Bioresource Technology 77 (2001) 183191

Molecular weight distribution of Pinus radiata kraft mill wastewater

treated by anaerobic digestion
G. Vidal a,b,*, S. Videla a, M.C. Diez a
a

Department of Chemical Engineering, University of La Frontera, P.O. Box 54-D, Avda. Francisco Salazar 01145, Casilla 54-D, Temuco, Chile
b
University of Concepci
on, Environmental Science Centre (EULA Chile), Casilla 160-C, Concepci
on, Chile
Received 23 August 1999; received in revised form 31 July 2000; accepted 8 September 2000

Abstract
Kraft mill is responsible for massive discharge of highly polluted euents. The main characteristics of this euent are high
toxicity and low biodegradability due to tannin, lignin and chlorophenol compounds. The composition may vary dramatically
depending, for instance, on the utilised feedstock and process. The purpose of this work was to investigate the molecular weight
distribution of Pinus radiata kraft pulping wastewater treated by anaerobic digestion by using two types of anaerobic reactors: xed
bed and sludge blanket.
Anaerobic sludge blanket (UASB) and anaerobic lter (AF) were operated. In both reactors, the total alkalinity ranged between
1.0 and 1.5 g CaCO3 =l, while the organic load rate (OLR) was increasing during operation from 1.2 to 3:3 gCOD=l d. COD and
total phenolic compounds UV215 removal ranged between 3050% and 1320%, respectively, while the BOD5 removal ranged 60
90%. However only a partial biodegradation (1043%) of tannin and lignin was observed. Results from ultraltration analyses
indicated that the fraction with a molecular weight (MW) <1000, COD and colour decreased after anaerobic treatment, but the total
phenolic compounds increased. In the 1000 < MW < 10; 000 fraction, there was no change in COD, UV215 and colour. In the
>10,000 MW fraction, colour and COD fraction increased by 14% and 5%, respectively, after anaerobic treatment. It can be
concluded from this study, that treatment with UASB or AF reactors is not enough, under the conditions tested, for a large COD
removal from Pinus radiata wastewater. 2001 Published by Elsevier Science Ltd.
Keywords: Anaerobic biodegradability; Pinus radiata mill euents; Phenolic compounds; Molecular weight distribution

1. Introduction
The application of anaerobic wastewater treatment
systems in the forest industry is a relatively recent development (Lettinga et al., 1991; Field et al., 1992). Anaerobic treatment not only provides saving in operation
cost and sludge disposal, but benets are also obtained
from the production of methane gas (Field et al., 1992).
The use of anaerobic systems was made possible by the
development of high-rate processes and the increased
understanding of factors controlling the anaerobic biodegradation (Rintala and Puhakka, 1994). The required
high sludge retention of modern high-rate anaerobic reactors is based on bacterial sludge entrapment, bacterial
immobilisation by an attachment mechanism or sludge
blanket reactors (Mendez and Lema, 1992; Fernandez
et al., 1995). The contact process, anaerobic lter (AF)
*

Tel.: +56-41-242465; fax: +56-41-242546.

E-mail address: glvidal@udec.cl (G. Vidal).

and upow anaerobic sludge blanket (UASB), can serve

as an example (Field et al., 1992; Rintala and Puhakka,
1994). Data from anaerobic treatment systems for kraft
bleaching euents obtained by other authors is summarised in Table 1. Data shows that the removal
eciencies vary from 50% to 80% of COD, depending
mostly on the biodegradability of the particular wastewater being treated. BOD removal eciencies are high,
in most cases between 75% and 99%, indicating that
anaerobic treatment is particularly useful for the elimination of readily biodegradable organic matter. Recalcitrant COD was observed in all these studies. It is
possible to attribute the non-biodegradable fraction to
the lignin presence. In fact, Soto et al. (1991) reported a
biodegradation of lignin compound determined as
UV280 up to 75%, while Kortekaas et al. (1998) reported
a COD eciency from 27.5% to 57.7%.
Forest industry euents contain numerous types of
phenolic compounds which range from simple monomers to high molecular weight (MW) polyphenolic

0960-8524/01/$ - see front matter 2001 Published by Elsevier Science Ltd.

PII: S 0 9 6 0 - 8 5 2 4 ( 0 0 ) 0 0 1 4 1 - 3

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G. Vidal et al. / Bioresource Technology 77 (2001) 183191

Notation
A
AF
AFB
AOX
BE
BOD5
COD
ECF
EEC
FPLC
EFBM
HRT
HSWBL
IA

attached biomass
anaerobic lter
anaerobic uidised bed
adsorbable organic halogens
bleaching extraction
biological oxygen demand
chemical oxygen demand
elemental chlorine free
elementary chlorine-containing (bleaching)
fast performance liquid chromatography
eucalyptus board manufacturing
wastewaters
hydraulic retention time
hemp stem wood black liquor
intermediate alkalinity

polymers. Generally, low MW phenols are biodegradable (Colberg, 1988; Field et al., 1988) including
monomers and chlorinated phenols derived from degradation of lignin in the bleaching process. Lettinga
et al. (1991) reported that as MW of phenolic compounds
increased, anaerobic biodegradability decreased. Early
experiments have shown that anaerobic treatment alone
can remove up to 86% of the total low molecular weight
organo-chlorides (MW < 1000) and more than 90% of
chlorophenols and chloroguaicols. The high MW fraction of total organic chlorides (MW > 1000) is only
slightly aected and the COD removal does not generally exceed 35% (Welander, 1988). However, other authors indicate that high MW lignin fragments and
tannins are simply not biodegradable in anaerobic environments (Field et al., 1992; Lettinga et al., 1991).
From a water quality viewpoint, the fraction with low
relative MW (MW < 1000) and, in particular, the
chlorinated compounds which may be able to penetrate
the biological cell membranes, should be regarded as the
most hazardous to aquatic organisms. However, high
MW (MW > 1000) chlorinated matter may be potentially hazardous due to degradation into smaller molecules in the receiving water body (Lindstr
om and

Osterberg,
1984).
Anaerobic treatment is adversely impacted by the
presence of toxic substances which can interfere with the
metabolism of readily biodegradable substrate (Vidal
et al., 1997a; Sierra-Alvarez et al., 1994). Wood resin
(McCarthy et al., 1990), tannins (Field et al., 1988) and
chlorinated phenolics (Salkinoja-Salonen et al., 1982;
Vidal et al., 1997a) are toxic to methanogenic bacteria.
The crude resin collected from pine trees causes 50%
inhibition of methanogenic activity at around 50 mg/l.
On the other hand, McCarthy et al. (1990) showed the

KBC
KBE
MW
MWCO
O
OLR
T
TA
TCF
TNK
TS
TSS
UF
UASB
UV
VFA
VS
VSS

kraft bleaching chlorination

kraft bleaching extraction
molecular weight
molecular weight cut-o
occluded biomass
organic load rate
temperature
total alkalinity
total chlorine free
total nitrogen kjeldahl
total solid
total suspended solid
ultraltration
upow anaerobic sludge blanket
ultraviolet absorbance
volatile fatty acid
volatile solid
volatile suspended solid

role of resinic acids in the methanogenic toxicity of

chemithermomechanical pulp, and found that at a resin
acid concentration of 300 mg/l there is 40% of methanogenic activity inhibition. In addition, Sierra-Alvarez
et al. (1994) showed that bark-condensed tannins as well
as hydrolyzable tannins are quite toxic to methane
bacteria; depending on the kind of tannin, the IC50 can
be between 350 and 3000 mg/l. Only lignin monomers
with alkyl side chains have a noteworthy toxicity.
Considering that the monomers only make up a small
fraction of the total lignin, they are probably of minor
importance to wastewater toxicity (Sierra-Alvarez et al.,
1994). Besides, Sierra-Alvarez and Lettinga (1990) show
that the methanogenic toxicity was only evident in the
lignin low MW fraction.
Anaerobic biodegradability and toxicity to methanogens are strongly dependent on the characteristics of
wastewater. On the other hand, characteristics of the
pulp and paper industry wastewaters vary depending on
the process technology, type of the feedstock used as a
raw material and the specic management of the process
water, particularly internal wastewater recycling. In fact,
in kraft mill euents, the bleached sequence process has
a real impact on organic matter, colour and indirectly
over high molecular weight compounds (Cates et al.,
1995; Dahlman et al., 1995). Composite euents from
total chlorine-free (TCF) bleaching had approximately
twice the COD in softwood (like pine) euent and
nearly three times the COD in hardwood (like Eucalyptus) euent relative to the elemental chlorine-free
bleaching sequences (ECF) composite euent (Cates
et al., 1995). Moreover, Cates et al. (1995) found that
the colours of the softwood and hardwood TCF euents
were 30% and 38% lower than the softwood and hardwood ECF counterparts, respectively. Vidal et al.

KBE

BE

KBC
KBE
HSWBL
EFBM

AFB

AF

UASB
UASB
UASB
UASB

0.30.7
0.50.9
5.8

0.58

0.40.5

0.40.8

BOD5

0.92.1
1.11.6
19.3
52

3.5

0.81.1

COD

47190
613

0.62.2

0.52.2

AOX

Wastewaters characteristics

113b
287c

UV

55
55
36
36

3538

36

36

(C)

9.920.6

1.11.5
1.11.2

0.33

0.80.1

(d)

HRT

0.61.9
0.61.9
3.73.8
0.51.6

1.42.1

1.52.5

(g=l d)

OLR

6278
8291

61

60

COD

98

78

40

5090

BOD5

Removal (%)
AOX

68

7090

5095

2858b
3875c

UV
Hakulinen and Salkinoja-Salonen (1982)
Hakulinen and Salkinoja-Salonen (1982)
H
aggblom and Salkinoja-Salonen (1991)
Rintala et al. (1992)
Rintala et al. (1992)
Kortekaas et al. (1998)
Soto et al. (1991)

References

Unit. OLR: g COD=l d, HRT: days, COD; g/l, BOD5 : g/l, AOX: mg Cl/l. Reactors. UASB: Upow Anaerobic Sludge Blanket. AFB: Anaerobic Fluidized Bed. AF: Anaerobic Filter. Wastewater.
KBC: Kraft Bleaching Chlorine. KBE: Kraft Bleaching Extraction. BE: Bleaching Extraction. HSWBL: Hemp Stem Wood Black Liquor. EFBM: Eucalyptus Fiber Board Manufacturing
b
UV280 : absorption unit 1, 1 cm.
c
UV215 : absorption unit 1, 1 cm.

KBC

AFB

Reactor

Table 1
Comparison of various anaerobic treatment process for dierent wastewaters from kraft mill industries

G. Vidal et al. / Bioresource Technology 77 (2001) 183191

185

186

G. Vidal et al. / Bioresource Technology 77 (2001) 183191

(1997a) showed that a large fraction of the elementary

chlorine-containing (ECC) bleaching euent COD was
anaerobically biodegradable, accounting for 67% of the
COD. This value largely exceeds the anaerobic COD
removal eciency (29%) found in softwood ECC
bleaching (Yu and Welander, 1994). Also, this study
(Vidal et al., 1997a) indicates that TCF bleaching enhances the anaerobic COD biodegradability up to 75%.
In this work the euent came from kraft mill processing in factories where chlorine dioxide is used at the
bleaching stage. Pinus radiata is used as feedstock. The
purpose of this study was to investigate the molecular
weight distribution of Pinus radiata kraft pulping
wastewater treated by anaerobic digestion using two
types of anaerobic reactors: xed bed and sludge blanket.
2. Methods
2.1. Wastewater
Wastewater was obtained from a modern bleached
kraft mill located in the South of Chile (Diez et al.,
1999), where only aerobic lagoons are used as biological
treatment.
Two
dierent
sequences
(D0 E0 D1 D2 ; C50% =DE0 D1 D2 ) are used for bleaching
softwood pulp. Both bleaching processes apply four
chemical steps, and only one bleaching sequence with
50% of chlorine substitution in the rst step. Pinus
radiata is the raw material used in the process.
2.2. Biomass characterisation
Initial and nal activities of biomass in both reactors
anaerobic lter (AF) and upow anaerobic sludge
blanket (UASB) were determined according to Soto
et al. (1993). At the end of the operation, the characterisation of AF reactors biomass was determined. Two
kind of biomass were considered: the attached biomass
(A) adhered to the support and the occluded biomass
(O), which remained as ocs or granules between the
rings. A previously developed method (Pu~
nal et al.,
1999) for separating the dierent types of biomass was
employed.
2.3. Continuous anaerobic treatment
The UASB (200 ml) and AF (200 ml) were operated
in a continuous ow system. The supporting material of
AF consisted of corrugated PVC Raschig rings of 1.4 cm
diameter with a specic surface of 449 m2 /m3 . Both reactors were inoculated with 20 gVSS/l of anaerobic
occulent sludge (0:9 g COD=g VSS d) and placed in
a thermostatic chamber at a constant temperature of
37C.

Both reactors were initiated with raw wastewater at

low organic load rate (OLR) and increased progressively. Nutrients were added to reach a COD/N/P ratio
of 200/7/1 using standard stock solutions (Sierra-Alvarez and Lettinga, 1991) and pH adjusted to 7:0  0:05
by 1M and 5M HCl or NaOH. Flow rate, pH, COD,
BOD5 , TS, colour, tannin and lignin, and total phenol
compounds (UV215 ) were measured during the operation
in both reactors. Also, total alkalinity (TA) and Alkalinity ratio were determined. Methane gas production
was also monitored during the operation in both reactors.
2.4. Analytical methods
Volatile suspended solid (VSS), total suspended solid
(TSS), total solid (TS), volatile solid (VS), chemical
oxygen demand (COD), biological oxygen demand
(BOD5 ), colour, phenols, total phenolic compounds
(UV215 ), total nitrogen kjeldahl (TNK), phosphate and
tannin and lignin fragments were measured by standard
methods (APHA, 1985). Samples for determination of
COD, BOD5 , colour, phenols, UV215 , phenolic and lignin degradation products were membrane ltered (0.45
lm). UV215 was determined in a 1 cm quartz cuvette by
diluting the samples to less than 0.8 absorbance units,
0.025M borate was used as buer providing a pH 9.1.
Total alkalinity and the alkalinity due to VFA (Intermediate Alkalinity, IA) were determined by titration
(Ripley et al., 1986). Its ratio (IA/TA) is a very useful
parameter because it allows the proper operation of the
digester to be checked. When this ratio remains below
0.30.4, it indicates that no accumulation of VFA occurs
(Fernandez et al., 1995). The produced biogas was periodically measured by volumetric displacement of an
alkaline solution (25 g NaOH/l) used to retain the produced CO2 .
Ultraltration was done at 20C using a 450 ml stirred cell (Micro-Prodicon Model UHP 75) with an exposed membrane surface area of 38.5 cm2 . Two cellulose
membranes were used, the nominal molecular weight
cut-os (MWCO) were 10,000 and 1000 Da. Nitrogen
was applied over the liquid in the stirred cell. Prior to
ultraltration, the pH of the wastewater was adjusted to
8.0 and subsequently the sample was ltered through a
0.2 lm membrane lter to fractionate the lignin, 80 ml
of wastewater sample was ltered through a 10,000 MW
membrane to give approximately 75 ml of permeate and
3 ml of retentate. This ltration was repeated after addition of 50 ml of NaHCO3 buer solution (0.025M, pH
8.0) to the retentate. Subsequently, the permeates were
collected and ltered through a 1000 MWCO to give a
retentate of 3 ml. After ultraltration three UF samples
were obtained: a 10,000 Da retentated sample, called as
``> 10; 000 Da''; another sample which passed by the
10,000 Da membrane, but was rejected by the 1000 Da

G. Vidal et al. / Bioresource Technology 77 (2001) 183191

membrane, denominated as ``10; 000 < MW < 1000'';

and a 1000 permeate sample, named as ``< 1000 Da''.
Raw wastewater and anaerobic euent were analysed
by Fast Performance Liquid Chromatography (FPLC)
with a LCC 501 Plus detector using a Superdex 75 HR
10/30 column (Pharmacia, Piscataway, USA) at a 0.4
ml/min ow rate and monitoring at 280 nm. 750 nM
NaCl and 10% (v/v) methanol were used as eluent
(Feijoo et al., 1995). The percentage of MW compound
degradation was calculated by comparing the areas under the curves corresponding to each chromatogram.
The biochemicals used for molecular weight markers
included Aprotinin (MW 6500 Da), Quimotripsinogen
A (MW 25,000 Da), Ovoalbumine (MW 43,000 Da)
and Ferritine (MW 440,000 Da).

3. Results and discussion

3.1. Operation and performance of the UASB and AF
reactors
The characteristics of the euents are shown in Table
2. pH ranged (3.56.0) from acidic euents in bleaching
sequences containing steps to neutral or alkaline values
in those utilising peroxide dioxide. Total and soluble
COD values were similar, indicating low suspended
solids in the euents. According to BOD5 values, only
25% of the organic matter was aerobically biodegradable. Total phenolic compounds ranged between 190
and 350 mg/l; 1823% corresponding to the tannin and
lignin degradation products and 0.340.47% corresponding to the phenolic compounds.
The feasibility of phenolic compound biodegradation
of Pinus radiata kraft mill wastewater was studied by
comparing the performance of a UASB reactor with an
AF reactor. A diluted inuent was fed to a UASB
(OLR: 1:7 g COD=l d) and to a AF (OLR: 1:2 g
COD=l d). In order to prevent methanogenic inhibition
in the start-up of the reactor and to promote gradual
biomass acclimation, the OLR was increased gradually

Table 2
Raw wastewater characteristics
Parameter

Range

pH
COD (mg/l)
BOD5 (mg/l)
Total phenolic compounds UV215 (mg/l)
Phenols (g/l)
Tannin and lignin (mg/l)
Colour (U Pt/Co)
TSS (g/l)
TNK (mg/l)
Phosphate (mg/l)

3.510.6
0.81.9
0.20.5
190.0350.0
0.91.2
44.064.0
9501400
1.23.1
3.09.0
0.41.3

187

to 3:1 g COD=l d and the TRH from 12 to 15 h. It is

well known that wastewater derived from Pinus radiata
is more toxic than those derived from hard wood (for
example, Eucalyptus). Previous research has shown that
methanogenic toxicity expressed as 50% IC (50% inhibiting concentration) is 0.6 g COD/l for Pinus radiata
kraft mill euent (Vidal et al., 1997b). This value is
relatively toxic compared with the debarking process,
for example (50% IC: 0.91.0 g COD/l) (Sierra-Alvarez
et al., 1994). This is not unexpected because of the larger
wood extract content in soft wood (in this case Pinus
radiata) as compared with hard wood (see Fig. 1).
In UASB reactor case, the operation was divided in
four stages according to Table 3 and Fig. 2; the average
OLR increased from 1.7 to 3:1 g COD=l d with an average of BOD5 elimination from 60% to 90%. A reduction in BOD5 was observed with increasing
operation time, possibly due to adaptation. The corresponding COD removal eciencies ranged between 30%
and 40%. The phenolic compounds removal (UV215 ) was
about 1030% which indicates only a minor removal of
these types of compounds. The total alkalinity (TA)
ranged between 1000 and 1500 mg/l. The alkalinity ratio
(IA/TA) was always below 0.3, indicating that no accumulation of VFA occurs (Fernandez et al., 1995). The
low COD removal eciency reects the fact that lignin
in these wastewater is not signicantly degraded by anaerobic bacteria. According to Sierra-Alvarez and
Lettinga (1990) only low MW lignin, which accounts for
a minor fraction of the total wastewater lignin, is biodegraded under anaerobic conditions. Sierra-Alvarez
and Lettinga (1990) also showed that 5085% of phenolic compounds were degraded under anaerobic conditions. On the other hand, only 1029% of tannin and
lignin was removed in this system (Table 3). Kortekaas
et al. (1998) reported a 43% lignin removal eciency
based on UV280 absorbance measurements in a combined anaerobicaerobic system. Moreover, the highest
(29%) tannin and lignin removal was simultaneous with
phenolic compounds. However, colour in the UASB
systems was not removed. According to Field et al.
(1992) colour is an important characteristic associated
with lignin and other high MW polyphenolics. Moreover, colour can be increased due to the low molecular
weight lignin polymerization (Kortekaas et al., 1998).
At the end of the operation (350 d), occulent sludge
in the reactor increased approximately 30% (5,7 g VSS);
however, the methanogenic activity decreased about
77% (0:2 g COD=g VSS d). No granular sludge was
obtained, due to the absence of recirculation in the reactor and due to this low operating upow velocity.
Kortekaas et al. (1998) showed that without recirculation, the methanogenic activity in a UASB dropped to
under 2.5% in the black liquor euent. They also reported that recirculation increased the methanogenic
activity by about 26.3%.

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G. Vidal et al. / Bioresource Technology 77 (2001) 183191

Fig. 1. A schematic presentation of the experimental set-up. (A) UASB reactor (1. Inuent, 2. Distributor, 3. Sludge bed, 4. Separation S/L/G, 5.
Biogas, 6. Euent). (B) AF reactor (1. Inuent, 2. Supporting material, 3. Biogas, 4. Euent).

Table 3
Average removal parameters in the UASB and AF reactors during the operation
OLR
(g=l h)

COD (%)

BOD5 (%)

UV215 (%)

Colour
(%)

Phenol
(%)

Tannin and lignin

(%)

UASB reactor
190
90160
160180
180215

1.7
2.1
2.8
3.1

33
32
39
36

60
85
90
90

31
17
20
13

)20
)25
)5
)90

50
80
85
50

10
12
29
19

AF reactor
185
86160
161295

1.2
2.2
3.3

50
42
49

87
85
77

21
13
15

)25
)23
)13

44
24
45

40
38
45

Period (d)

The average OLR in the anaerobic lter increased

from 1.2 to 3:3 g COD=l d, over a period of 295 days of
operation are illustrated in Table 3 and Fig. 3. The
operation and performance of the AF reactor. Results
from the AF reactor were similar to UASB studies.
Removal of phenolic compounds such as lignin was low
(3845%) and the colour increased during the operation
()13%, )90%). The eciency of removal of both, BOD5
and colour decreased as the OLR increased from 1.2 to
3:3g=l h). Biodegradable matter removal in the AF
reactor ranged between 77% and 87%, while the COD
removal was slightly higher than the UASB reactor
(until 49%). However, tannin and lignin removal varied
between 38% and 45% during reactor operation. Those
results can be explained by the biolm formation in an
inert support material (total biomass: 0.13 g VSS attached in the 42 Rasching rings). The biomass activity
decreased during the operation of the reactor; in fact,

the specic methanogenic activity of the attached biomass was only 4:8 mg COD=g VSS d and occluded
biomass (3.7 g VSS) 0:52 g COD=g VSS d. The attached biomass was less active than the occluded mass,
which agrees with previous reports made by other authors (Pu~
nal et al., 1999, Tilche and Vieira, 1991). In this
case, the activity loss can be related to the presence of
the increasing toxic concentrations (OLR: 1.2 to
3:3 g COD=l d). However, biomass adaptation to the
toxic compounds was also observed (see COD, UV215 ,
phenol removal parameters for 2.2 and 3:3 g=l h). The
stable operation of the AF reactor can be attributed to
the increasing biomass from 2.44 g VSS until 3.86 g VSS
at the end of the operation.
According to this study, the anaerobic system cannot
biodegrade the Pinus radiata kraft mill pulping wastewater completely and, consequently, there were recalcitrant compounds in the anaerobic euent. Vidal et al.

G. Vidal et al. / Bioresource Technology 77 (2001) 183191

Fig. 2. Performance of UASB reactor. (a) OLR values, (b) Alkalinity

ratio, (c) Percentage of BOD5 j and phenol  removal, (d) Percentage of COD ( ) and colour (- - - -) removal.

(1997b) also noted that the best COD removal was 42%
at a 10% dilution of the wastewater with tap water.
The eect of biological treatment on the molecular
weight distribution of wastewater lignin was studied by
a combination of ultraltration (UF) and gel permeation chromatography (FPLC) techniques. The UF of
raw wastewater and anaerobic euent (mixed UASB
and AF euent) is shown in Fig. 4. Because membrane
lters of 10,000 and 1000 Da were used, raw wastewater
and anaerobic euent were divided into three fractions.
In order to compare each fraction, COD, colour and
UV215 were measured. By comparing raw wastewater
and anaerobic euent, it can be observed that COD and
colour were reduced in the fraction below 1000 Da as a
consequence of anaerobic treatment. However, total
amount of phenolic compounds (UV215 ) increased approximately 25%. In the fraction 1000 < MW <
10; 000 Da, it was observed that COD, UV215 and colour almost no change. In addition, colour and COD in
MW > 10; 000 Da fraction after anaerobic treatment
increased approximately 14% and 5%. Compounds
above 10,000 Da MW are lignin compounds, which
according to Kortekaas et al. (1998) could be increasing
due to the low lignin polymerisation. In fact, they reported polymerisation from 2200 Da lignin to lignin of

189

Fig. 3. Performance of AF reactor. (a) OLR, (b) Total alkalinity ()

and alkalinity ratio (} ), (c) Percentage of COD ( ) and UV215
(- - - -) removal.

more than 34,000 Da. The polymerisation darkened

colour of lignin is also supported by the results shown in
Table 3. On the other hand, the above mentioned results
explains the recalcitrant COD in the anaerobic euent
(almost 60% of COD in UASB and 50%, approximately,
in AF system). Moreover, only low MW lignin, which
accounts for a very minor fraction of the total wastewater lignin, is actually degraded by anaerobic bacteria
(Field et al., 1992). In fact, wastewater such as black
liquors and bleaching euents, in which lignin can account for 50% of the COD are generally only 50% biodegradable or less (Sierra-Alvarez et al., 1991).
Similarly, semichemical and chemo-thermo-mechanical
pulping liquors contain signicant fraction of lignin and
are so not fully biodegradable (Lettinga et al., 1991).
The relationship between COD, UV215 and colour is
shown in Table 4. Colour bearing properties of lignin
changed from 0.8 colour/UV215 for the raw wastewater
to 1.3 for anaerobically treated. Colour in the
MW < 1000 retentate and MW > 10; 000 Da samples
contained more colour that the lignin in the
1000 < MW < 10; 000 Da permeate sample. The main
dierence was between three UF samples in the parameter colour/COD, with respect to the MW < 1000
fraction, which changed from 19.2 colour/COD for the

190

G. Vidal et al. / Bioresource Technology 77 (2001) 183191

Fig. 5. MW distribution of the raw wastewater and anaerobic euent.

Raw wastewater ( ) Anaerobic euent (- - - -).

Fig. 4. Ultraltration of the raw wastewater and anaerobic euent. (a)

Raw wastewater, (b) Anaerobic euent (number of used replicates: 4).

raw wastewater to 0.15 colour/COD for anaerobic efuent. As suggested by Field et al. (1992), this may be
due to the ability of the anaerobic consortium to biodegrade low MW compounds.
The MW distribution of raw wastewater and anaerobic euent is shown in Fig. 5. The behaviour of the low
MW compounds cannot be observed because the column used could only separate compounds with molecular weight higher than 15,000 Da with reasonable
resolution. It is evident that anaerobic treatment is unable to biodegrade compounds with a MW of 25,000
Da. Moreover, these types of compounds are unable to
penetrate through the cell walls of the outer membrane
or the slime layers surrounding bacteria. In addition,

high MW compounds, such as lignin and polymerised

tannins, were found to be relatively non-toxic for
methanogens (Sierra-Alvarez et al., 1995). Field et al.
(1990) showed that the compounds greater than 1000
3000 g/mol are too large to cause bacterial inhibition.
The chlorophenols therefore are not the only source of
methanogenic toxicity in bleaching euents. Further
studies are needed to determine the importance of the
other low MW organohalogens, such as low MW fractions of chlorolignin, chloroform and chlorinated resin
acids (Sierra-Alvarez et al., 1995).
Based on results obtained in this study, UASB and
AF reactors do not work very well for treatment of
Pinus radiata wastewater. In fact, we have observed a
much lower biodegradation percentage than those reported by Soto et al. (1991) and Kortekaas et al. (1998).
One of the reasons for this dierent behaviour is the type
of kraft mill euent utilised: Eucalyptus in these two
previous studies and Pinus radiata in our case.
Results from this study suggest that anaerobic bacteria are unable to degrade the lignin chromophoric
structure. The polymerisation of lignin may oer some
benets for subsequent physico-chemical removal. Precipitation of lignin with lime or sodium sulphate is

Table 4
Characteristics of UF fractionated samples of raw wastewater and anaerobically treateda
Sample
Raw wastewater
UV215 /COD
Colour/COD
1000)
Color/UV215
1000
Anaerobic euent
UV215 /COD
Colour/COD
1000
Colour/UV215
1000
a

Total

MW < 1000

100010,000

MW > 10; 000

0.19
19.2
0.8

0.36
0.14
0.38

0.47
0.49
1.02

0.34
0.27
0.80

0.13
0.15
1.28

0.42
0.20
0.47

0.52
0.76
1.48

0.39
0.50
1.30

COD, Colour and UV215 of the dierent UF samples are expressed as a percentage of the concentration in the raw wastewater.

G. Vidal et al. / Bioresource Technology 77 (2001) 183191

known to be more eective for high MW lignin (Kortekaas et al., 1998). Moreover, combined technologies,
including physico-chemical and enzymatic or fungal
treatments (Feijoo et al., 1995) may be considered to
remove the colour which is resistant to anaerobic
wastewater treatment (Field et al., 1992). A combination
of these technologies could possibly allow complete biological removal of organic matter in forest industry
wastewater.
Acknowledgements
This work was supported by FONDECYT 1970868
grant. The authors wish to thank the Biotechnology
Group belonging to Chemical Engineering Department
of the University of Santiago de Compostela for the
FPLC analysis.
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