .
ACKNOWLEDGEMENTS
This wo k was suppo ed by Fondecy 1010644.
G. Vidal hanks o Di ección de In es igación,
Uni e sidad de Concepción (Chile) by suppo ing
he s ays a he Bio echnology G oup, Uni e sidad
de San iago de Compos ela (Spain).
RESUMO
E luen es indus iais al amen e colo idos são
di íceis de deg ada u ilizando as ecnologias
isico-químicas e bac e ianas exis en es
a ualmen e. O sis ema enzimá ico ex acelula
sec e ado pelo ungo de pod idão b anca ap esen a
um g ande po encial oxida i o, capaz de da início
a decomposição das es u u as esiduais dos
compos os esponsá eis pela co des es e luen es.
Aspec os isiológicos o am a aliados pa a
op imiza as condições de descolo amen o de um
co an e sin é ico, Poly R-478, pelo ungo de
pod idão b anca T ame es e sicolo # 52J. Os
expe imen os de descolo amen o o am ealizados
em ope ação semi-con ínua du an e ês ciclos
pa a melho a a e iciência do p ocesso. As
melho es e iciências de descolo amen o (65 % a
80%) o am ob idas em cul u as de ungos a adas
em condições limi adas de ni ogênio em
a mos e a de a .
REFERENCES
Bumpus, J. A.; Tien, M.; W igh , D. and Aus , S. D.
(1985), Oxida ion o pe sis en en i onmen al pollu an s
by a whi e o ungus. Science, 228, 1434-1436.
Dawson, D. I. (1981), Polyme ic dyes. Ald ichim Ac a,
14, 23-29.
Field, J. A.; Jong, E.; Feijoo-Cos a, G. and Bon , J. A.
M. (1993), Sc eening o ligninoly ic ungi applicable
o he biodeg ada ion o xenobio ics. T ends
Bio echnol., 11, 44-49.
Ghose, T. K. (1987), Measu emen o cellulase. Pu e
Appl. Chem., 59, 257-268.
Glenn, J. K. and Gold, M. H. (1983), Decolo iza ion o
se e al polyme ic dyes by he lignin-deg ading
basidiomyce e Phane ochae e ch ysospo ium. Appl.
En i on. Mic obiol., 45, 1741-1747.
Lema, J. M.; Mo ei a, M. T.; Feijoo, G.; Mielgo, I.
and López, C. (2002), Enzima ic memb ane eac o s
o biodeg ada ion o ecalci an compounds. J.
Bio echnol., 99, 249-257.
Mes e , T.; Peña, M. and Field, J. A. (1996), Nu ien
egula ion o ex acellula pe oxidases in he whi e
o ungus Bje kande a sp. s ain BOS55. Appl.
Mic ob. Bio echnol., 44, 778-784.
Mielgo, I.; Mo ei a, M. T.; Feijoo, G. and Lema, J. M.
(2002), Biodeg ada ion o a polyme ic dye in a
pulsed bed bio eac o by immobilised Phane ochae e
ch ysospo ium. Wa e Res., 36, 1896-1901.
Mo ei a, M. T.; Feijoo, G.; Sie a-Al a ez, R.; Lema, J.
M. and Field, J. A. (1997), Biobleaching o oxygen
deligni ied k a pulp by se e al whi e o ungal
s ains. J. Bio echnol., 53, 237-251.
Palle la, S. and Chambe s, R. P. (1996), New u e hane
p epolyme inmobilized ungal bio eac o o
decolo iza ion and dechlo ina ion o k a bleach
e luen s. Tappi J., 79, 155-161.
Roye , G.; Ye ushalmi, L.; Rouleau, D. and Des oche s,
M. (1991), Con inuous decolo iza ion o bleached
k a e luen s by T ame es e sicolo in he o m o
pelle s. J. Ind. Mic obiol., 7, 269-278.
Tien, M.; Ki k and T. K. (1988), Lignin pe oxidase o
Phane ochae e ch ysospo ium. Me hods Enzymol.,
161, 238-249.
Young, L. and Yu, J. (1997). Ligninase-ca alyzed
decolo iza ion o syn he ic dyes. Wa . Res., 31,
1187-1193.
Recei ed: Janua y 28, 2002;
Re ised: Decembe 27, 2002;
Accep ed: Decembe 18, 2003.
400 500 600 700
Wa eleng h (nm)
Abso bance
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
In luen
E luen
