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Biodegradability of kraft mill TCF biobleaching effluents:Application of enzymatic laccase-mediator system
P.G. Monje a, S. Gonzalez-Garcıa a,*, D. Moldes b, T. Vidal b, J. Romero c, M.T. Moreira a,G. Feijoo a
a Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, 15782 Santiago de Compostela, Spainb Textile and Paper Engineering Department, ETSEIAT, Universitat Politecnica de Catalunya, Colom 11, E-08222 Terrassa, Spainc ENCE, Marisma de Lourizan s/n, 36153 Pontevedra, Spain
a r t i c l e i n f o
Article history:
Received 21 September 2009
Received in revised form
18 December 2009
Accepted 28 December 2009
Available online 7 January 2010
Keywords:
Aerobic biodegradability
Anaerobic biodegradability
Toxicity
Bleaching effluents
L-stage
Pulp and paper industry
* Corresponding author. Tel.: þ34 981563100xE-mail address: [email protected] (S.
0043-1354/$ – see front matter ª 2010 Elsevidoi:10.1016/j.watres.2009.12.047
a b s t r a c t
The great amount of pollutants released from kraft pulp processes, mainly from cooking
and bleaching stages, is one of the most relevant environmental problems in this type of
industry. New bleaching sequences are being studied based on the use of oxidative
enzymes from fungal cultures. In this study, the bleaching systems consisting of Laccase
and different mediators such as 1-hydroxybenzotriazole, violuric acid, syringaldehyde and
methyl syringate in the bleaching sequence of Eucalyptus globulus kraft pulp were applied.
The main objective of this study is to evaluate the aerobic and anaerobic biodegradability
and toxicity to Vibrium fischeri of generated L-stage and total bleaching sequence effluents.
The highest levels of aerobic and anaerobic degradation of the generated effluents were
achieved for treatments with laccase plus violuric acid, with 80% of aerobic degradation
and 68% of anaerobic biodegradation. V. fischeri toxicity was remarkably reduced for all the
effluents after aerobic degradation.
ª 2010 Elsevier Ltd. All rights reserved.
1. Introduction reagents (ECF): ClO2 and NaOCl (Folke et al., 1993), or by totally
Pulp and paper industry is traditionally known to be a large
contributor to the environmental impact due to its large
consumption of energy and chemicals, and also to the
generation of effluents with high concentrations of suspended
solids, organic load, as well as toxicity (Berube and Kahmark,
2001; Kahmark and Unwin, 1999; Srinivasan and Unwin, 1995).
Pulp bleaching sequences are especially problematic due to
the presence of organohalogens and wood extractives in the
effluents (Xavier et al., 2005; Jokela and Salkinoja-Salonen,
1992). Chemical bleaching of pulp was initially carried out by
using chlorine (Cl2), later replaced by elemental chlorine-free
16020; fax: þ34 98154716Gonzalez-Garcıa).er Ltd. All rights reserved
chlorine-free (TCF) reagents, such as H2O2, O2 and O3 (Bajpai,
2004; Nelson et al., 1998). Regarding toxicity levels, softwood
and hardwood TCF effluents are less toxic than ECF effluents,
being Cl2 bleaching effluents the most toxic wastewaters
based on EC50 values (Tarkpea et al., 1999; Cates et al., 1995).
Aerobic and anaerobic treatments of the bleaching efflu-
ents have been applied to reduce their environmental impact,
in particular, their organic load and toxicity (Freitas et al.,
2009; Mounteer et al., 2002; Tarkpea et al., 1999; Ahtiainen
et al., 1996). Aerobic processes ranging from activated sludge
to aerated lagoons were successfully applied in the treatment
of high strength wastewaters and chlorinated bleaching kraft
8.
.
Nomenclature
L-stage pulp bleaching step with laccase and/or mediator
LMS laccase-mediator system
LMediator LMS-stage bleaching effluent
LMediatort LMS-stage combined with TCF bleaching sequence
effluent (L-O-Q-PoP)
HBT 1-hydroxybenzotriazole
VA violuric acid
SyAl syringaldehyde
MetSyr methyl syringate
O-stage oxygen delignification
Q-stage treatment with chelating agents
PoP-stage oxygen-reinforced hydrogen peroxide
bleaching followed by a depressurization to
remove the oxygen
CODt, CODs total and soluble chemical oxygen demand
CODCH4 methanized chemical oxygen demand
BOD5, BOD30 biochemical oxygen demand after 5 and 30 d
respectively
SS, TSS, VSS suspended solids, total and volatile
suspended solids respectively
VFA volatile fatty acids
EC50 effective concentration of the sample that
causes a 50% reduction in the light output of
the Microtox test microorganism.
UASB reactor upflow anaerobic sludge blanket reactor
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 2 2 1 1 – 2 2 2 02212
effluents (Pokhrel and Virarahghavan, 2004). Anaerobic
biodegradability and toxicity to methanogens are strongly
dependent on the wastewater characteristics, which depend
on the processing technology, the feedstock used as raw
material and the internal wastewater recycling (Vidal and
Diez, 2003; Cates et al., 1995). In theory, the anaerobic process
presents significant advantages in comparison with the
aerobic alternative, such as considerably lower energy
consumption, production of biogas and low production of
sludge. However, the anaerobic treatment presented limited
efficiency when applied to the decontamination of pulp mill
effluents (Bajpai, 2000). The organohalogens and extractive
compounds presented in bleaching wastewaters showed to be
inhibitory to methanogenic bacteria (Sierra-Alvarez et al.,
1994). The effluents from Cl2 and ECF bleaching effluents
presented high methanogenic toxicities and only TCF
bleaching effluents were less toxic (Vidal et al., 1997).
Nowadays, the enzymatic bleaching could be an alterna-
tive for a cleaner pulp production (Fu et al., 2005; Skals et al.,
2008). Treatment with xylanases boosts overall bleaching
process by improving subsequent stages (Valls and Roncero,
2009; Shatalov and Pereira, 2007; Roncero et al., 2003) and has
even been incorporated into bleaching sequences in some
pulp mills (Bajpai, 2004; Popovici et al., 2004). The enzymatic
treatment with oxidoreductases such as laccases is a prom-
ising alternative under intensive research. These enzymes are
capable of oxidizing phenolic units and amine compounds in
lignin (Higuchi, 2004). In combination with redox mediators,
laccases can expand their action to non-phenolic substrates
(Freudenreich et al., 1998). Several studies have confirmed the
potential of the so-called laccase-mediator system (LMS) for
the bleaching of different types of pulp (Ibarra et al., 2006;
Sigoillot et al., 2005; Camarero et al., 2004; Nelson et al., 1998).
Laccase-mediators can be synthetic compounds, such as 2,20-
azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 1-
hydroxybenzotriazole (HBT), violuric acid (VA) or natural
ones, such as syringaldehyde (SyAl), methyl syringate (Met-
Syr) and p-coumaric acid. Laccase-HBT was found to be one of
the most promising combinations for delignification of kraft
pulp in mill applications (Sigoillot et al., 2005; Camarero et al.,
2004; Garcıa, 2003) as well as laccase-VA (Moldes et al., 2008).
The scale-up of this technology must overcome two main
challenges: (i) the efficient production of enzyme and media-
tors at low cost and (ii) the use of eco-friendly mediators in the
biobleaching stage, which assures minimal environmental
pollution.
The goal of this paper is to determine the anaerobic and
aerobic biodegradabilities as well as the Microtox� toxicity of
effluents from L-stage and from the combination of the LMS
with the entire TCF bleaching sequence.
2. Materials and methods
2.1. Effluents
Eucalyptus (Eucalyptus globulus) kraft pulp was washed and
enzymatically treated (L-stage) at lab-pilot scale according to
the following conditions: laccase (20 U/g of dry pulp) with
mediators (1.5% of dry pulp) were added to 200 g of dry pulp at
10% consistency (dry pulp mass/total mass), pH 5, 50 �C and
6 bar O2 pressure for 2 h. The mediators used to boost the
laccase effect were: 1-hydroxybenzotriazole (HBT), violuric
acid (VA), syringaldehyde (SyAl) and methyl syringate (Met-
Syr). In addition, control experiments were performed without
mediator and without mediator and enzyme.
After L-stage, the following bleaching step was an oxygen
delignification (O-stage) with the addition of a solution of
NaOH (1.5%) and a solution of MgSO4 (0.5%) at 6 bar O2 and
98 �C for 1 h. The third step corresponded to a Q-stage,
a treatment with chelating agents in order to avoid metal
pollution, with the addition of 0.3% DTPA solution at 40%, pH
5.5–6, 85 �C and 1 h. The last step of the TCF sequence was the
PoP-stage, an oxygen-reinforced hydrogen peroxide bleaching
followed by a deppressurization where oxygen is removed.
The PoP-stage included the addition of NaOH (1.5%), MgSO4
(0.1%), SiO3Na2 (0.5%), H2O2 (3%), Busperse (0.033%) at a pres-
sure of 6 bar O2 and 105 �C for 140 min. Thereafter, pressure
was released and the temperature was maintained at 98 �C for
3 additional hours.
The conventional TCF bleaching sequence referred as
O-O-Q-PoP was also compared with the combination of the
enzymatic stage in replacement of the first oxygen stage in the
conventional TCF sequence, that is, L-O-Q-PoP, where L
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 2 2 1 1 – 2 2 2 0 2213
corresponds to the enzymatic stage with and without the
previous mentioned mediators: HBT, VA, SyAl and MetSyr.
After each bleaching stage, pulp was washed to determine
physico-chemical properties (kappa number, ISO brightness,
viscosity) and the generated wastewaters collected for anal-
ysis. The bleaching effluents of the entire TCF sequences were
a mixture of the wastewaters generated in each single stage of
the bleaching sequence in relative proportions of 25%.
Therefore, twelve different effluents were evaluated: 6
L-stage effluents and 6 total bleaching sequence effluents. The
effluents from the L-stage were: an effluent with no enzyme
and mediator (Control), an effluent from the enzyme treat-
ment with no mediator (L) and four different wastewaters
with LMediator systems (LHBT, LVA, LSyAl, LMetSyr). Total TCF
bleaching effluents were: effluent from the pulp mill bleaching
sequence (O-O-Q-PoP), and five total enzymatic bleaching
sequence effluents (L-O-Q-PoP) where the difference was the
L-Stage (Lt, LHBTt , LVA
t , LSyAlt , LMetSyr
t ).
2.2. Analytical methods
Chemical oxygen demand (COD) and total and volatile sus-
pended solids (TSS and VSS) were measured as described in
Standard Methods (APHA-AWWA-WPCF, 1992).
2.3. Aerobic biodegradability assays
The aerobic biodegradability assays were determined by using
Manometric BOD Measuring Devices Oxitop IS 12. The
measurement was carried out according to the Standard
Methods (APHA-AWWA-WPCF, 1992). In this study, BOD was
measured during 30 d at 20 � 1 �C once for each effluent.
L
Kraft Pulp stream
Lacasse + Mediator
Oxygen-reinforcedNaOHMgSO4
L - stage effluent ( L Mediator )
O
CH4Anaerobic
EffluentAerobic Effluent
Anaerobic Reactor Aerobic Reactor
Aerobic/ Anaerobic
Enzymatic TCF blea
Fig. 1 – Schematic diagram of the studied system composed by
treatment of the generated wastewaters (aerobic and anaerobic
2.4. Anaerobic biodegradability assays
The anaerobic biodegradability assays were performed in
closed dark-glass serum flasks of 125 ml with a liquid volume
of 100 ml in a thermostatic bath at 30 � 2 �C. Vials were con-
nected to inverted Mariotte flasks containing an alkaline
solution of NaOH (25 g/l), which allowed the measurement of
methane production by liquid displacement (Feijoo et al.,
1995). Alkaline solution neutralizes CO2 produced in vials and
allows methane to reach the empty space of Mariotte flask.
The assay medium was prepared by adding 2 g-VSS/l of
anaerobic sludge, 5 g/l NaHCO3, and 10 ml of a nutrient stock
solution (�10) containing inorganic macro-nutrients (mg/l):
NH4Cl (280), KH2PO4 (250), MgCl2$6H2O (78), CaCl2$2H2O (10),
and the following micro-nutrients (mg/l): FeCl2$4H2O (2000),
CoCl2$6H2O (2000), MnCl2$4H2O (50), AlCl3$6H2O (90),
CuCl2$2H2O (30), ZnCl2 (50), H3BO3 (50), (NH4)6Mo7O2$4H2O (90),
Na2SeO3$5H2O (100), NiCl2$6H2O (50), EDTA (1000) and 36% HCl
(1 ml/l). The bleaching effluents (70 ml) and 2 ml of distilled
water were added to the dark-glass flasks. In order to obtain
a reducing medium, 4 g/l Na2S$9H2O were added. Finally, pH
was adjusted to 7.3 � 0.1 by using diluted HCl or NaOH solu-
tions and N2 gas was bubbled up into the vial for 6 min (3 min
in liquid zone and 3 min in air zone) in order to remove air
from the head space and to get an adequate activity of the
biomass. Experiments were carried out in triplicate. Results
will be shown as media and standard deviation.
The specific methanogenic activity of the sludge was
carried out in duplicate using the same procedure as described
for anaerobic biodegradability assays. The L-stage effluents
were substituted by distilled water. A neutralized mixture of
VFA stock solution was used as substrate to provide a final
concentration of 2 g COD/l (Soto et al., 1993).
PoP
DTPAsolution
Oxygen-reinforcedH2O2 Bleached Pulp
Total TCF bleachingEffluent ( L Mediator
t )
Q
Biodegradation
ching sequence
CH4Anaerobic
EffluentAerobic Effluent
Anaerobic Reactor Aerobic Reactor
an enzymatic TCF bleaching sequence (L-O-Q-PoP) and the
biodegradation).
Table 1 – Pulp properties before and after L-stage.
Sample Kappanumber
Viscosity(ml/g)
ISO brightness(%)
Initial pulp 13.3 1087 43.11
Control 12.2 1076 43.96
L 12.2 1078 43.03
LHBT 9.7 1071 45.68
LVA 9.0 1018 43.63
LSyAl 11.1 1109 38.85
LMetSyr 12.3 1109 36.88
Table 3 – Characteristics of aerobically biodegradedL-stage effluents.
Sample BOD5
(mg/l)BOD30
(mg/l)CODs initial
(mg/l)CODs
finala
(mg/l)
CODs
degradation(%)
Control 50 250 2575 2570 0.19
L 400 1800 4160 2295 44.83
LHBT 600 2600 5360 3245 39.46
LVA 1400 3100 4475 1350 69.83
LSyAl 300 1400 4515 3645 19.27
LMetSyr 300 2100 6820 4380 35.78
a After 30 days of aerobic biodegradation.
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 2 2 1 1 – 2 2 2 02214
2.5. Microtox test
Microtox� toxicity assays were performed by using a Microtox
model 500 Analyzer. A luminescent marine bacterium,
Vibrium fischeri, was the bioassay organism used in these
experiments. The tests exposed the bacteria to every bleach-
ing effluent before any treatment and measured the toxic
effect on the organism, after 5 and 15 min. The obtained
results were expressed as EC50 value, which corresponds to
the effective concentration of a sample that causes a reduc-
tion in the light output of the Microtox test organism by 50%.
The EC50 values were estimated automatically according to
the ‘‘Basic Test’’ protocol of the Microtox software (Microbics
Corporation, 1992). The toxicity of the 12 biobleaching
wastewaters after aerobic degradation was also measured.
2.6. Aerobic and anaerobic biomass
Flocculent aerobic sludge was obtained from a municipal
wastewater treatment plant. The aerobic biomass was
4 g-VSS/l and 4.8 g TSS/l. Sludge was refrigerated at 4 �C until
its use in aerobic assays.
Granular anaerobic sludge was collected from an UASB
reactor treating effluents from a brewery and used in the
anaerobic biodegradability assays. The specific methanogenic
activity of this sludge, determined from VFA as a substrate
was 0.100 � 0.004 g-CODCH4/g-VSS$d. The biomass was
previously conditioned to biobleaching effluents for 30 d and
then the sludge was maintained refrigerated at 4 �C until use
in described experiments.
2.7. Pulp properties
Brightness, kappa number (indicator of lignin content) and
viscosity of pulp were determined according to ISO 3688, ISO
302 and ISO 5351-1 respectively (ISO), 1998.
Table 2 – Characteristics of the different biobleaching effluents
Control L LH
Enzyme None Laccase Lac
Mediator None None HBT
pH 8.24 7.56 5.34
CODt (mg/l) 2820 4370 641
CODs (mg/l) 2575 4160 536
TSS (mg/l) 80 130 150
VSS (mg/l) 80 130 150
3. Results
3.1. Evaluation of the potential biodegradability andtoxicity of the L-stage
Fig. 1 shows a flowchart with a detailed indication of the
different streams. Pulp properties before and after the L-stage
are presented in Table 1. The most effective mediators in
delignification were the synthetic ones: HBT and especially
VA, as they showed the lowest kappa number values. These
mediators also attained the highest ISO brightness values. The
viscosity values obtained for the synthetic mediators suggest
a negligible decrease of cellulose integrity. Regarding natural
mediators, LSyAl and LMetSyr systems improved slightly the
kappa number compared with the initial pulp before any
bleaching stage. However, the treatment LMetSyr did not show
better results than the bleaching stage with laccase and no
mediator, unlike the LSyAl system. Both natural mediators
presented worse results for ISO brightness compared to initial
pulp or the processes without mediator and/or enzyme.
The characteristics of the different L-stage effluents
collected are shown in Table 2. The pH ranged from alkaline
values in biobleaching wastewaters with no mediators
(Control and L) to acidic values in LMediator systems. The ratio
between soluble and total COD was about 0.85–0.90 for all
biobleaching wastewaters except the sample obtained from
enzymatic treatment with SyAl: 0.75. Bleaching effluents
generated from LMediator systems showed the highest COD
values. The control sample had the lowest COD value. This
was the consequence of not adding laccase either mediator.
The addition of enzyme increased the chemical oxygen
demand but not with the levels of bleaching effluents from
LMediator systems.
from the L-stage.
BT LVA LSyAl LMetSyr
case Laccase Laccase Laccase
VA SyAl MetSyr
5.45 4.30 5.34
0 4805 5540 7950
0 4475 4515 6820
130 460 160
130 460 150
0
20
40
60
80
100
120
a
0 100 200 300 400 500 600
HC
emulo
V4
)L
m(
0
20
40
60
80
100
120
0 100 200 300 400 500 600
HC
emulo
V4
)L
m(
Time (h)
b
Fig. 2 – Anaerobic biodegradability of L-stage effluents at
maximum concentration (70%) without adding VFA (except
blank), expressed as cumulative methane production.
Samples: (C) Blank; (B) Control; (>) L; (,) LHBT; (-) LVA;
(6) LSyAl; (:) LMetSyr.
Table 5 – Microtox toxicity of initial L-stage effluents andwastewater streams after aerobic biodegradation process(based on Microtox, 5-min and 15-min EC50 values (%)).
Sample Effluents After aerobic biodegradation
EC50
5-minEC50
15-minEC50
5-minEC50
15-min
Control 245 271 280 184
L 38 40 185 102
LHBT 5 6 21 25
LVA 5 5 58 44
LSyAl 0 0 3 3
LMetSyr 3 3 45 38
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 2 2 1 1 – 2 2 2 0 2215
At this stage of the experimental set-up, only the effluents
generated in the L-stage were evaluated for their aerobic or
anaerobic biodegradability. The results of the aerobic degra-
dation of the L-stage effluents are shown in Table 3. BOD5 and
BOD30 are important parameters indicative of short-term and
long-term aerobic biodegradation. According to these
parameters, the most aerobically biodegradable effluent was
LVA wastewater, followed by LHBT effluent. The other L-stage
wastewaters analyzed were slowly biodegraded and the
Control assay reached very low values for BOD5 and BOD30 due
to the low concentration of organic matter. The COD values
Table 4 – Results of anaerobic biodegradability assays of the dif(% of methanization and degradation).
Sample CODtinitial(mg/l)
CODs initial(mg/l)
CODs final(mg/l) degr
Control 1975 1800 570
L 3060 2690 540
LHBT 4485 3750 2200
LVA 3365 3130 1350
LSyAl 3880 3160 2360
LMetSyr 5565 4775 2560
before and after aerobic biodegradation are also shown in
Table 3. The mass balance points out that the value of initial
soluble COD is the same as the one calculated from the sum of
the final BOD30 and the final soluble COD. Every effluent met
the mass balance with a maximum deviation of 10%. LVA
wastewater showed the highest value of biological oxygen
demand after 5 and 30 days. The removal of soluble organic
matter with an aerobic treatment for LVA effluent was 69.8%.
This effluent was found to be about 30–35% more biodegrad-
able than the other L-stage wastewaters.
The methane production of the anaerobic biodegradability
assays for the bleaching effluents from the L-stage are shown
in Fig. 2. The blank sample, containing distilled water with
VFA mixture, was totally biodegraded by the anaerobic sludge.
The specific methanogenic activity of the sludge was
0.10 g-CODCH4/g-VSS$d, which points out the rate of metha-
nization of the organic matter by the anaerobic biomass. No
inhibitory effect of the methanogenic activity was observed
for the effluents generated in the enzymatic treatment. LMetSyr
effluent showed the highest cumulative methane production.
However, L wastewater presented higher methane production
than the other LMediator effluents although the organic matter
content was lower. The results of the anaerobic biodegrada-
tion of the bleaching wastewaters at maximum concentration
are presented in Table 4. They showed that the highest soluble
degradation was accomplished for L and Control assays. The
low soluble degradation reached for the LSyAl effluent is also
outstanding. Focusing on methanization, non-mediator
wastewaters reached the highest values, followed by LVA
effluent.
The short-term (5 min) and long-term (15 min) EC50 values
for effluents from the L-stage and after aerobic biodegradation
ferent biobleaching effluents from the L-stage after 24 days
Solubleadation (%)
CH4 (ml) CODCH4
(mg/l)CODt
Methanization (%)
68.33 67.7 1671.6 84.64
82.35 89.2 2202.5 71.98
41.33 87.5 2160.5 48.17
56.87 75.6 1866.7 55.47
25.32 71.2 1758.0 45.31
46.39 108.3 2674.1 48.05
Table 6 – Pulp properties after enzymatic TCFbiobleaching sequence.
Sample Kappanumber
Viscosity(ml/g)
ISO brightness (%)
O-O-Q-PoP 5.6 763 88.83
L-O-Q-PoP
Lt 5.6 781 88.55
LHBTt 5.0 761 90.15
LVAt 5.1 800 89.90
LSyAlt 5.7 805 87.38
LMetSyrt 5.6 780 88.26
t Total TCF bleaching effluent with the specified L-stage.
0
200
400
600
800
1000
1200
1400
0 5 10 15 20 25 30
Og
m(D
OB
2)
L/
Time (d)
Fig. 3 – Aerobic biodegradability of total TCF bleaching
sequence effluents expressed as Biochemical Oxygen
Demand (BOD). Samples: (B) O-O-Q-PoP; (>) Lt; (,) LHBTt ;
(-) LVAt ; (6) LSyAl
t ; (:) LMetSyrt .
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 2 2 1 1 – 2 2 2 02216
are shown in Table 5. Toxicity of L-stage effluents was very
high, especially for the LSyAl effluent, while those effluents
with no mediator (Control and L wastewaters) showed lower
values of toxicity in the marine bacterium. The aerobic
biodegradation reduces the toxicity to V. fischeri in all effluents,
with a very remarkable increase of EC50 values for the LVA
effluent. The Microtox toxicity assays were only carried out for
L-stage effluents before and after aerobic degradation because
of the higher organic matter degradation accomplished by the
aerobic processes in comparison with anaerobic ones.
3.2. Evaluation of the potential biodegradability andtoxicity of a TCF bleaching sequence implemented witha L-stage
The properties of the pulp after total TCF bleaching sequence
are shown in Table 6. The best TCF bleaching sequences for
delignification were those where the L-stage was carried out
with synthetic mediators (LVAt and LHBT
t ) because of the low
kappa number values reached and the ISO brightness values.
The highest viscosity values for LVAt and LSyAl
t bleaching
sequences point out better properties related to cellulose
integrity.
In Table 7 the characteristics of total TCF bleaching
sequence effluents are shown. The pH was alkaline in all the
bleaching wastewaters although the pH was lower for effluents
from sequences with enzymatic treatments and addition of
mediators (LMediatort ) compared to pulp mill-bleaching effluent
(O-O-Q-PoP). Soluble and total COD values were very similar,
with ratios of CODs/CODt higher than 0.94. The conventional
bleaching effluent showed the lowest COD value as no L-stage
was performed, which increases the content of organic matter
Table 7 – Characteristics of total TCF bleaching sequence efflu
O-O-Q-PoP Lt
Enzyme (L-stage) None Laccase
Mediator (L-stage) None None
pH 11.33 10.44
CODt (mg/l) 1630 1935
CODs (mg/l) 1615 1925
TSS (mg/l) 20 10
VSS (mg/l) 20 10
in the effluents especially for LMediator systems. The concen-
trations of TSS and VSS were very low for all the bleaching
sequence wastewaters.
The aerobic biodegradability of the total TCF bleaching
effluents was expressed as BOD (mg O2/l), as shown in Fig. 3.
The highest aerobically biodegradable effluent was LVAt
wastewater, followed by LHBTt and LMetSyr
t effluents. The other
total TCF bleaching wastewaters analyzed were slowly bio-
degraded. These trends were confirmed by the results of total
bleaching sequence effluents shown in Table 8. The
maximum degradation of soluble organic matter: 80% was
reached for LVAt effluent. This turned out to be 15–25% more
biodegradable than other wastewaters. The pulp mill bleach-
ing effluent, O-O-Q-PoP, showed one of the lowest soluble
COD values after aerobic biodegradation. However, O-O-Q-PoP
also presented the poorest percentage of soluble organic
matter aerobically biodegraded due to the reduced amount of
organic matter content in the effluent.
In Fig. 4 the cumulative methane production of total TCF
bleaching sequence effluents is shown. No inhibitory effect of
the methanogenic activity was detected for any total bleach-
ing effluent. The highest productions of methane were found
to be LVAt and LSyAl
t effluents. After 500 h of anaerobic biodeg-
radation all the curves stabilized, so the methanization was
complete. The results of the anaerobic biodegradation of total
TCF bleaching sequence effluents are shown in Table 9. The
highest soluble degradation of organic matter was shown by
O-O-Q-PoP and Lt effluents, and also the lowest COD values
after anaerobic biodegradation. LHBTt presented the worst
ents.
LHBTt LVA
t LSyAlt LMetSyr
t
Laccase Laccase Laccase Laccase
HBT VA SyAl MetSyr
10.01 9.93 8.94 9.23
2600 1935 2295 2830
2450 1925 2280 2820
50 10 0 30
50 10 0 30
Table 8 – Characteristics of aerobically biodegraded totalTCF bleaching sequence effluents.
Sample BOD5
(mg/l)BOD30
(mg/l)CODs initial
(mg/l)CODs
finala
(mg/l)
CODs
degradation(%)
O-O-Q-PoP 0 400 1615 710 56.0
L-O-Q-PoP
Lt 0 600 1925 770 60.0
LHBTt 150 800 2450 820 66.5
LVAt 300 1150 1925 385 80.0
LSyAlt 100 600 2280 955 58.1
LMetSyrt 100 800 2820 1185 58.0
a After 30 days of aerobic biodegradation.
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 2 2 1 1 – 2 2 2 0 2217
soluble degradation results. LVAt and O-O-Q-PoP effluents were
completely methanized, as shown in Fig. 4. The methaniza-
tion of the other total enzymatic bleaching sequence waste-
waters was around 40% lower.
The V. fischeri toxicity for total TCF bleaching sequence
effluents is shown in Table 10, as well as the toxicity of these
wastewaters once they were aerobically biodegraded. EC50
values were similar for O-O-Q-PoP and LMediatort effluents,
showing the lowest toxicity Lt. Microtox toxicity for total
bleaching effluents decreased after aerobic biodegradation,
reaching the best results in O-O-Q-PoP and LMetSyrt wastewaters.
0
20
40
60
80
100
0 100 200 300 400 500
HC
emulo
V4
)L
m(
0
20
40
60
80
100
0 100 200 300 400 500
HC
emulo
V4
)L
m(
Time (h)
a
b
Fig. 4 – Anaerobic biodegradability of total TCF bleaching
sequence effluents at maximum concentration (70%)
without adding VFA (except blank), expressed as
cumulative methane production. Samples: (C) Blank;
(B) O-O-Q-PoP; (>) Lt; (,) LHBTt ; (-) LVA
t ; (6) LSyAlt ; (:) LMetSyr.
t.
4. Discussion
The synthetic mediators violuric acid (VA) and 1-hydroxy-
benzotriazole (HBT), were found to be the most effective
mediators for boosting the laccase-assisted biobleaching of
eucalyptus kraft pulp (Moldes et al., 2008; Camarero et al.,
2007). This trend is supported by this work according to the
lowest kappa numbers and the highest brightness values
reached by the L-stage and the total bleaching sequences with
synthetic mediators. They are better than the kappa number
and the ISO brightness value for the pulp mill TCF bleaching
sequence obtained in this work and the values are similar to
those reported in other studies (Ibarra et al., 2006). These
results also suggest a low content of lignin in the pulp since
the lignin is a high molecular weight, three-dimensional
polymer with a structural role in wood cells that decreases the
brightness of pulp due to the presence of chromophoric
groups. However, natural mediators permitted a prolonged
use of the enzyme because of the low laccase inactivation
during the biobleaching stage (Moldes et al., 2008). The enzy-
matic treatment of kraft pulp with SyAl and MetSyr increased
kappa number and decreased the brightness for the L-stage
and L-O-Q-PoP bleaching sequence. The behaviour of these
two natural mediators in the L-stage and in the combined
bleaching sequence was the consequence of coupling-poly-
merisation reactions on lignin rather than direct oxidative
degradation (Moldes et al., 2008).
If a new bleaching sequence is intended to be imple-
mented, comprehensive studies on the potential degradability
and toxicity of the generated effluents are required. Regarding
the characteristics of the different wastewaters, TCF bleach-
ing sequences using enzymes and oxygen-based chemicals
significantly increased COD values in the bleaching effluents
compared to ECF sequences. This is due to the presence of
carbohydrates released by enzymatic treatment and the non-
specific action of ozone and hydrogen peroxide in TCF
sequences (Cates et al., 1995). The higher COD of LMediator and
LMediatort effluents was caused by the oxidation and release of
lignin and other components from pulp during the L-stage
performed in presence of mediators, and also by the presence
of laccase and mediator themselves. However, LMediatort efflu-
ents showed lower concentrations of organic matter than
LMediator wastewaters because of the dilution carried out after
L-stage (Aracri et al., 2009). Total and soluble COD were similar
for each wastewater and showed low amounts of suspended
solids in the effluents. Concentrations of TSS and VSS of
effluents were similar suggesting that all solids were organic.
LMediator effluents showed low levels of aerobic biodegra-
dation; only LVA wastewater attained levels comparable to the
BOD of municipal wastewaters (Metcalf and Eddy, 1995). Many
authors reported different BOD5 values for bleaching effluents
before any treatment with values less than 1000 mg/l (Pokhrel
and Virarahghavan, 2004). BOD5 values obtained for LMediator
effluents were in the typical range for all wastewaters, but LVA
effluent showed higher biochemical oxygen demand. This
wastewater was more biodegradable and had less toxic
compounds to aerobic microorganism introduced as inoc-
ulum than the other LMediator effluents. BOD5 values obtained
for LMediatort and O-O-Q-PoP effluents were lower than
Table 9 – Results of anaerobic biodegradability assays of the total TCF bleaching sequence effluents after 24 days (% ofmethanization and degradation).
Sample CODtinitial(mg/l)
CODs initial(mg/l)
CODs final(mg/l)
Solubledegradation (%)
CH4 (ml) CODCH4
(mg/l)CODt
methanization (%)
O-O-Q-PoP 1140 1130 215 81.0 46.8 1155.6 101.36
L-O-Q-PoP
Lt 1355 1345 315 76.6 34.0 839.5 61.96
LHBTt 1820 1715 950 44.6 46.9 1158.0 63.63
LVAt 1355 1345 425 68.4 60.4 1491.4 110.06
LSyAlt 1605 1595 435 72.7 55.6 1372.8 85.53
LMetSyrt 1980 1975 715 63.8 42.8 1056.8 53.37
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 2 2 1 1 – 2 2 2 02218
wastewaters from the L-stage because of the dilution and the
lower amount of organic matter. These values were found in
the typical range defined by Pokhrel and Virarahghavan
(2004). Moreover the aerobic biodegradation of LMediatort and
O-O-Q-PoP effluents was around 15–35% higher for every
effluent compared to LMediator wastewater. The dilution effect
was clearly shown by LSyAlt , with the greatest increase of the
aerobic biodegradability, 38.8%. The LVAt effluent was also the
most biodegradable wastewater studied, similar to urban
wastewaters.
The anaerobic biodegradability and toxicity to metha-
nogens are strongly dependent on wastewater characteristics
(Cates et al., 1995). Chlorine bleaching effluents are problem-
atic for anaerobic wastewater treatment due to their high
methanogenic toxicity and low biodegradability, as a conse-
quence of producing chlorinated phenols (Sierra-Alvarez
et al., 1994). ECF bleaching effluents were not less toxic than
chlorine bleaching wastewaters, while TCF effluents showed
the lowest methanogenic toxicity. The fact that ECF effluents
were not less toxic than conventional ones (bleaching with
chlorine), as well as the residual toxicity of TCF, indicate that
other substances besides organohalogens contribute to the
high methanogenic toxicity in bleaching effluents (Vidal et al.,
1997). Wood resin compounds released by alkaline extraction
stages might be important inhibitory substances in all
bleaching processes. In the L-stage effluents (LMediator waste-
waters) many of these substances, such as organohalogens
and wood resin compounds, were not present due to the
effluents’ origin. However, LMediatort effluents showed gener-
ally a higher soluble organic degradation and methanization
Table 10 – Microtox toxicity of initial total TCF bleachingsequence effluents and wastewater streams after aerobicbiodegradation process (based on Microtox, 5-min and15-min EC50 values (%)).
Sample Effluents After aerobic biodegradation
EC50
5-minEC50
15-minEC50
5-minEC50
15-min
O-O-Q-PoP 73 73 223 171
L-O-Q-PoP
Lt 135 115 97 90
LHBTt 48 43 78 80
LVAt 88 79 133 97
LSyAlt 49 48 125 107
LMetSyrt 89 105 226 234
than LMediator wastewaters. High load wastewaters were
difficult to be anaerobically biodegraded, especially by the
presence of mediators used in the L-stage. Dilution of LMediator
effluents triggers anaerobic biodegradation. This is also the
case for LMediatort .
Regarding Microtox toxicity, results revealed that for LMediator
effluents before any treatment the toxicity raised high values,
probably because of the presence of an enzyme and a mediator,
forming a couple with high oxidation power. All the LMediator
effluents showed an elevated toxicity to luminescent marine
bacterium, but the use of natural mediators in laccase-mediator
systems (LMS) increased the toxicity to V. fischeri compared to
synthetic ones, although the difference between them was
small. LMediatort effluents showed higher EC50 values than
wastewaters from the L-stage, indicating lower Microtox
toxicity. The first stage of the pulp mill TCF bleaching sequence,
an O-stage, had lower contribution to the overall Microtox
toxicity effect than using a synthetic or natural mediator in the
L-stage. Long-term Microtox toxicity increased compared to
short-term ones as a consequence of longercontact between the
effluent and V. fischeri. Many researchers have found that
secondary treatment considerably reduces the toxicity (Ahtiai-
nen et al., 1996), such as biological treatment with Pleurotus
sajor caju (Freitas et al., 2009) or aerated lagoons (Tarkpea et al.,
1999). This study backed up the reduction of Microtox toxicity
to V. fischeri after aerobic biodegradation for LMediator and
LMediatort effluents. LVA wastewater showed the better Microtox
toxicity results and LSyAl effluent the highest ones after aerobic
degradation and before any treatment. However, toxicity to
V. fischeri after aerobic biodegradation was also lower for
LMediatort effluents than effluents from the L-stage. Effluents from
the L-O-Q-PoP sequence showed higher toxicity than pulp mill
TCF bleaching sequence, except LMetSyrt wastewater that was
similar. This is due to the mediator oxidation by the enzyme and
the formation of stable radicals.
5. Conclusions
Synthetic mediators, VA and HBT, in combination with lac-
case improved the kappa number index and brightness after
the L-stage and enzymatic TCF bleaching sequence. LVA and
LVAt effluents generated from the L-stage and from the entire
bleaching sequence, respectively, were found to be aerobically
biodegraded, similar to urban wastewaters. They were about
30% and 15% more biodegradable than the other LMediator and
LMediatort effluents.
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 2 2 1 1 – 2 2 2 0 2219
Kraft pulp bleaching with laccase and no mediator produced
effluents (L, Lt) that were highly biodegraded by anaerobic
bacteria, as well as pulp mill bleaching sequence wastewater.
The addition of natural or synthetic mediator reduced anaer-
obic biodegradability. The LVAt and the O-O-Q-PoP effluents
were completely methanized.
Microtox toxicity of effluents from the L-stage to V. fischeri
was extremely high because of the oxidized mediators and the
formation of stable radicals. The dilution effect of the bleaching
sequence reduced the Microtox toxicity of LMediatort effluents.
Aerobic treatment decreased Microtox toxicity of every gener-
ated effluent from L-stage and the TCF bleaching sequence.
Acknowledgements
This work has been partially financed by the European Project
NMP2-CT-2006-026456 BIORENEW and the Xunta de Galicia
(Project Reference: PGIDIT09MDS010262PR).
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