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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: sara.gonzalez@usc.es (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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