Integrated biological treatment of recalcitrant effluents

Water Science and Technology Vol 50 No 3 pp 145156 IWA Publishing 2004

from pulp mills
A. Ortega-Clemente*, C. Estrada-Vzquez*, F. Esparza-Garca*, S. Caffarel-Mndez**,
N. Rinderknecht-Seijas*** and H.M. Poggi-Varaldo*
* CINVESTAV-IPN, Dept. Biotechnology, Environmental Biotechnology R&D Group, P.O.Box 14-740,
Mxico D.F., 07000, Mxico. (E-mail: hectorpoggi2001@yahoo.com)
** TESE, Ecatepec Institute of Technology, Edo. de Mex., Mexico
*** ESIQIE-IPN, Division of Basic Science, Campus Zacatenco, Mxico D.F., Mxico

Abstract This work aimed at determining the degree of depuration of a recalcitrant effluent (weak black
liquor, WBL) achieved in a series treatment consisting of a first stage methanogenic fluidised bed reactor
followed by a second stage aerobic, upflow reactor packed with biocubes of Trametes versicolor
immobilised onto small cubes of holm oak wood. The mesophilic, lab scale methanogenic fluidised bed
reactor contained a microbial consortium immobilised onto granular activated carbon 500 m average size.
The process removed decreasing amounts of organic matter at decreasing hydraulic retention times (HRT),
eventually reaching an average of 50% at 0.5 day HRT. Colour and ligninoid removals also decreased with
decreasing HRT. Although the methanogenic fluidised bed reactor provided an effective treatment for the
degradable organic matter, important concentrations of recalcitrant organic matter and colour still remained
in the anaerobic effluent. This anaerobic effluent was fed to the aerobic packed bed reactor. Two HRT were
tested in this unit, namely 5 and 2.5 days. The reactor averaged an organic matter removal in the range of
32% COD basis, during an experimental run of 95 days. Colour and ligninoid contents were removed in high
percentages (69% and 54%, respectively). There was no significant difference in reactor performance at 5-
and 2.5-day HRT. There was a positive correlation between pollutant removal efficiencies and Laccase
activity in crude extracts of the reactor liquor. No supplemental soluble carbohydrate was required to sustain
the fungus activity and the consistent reactor performance. Overall, the two-stage treatment achieved
approximately a 78% removal of the original organic matter of the WBL (COD basis) and ca. 75% of colour
and ligninoid contents.
Keywords Aerobic; anaerobic; fluidised bed; Kraft wastewater; ligninolytic fungus; packed reactor; series
treatment

Introduction
The Mexican pulp and paper industry (PPI) manufactures ca. 700,000 tonnes of pulp/yr and
2,900,000 tonnes of paper/yr. Approximately half of the pulp production is made by the
Kraft process. Nearly 60% of the Kraft mills are non-integrated factories, i.e., they manu-
facture non-bleached pulp (CNICP, 1993). The PPI contributes with a 2.1% share of the
industrial GIP and it directly employs 31,000 workers. The installed production capacity is
distributed among sixty-seven plants (Poggi-Varaldo, 1994a). The PPI discharges approxi-
mately 100 million m3/yr of wastewater, which represents ca. 12% of the annual industrial
discharges and the sector holds the second position in the ranking of main water industrial
polluters in Mxico (Poggi-Varaldo, 1994b). Currently, the PPI is facing more stringent
standards for wastewater discharges and associated permits.
Application of anaerobic treatment to the forest industry effluents has noticeably
increased in the last twenty years. This technology, particularly the immobilised biomass
reactors, offers clear advantages over aerobic and physicalchemical treatment processes
(Poggi-Varaldo et al., 1990; Lettinga et al., 1991; Poggi-Varaldo, 1993). Much research
has been conducted on anaerobic treatment of the PPI wastewaters. Outstanding advances 145
 have been made by European, North-American, and Mexican research teams in the last two
decades on the application of anaerobic processes to a variety of PPI effluents (Norrman et
al., 1984; Poggi-Varaldo et al., 1990, 1994; Hggblom and Salkinoja-Salonen, 1991;
Parker et al., 1993; Kortekaas et al., 1994; Poggi-Varaldo, 1994c; Poggi-Varaldo and
Rinderknecht-Seijas, 1996). One of the most challenging issues has been the treatment of
the wastewaters polluted by spent pulping liquors. These effluents are quite toxic to aquatic
fauna and microbes and contain considerable amounts of recalcitrant organic matter
A. Ortega-Clemente et al.

(Poggi-Varaldo, 1994a, 1995; Ali and Sreekrishnan, 2001). Mexican pulp mills have
implemented black liquor recovery and re-utilization. However, spills from digesters
washings and black liquor recovery systems are frequent. As a result, black liquor spills are
the main water pollutant in Mexican, non-integrated Kraft pulp mills. Previous work has
demonstrated that soda pulping liquor and wastewaters were very recalcitrant and toxic to
anaerobic consortia (Sierra-Alvarez et al., 1990, 1991; Kortekaas et al., 1994). However,
little is known about anaerobic treatment application to wastewaters contaminated with
Kraft black liquor.
It is well known that fungi belonging to the basidiomycetes (such as Trametes versicol-
or, Phanerochaete chrysosporium) are microorganisms with a demonstrated capability for
degrading lignin and its derivatives using a powerful and diverse group of enzymes (Coll et
al., 1993). We hypothetized that immobilised Trametes versicolor could be an interesting
alternative for post-treating and degrading the remaining recalcitrant organic matter of the
anaerobic effluent coming out of the anaerobic reactor. So, the objective of this work was to
determine the degree of depuration of a recalcitrant wastewater (hereinafter weak black
liquor, WBL) achievable in a series treatment consisting of a first stage methanogenic flu-
idised bed reactor followed by a second stage aerobic, upflow reactor packed with
biocubes of Trametes versicolor (ligninolytic fungus immobilised onto small cubes of
holm oak wood).

Materials and methods

Anaerobic batch assays
Ultimate anaerobic degradability (UAD) and toxicity assays of the weak black liquor
(WBL) were conducted according to procedures described elsewhere (Owen et al., 1979;
Shelton and Tiedje, 1983). The WBL was used as both the carbon and toxicity source; no
volatile organic acids were added. Non-acclimated inoculum for the assays came from a
mesophilic complete mix digester fed with synthetic wastewater and operated at 20 days
HRT (Estrada-Vzquez et al., 2001).

Series anaerobic/aerobic treatment: experimental design

Figure 1 displays a scheme of the series anaerobic/aerobic process configuration. Water
contaminated with black liquor spills (WBL) was fed to an anaerobic fluidised bed reactor
(RANLEF; Figure 1). The WBL was supplied by a Mexican Kraft pulp mill which used
pine as wood stock. After a cautious and conservative start-up and acclimation period, the
hydraulic retention time (HRT) was varied from 5 to 0.5 day in 23 consecutive pseudo-
steady states. The acclimation was carried out in 7 stages 20 days duration each; a mixture
of decreasing synthetic wastewater and increasing WBL proportions was fed into the reac-
tor. The bench scale reactor was a glass column 3.0 L total geometric volume containing 1 L
granular activated carbon (GAC) 30/40 mesh. More details on the reactor features and the
bench scale setup can be consulted elsewhere (Poggi-Varaldo et al., 1990, 1994). The treat-
ed effluent of RANLEF (named anaerobic effluent; EAn) from the stage at 1 day HRT was
fed to up-flow aerobic packed bed reactor (RAELEMP). Two HRT were tested in this unit,
146 namely 5 and 2.5 days. The reactor operated 60 days at 5 day HRT and 35 days at 2.5 day
 A. Ortega-Clemente et al.
Figure 1 Series Anaerobic/Aerobic process configuration. GAC: granular activated carbon; RANLEF:
Anaerobic Fludized Bed Reactor; RAELEMP: Packed bed reactor (wood cubes with immobilised Trametes
versicolor)

HRT. The aerobic packed bench scale reactor was a glass column 1.5 L total geometric
volume containing 0.75 L biocubes (active volume), corresponding to 150 units of
Trametes versicolor immobilised onto holm oak wood small cubes of 5 mm side. The
fungus immobilization was carried out in a two-stage process consisting of fungal colo-
nization of the cubes in Petri dishes followed by an affirmation step in shake flasks with
mycological broth (Ortega-Clemente et al., 2002). The reactor was operated at room
conditions (25C). The pH of the EAn was adjusted to 4.5 before feeding; no carbohydrate
nor other soluble carbon source was supplemented. The total fungal biomass in the packed
bed of the reactor was 1,200 mg as dry weight. A sterile, control reactor (packed with sterile
biocubes and fed with sterile anaerobic effluent) was operated in parallel to the main fungal
reactor.

Analysis
In the anaerobic treatment, the influent and effluents were analyzed for pH, alkalinity,
volatile organic acids (VOA) by steam distillation method, chemical oxygen demand
(COD), biochemical oxygen demand (BOD7), total solids, volatile solids, total suspended
solids, and volatile suspended solids. The parameter, which is approximately proportion-
al to the volatile organic acid concentration, was based on alkalinity measurements (Ripley
et al., 1986). Acute toxicity tests were carried-out in the influent and effluent using
Daphnia magna as test organism. All analyses were performed according to the Standard
Methods (1985), except Daphnia magna bioassays, and biochemical oxygen demand. The
first followed the Standard Methods with modifications as in Dutka (1992). Biochemical
oxygen demand was determined at 7 days incubation time according to Scandinavian stan-
dard (SFS 3019, 1979). Colour and ligninoid concentration were determined in spectropho-
tometer at wavelengths of 465 (visible) and 280 nm (UV) respectively. A 1 cm width quartz
cuvette was used; absorbance was measured previous dilution down to an absorbance value
nearly 0.3. Sample pH was adjusted to 7.0 with 1 mL of a phosphate buffer; this buffer con-
tained 305 mL of Na2HPO4 0.2 M and 195 mL of NaH2PO4 0.2 M in a 1,000 mL total vol-
ume. Biogas volume was measured by acid brine displacement, volumes reported here
were corrected at 0C and 103 kPa (referred to as NL). The composition of biogas was 147
 analyzed for methane and carbon dioxide by gas chromatography (Poggi-Varaldo et al.,
1997).

Formulae
Detoxification due to the treatment was reported using a dimensionless detoxification fac-
tor (DF), defined in Eq. (1) (Campos-Velarde et al., 1997) or in terms of the toxicity
removal efficiency detox (Eq. (2)):
A. Ortega-Clemente et al.

DF = LC50effluent/LC50influent (1)

detox = 100(1 1/DF) (2)

where LC50 is the concentration of the toxic wastewater at which half of the test organism
population is killed in the D. magna bioassays. For instance, the higher the DF, the more
detoxification is achieved by the treatment.
The specific, unit, net removal efficiencies (sun) of colour, ligninoids and chemical
oxygen demand (COD) were determined with the Eq. (3):

sun = [(Pi Pf) (Pic Pfc)]/(Pi * B) (3)

where Pi = initial value of the parameter in the experimental unit; Pf = final value of the
parameter, at the end of the incubation cycle; Pic = initial value of the parameter in the con-
trol flask; Pfc = final value of the parameter in the control flask; B = Biomass in the flask.
The efficiency defined in this way is expressed per unit of biomass (specific), per unit of ini-
tial pollutant concentration (unit) and it discounts the abiotic removal of the parameter
(net).
The overall, combined removals (o) of colour, ligninoids and chemical oxygen demand
(COD) were determined according to the Eq. (4):

O = 1 + 2 (1 * 2) (4)

where 1 = conventional removal efficiency in the anaerobic stage (RANLEF); 2 = con-

ventional removal efficiency in the fungal, aerobic stage (RAELEMP). The three efficien-
cies are in decimals (0 < j < 1).

Results and discussion

Wastewater characteristics
The influent WBL was a medium organic strength, alkaline (pH 8.8), highly coloured
wastewater. The organic matter content was 2,255 mg/L as COD; total solids, total sus-
pended solids and volatile suspended solids were 2,543, 2,132, and 128 mg/L, respectively.
The average volatile organic acid concentration was 210 mg/L. Colour and ligninoids con-
tents were 1.03 and 46.28 as absorbances at 465 and 254 nm, respectively.
The UAD was approximately 55% of the total COD, which suggested the presence of a
considerable amount of recalcitrant organic matter (ca. 45%). The latter was probably due
to ligninoid substances originated during the cooking of the wood (the Kraft chemical
digestion is one with the highest negative yields, i.e., 200 to 500 kg dissolved wood as
COD/tonne processed wood). The WBL-UAD was similar to that of the soda pulping
liquors (pine, spruce, wood hemp) which were in the range 45 to 59% COD but lower than
148 that of TMP liquor (67 to 78% COD; Kortekaas et al., 1994; Sierra-Alvarez et al., 1991).
Anaerobic toxicity of the weak black liquor (WBL)
The anaerobic toxicity tests results are depicted in Figure 2. At low WBL levels (10 to 50%,
i.e., 0.2 to 1 g COD/L), the organic matter was degraded with no inhibition effects. Thus,
the activity proportionally increased to WBL concentration. From 50% to 100% WBL pro-
portion, the toxic or inhibitory effect of the WBL predominated over the degradation
process, as expressed by the decrease on activity. The IC50 (concentration of WBL which
effected a 50% inhibition of the maximum activity) was approximately 90%, which trans-

A. Ortega-Clemente et al.
lates into 1.8 g COD/L. The toxicity to methanogenic consortia of WBL in this work was
comparable to that of soda pulping liquor. Sierra-Alvarez et al. (1991) determined that the
IC50 of soda pulping liquors (pine) was in the range 2.02.1 g COD/L. According to former
reports, the toxicity seemed to depend both on the pulping process and the type of wood
stock. For instance, Kraft and alkaline liquors resulted more toxic than thermo-mechanical
pulping (TMP) liquors; for a given pulping process, liquors from coniferous wood process-
ing seemed to give the most toxic liquors (2.0 COD/L versus 4.8 for spruce and 4.5 for
hemp; Sierra-Alvarez et al., 1991; Kortekaas et al., 1994). The wood extractives (resin
acids, terpenes, long-chain fatty acids) are reported to be the main toxicity source in pine-
derived black liquors. In this regard, it is known that resin acids have an acute toxicity to
fish comparable to chlorophenol compounds, with LC50 ranging from 0.2 to 1.7 mg/L
(Zender et al., 1994). Conifers contain 3 to 8% of extractives which were demonstrated to
be inhibitory to methanogenic consortia (Richardson et al., 1991; Zender et al., 1994).
Resin acids and related compounds are easily extracted by alkaline chemicals used in Kraft
and soda pulping processes and therefore, their concentration in black liquors may be
considerable.

Anaerobic fluidised bed reactor performance

The anaerobic treatment results are displayed in Figure 3.
The COD removal efficiency (total COD basis) varied in the range 80 to 48% when the
HRT was decreased from 5 days to 0.5 days, Figure 3A. The COD reductions were in the
range 87 to 96% expressed on biodegradable COD basis at the shortest HRT of 0.5 and 0.75
days. The highest COD removals during the last stages of the acclimation and the first
HRTs could be partially due to the adsorption capacity of the GAC carrier. As the experi-
ment continued at lower HRTs, the adsorption capability could become exhausted and only
the background biological COD removal remained. COD reductions in this work were
somewhat higher than that found by Kortekaas et al. (1994) for the anaerobic treatment of
wood hemp soda black liquors in UASB (42.3% at 9.218.4 gCOD/L.day) but similar to the
COD elimination in UASB treatment of pine and spruce soda pulping black liquors (51% at

125

100
ACTIVITY (%)

75

50

25

0
10% 30% 50% 80% 100%

-25
%DL

Figure 2 Activity of methanogenic inocula vs. dilute liquor concentration 149

 (A)
120 ACCLIMATION STEADY STATES
1.2

100
0.9

Yg (Lbg/g CODrem)
80

Ig (Lbg/[LR*d])
COD (%)
60 0.6

40
0.3
A. Ortega-Clemente et al.

20

0 0.0

T 75
d
T .5

HR T = d
T 1d

5
1
2
3
4
5
6
7

0.
5
HR = 3
HR = 2

HR 0.
=

=
=
T
T
HR
HR
STAGE
COD Rem Eff Yg Ig

(B)
2,000 1.5
Steady states Steady state
Start-up
5, 3.5, 2, 1, 0.75 d HRT = 0.5 d

1,600 1.2
TOTAL ALK. (mgCaCO3/L)

1,200 0.9

Acclimation
800 Reactor 0.6
complete
spill
400 0.3

0 0
0 50 100 150 200 250
TIME (days)

Total Alk (mg CaCO3) ALPHA

Figure 3 Fluidized Bed Anaerobic Reactor Performance: (A) average COD removal efficiency, average
biogas yield Yg,v and average biogas productivity Ig,v; (B) Alkalinity and

loading rates of 3.16.5 g COD/L.day; Sierra-Alvarez et al., 1994). Vidal et al. (2001)
found removals of 3050% and 6090% of COD and BOD, respectively, in the anaerobic
treatment of a Pinus radiata Kraft pulping wastewater in a UASB and anaerobic filter dur-
ing operation at 1.2 to 3.3 gCOD/(L d). Also, they observed a poor removal (1043%) of
tannin and lignin.
Biogas yield (Figure 3A) suffered a sharp drop in the first two pseudo-steady states (5
and 3.5 days HRT); it stabilized around 0.15 NL/gCODrem for the lower HRTs, and sur-
prisingly, it increased up to 0.21 NL/gCODrem at the shortest HRT (0.5 day). The low val-
ues of biogas yield strongly suggested a partial inhibition. However, its recovery at 0.5 day
HRT seemed to indicate that some type of acclimation eventually occurred. This was unex-
pected, because previous research on soda black liquors indicated that the methanogenic
activity of the exposed inocula did not increase in consecutive batch assays (Sierra-Alvarez
et al., 1991). Despite the partial inhibition symptoms, other treatment performance param-
eters showed values typical of a normal methanogenic process. For instance, no acidogene-
sis was detected: the pH was in the range 6.97.8, while the a parameter was below 0.5
(Figure 3B), suggesting a low concentration of volatile organic acids (Ripley et al., 1986).
Colour and ligninoids removals are listed in Table 1. They seemed to be significant for
the first pseudo-steady states.
Both removals dropped when the HRT decreased, suggesting that the adsorption mecha-
150 nism was an important factor at the beginning of the treatability study when the adsorption
Table 1 Removal efficiencies: colour and lignins

HRT (d) colour (%) lig (%)

5.0 61.4 69.6

3.5 52.8 64.2
2.0 46.2 57.0
1.0 30.7 41.2
0.75 17.9 27.5

A. Ortega-Clemente et al.
capacity of the GAC was not exhausted. Their removals were higher than those reported in
UASB reactors treating hemp black liquors (Kortekaas et al., 1995; 1012% ligninoids,
59% colour). The colour and UV reduction can be attributed to low molecular weight
lignins removal. The adsorptive capacity and eventual bioregeneration of the GAC carrier
of the RANLEF could provide an advantage for the reduction of these species (Poggi-
Varaldo and Rinderknecht-Seijas, 1995; Poggi-Varaldo et al., 1991) when compared with
other anaerobic reactor types.
The anaerobic treatment effected a moderate toxicity reduction (data not shown);
approximately 47% of the toxicity to D. magna was removed (DF = 1.88). This extent of
detoxification, although significant, was not sufficient to give a water quality suited for dis-
charging in water bodies. Kortekaas et al. (1994) reported similar detoxification factors for
anaerobic treatment of hemp black liquors, ranging from 1.55 to 1.82 (the test organism
was a methanogenic consortium). The detoxification can be attributed to the elimination of
low molecular weight lignins, which impart mild toxicity and were found to be degraded by
methanogenic consortia (Rintala and Lepisto, 1993). On the other hand, the toxic wood
extractives are not significantly degraded in anaerobic processes (Lettinga et al., 1991).
In general, the continuous anaerobic treatment of WBL in the RANLEF gave satisfacto-
ry results. However, anaerobic effluent post-treatment should be required for final dis-
charge to water bodies. Aerobic post-treatment in a Rotating Biological Contactor did not
significantly remove recalcitrant organic matter or toxicity (D. magna) (Poggi-Varaldo et
al., 1995, 1996); coagulation-flocculation with alum reduced 70% of the organic matter as
COD but the toxicity increased. Batch post-treatment using Lentinus edodes, a ligninolytic
fungus, showed promise for removing COD, colour and ligninoids from the anaerobic
effluent (Estrada-Vzquez et al., 1998).

Up-flow aerobic packed bed reactor performance

The aerobic treatment results are displayed in Figures 4 and 5. The COD removal efficien-
cies (Figure 4A) ranged between 30% to 60% for the two HRT tested (5 and 2.5 days). On
the other hand, colour removal efficiencies (Figure 4B) ranged between 40% to 80% for
HRT = 5 days, whereas for HRT = 2.5 days efficiencies ranged between 60% to 100%.
Lignins removal efficiencies (Figure 4B) ranged between 20% to 70% for HRT = 5 days,
and for HRT = 2.5 days efficiencies ranged between 40% to 70%. Colour and ligninoids
removal efficiencies were higher than the COD removal efficiencies. For the three parame-
ters, there is a first subperiod (the initial 12 days at 5-day HRT) where their removal effi-
ciencies were very poor. This pattern could be due to the release of organic matter from the
wood cubes since the Trametes versicolor on the cubes, previously incubated in mycologi-
cal broth, had to acclimate to the anaerobic effluent.
Figure 5 displays the enzyme activities measured in crude extracts of the reactor. The
highest activity of Laccase was observed in the period corresponding to HRT = 5 days (10
to 70 U Lac/mL), whereas for the other HRT (2.5 days) the enzyme activity was much
lower and ranged between 5 to 20 U Lac/mL. 151
 (A) (B)
90 100 120
HRT = 5 days HRT = 2.5 days
HRT = 5 days HRT = 2.5 days

80 100

REM Lignins (%)

REM Colour (%)
80

REM COD (%)

60
60
60
40
30 40
20 20
A. Ortega-Clemente et al.

0 0 0
0 20 40 60 80 100 0 20 40 60 80 100
TIME (days)
TIME (days)

Lignins Colour

Figure 4 Removal efficiencies of COD (A), colour and lignins (B)

(A) (B)
80 0.5 0.5
HRT = 5 days HRT = 2.5 days HRT = 5 days HRT = 2.5 days

Enzyme Act (U Mn-P/mL)

Enzyme Act (U Li-P/mL)
Enzyme Act (U Lac/mL)

60 0.4 0.4

0.3 0.3
40

0.2 0.2
20
0.1 0.1
0
0 20 40 60 80 100 0.0 0.0
TIME (days) 0 20 40 60 80 100
TIME (days)
Li-P Mn-P

Figure 5 Enzyme activities vs time: Laccase (A), Manganese peroxidase and Lignin peroxidase (B)

Lignin peroxidase (Li-P) and Manganese-peroxidase (Mn-P) activities were signifi-

cantly lower than that of the Laccase.
Table 2 shows the average removal efficiencies () and specific unit net efficiencies
(sun) determined for both HRTs of reactor operation. Only a few reports obtained good
removals of pollutants from PPI wastewaters using ligninolytic fungi with no carbon
source supplementation; yet, the incubation lasted only 7 days or less (Esposito et al., 1991;
Estrada-Vzquez et al., 1998; Estrada-Vzquez, 1997). Our fungal reactor was capable of
sustaining removal activity for 95 days without the supplementation of a soluble carbon
source such as carbohydrates. Presumably, the T. versicolor might have used the cellulose
of the wood support as a carbon source in our experiment. Colour and ligninoid removals in
our reactor compare favourably with reported efficiencies of 67.7% and 59.1% in a contin-
uous lab scale fluidised bed containing Lentinus edodes immobilized in calcium alginate
beads treating the same anaerobic effluent supplemented with 3,000 mg/L of maltose
(Ramrez-Canseco et al., 2000). They also reported results for a control fungal fluidised
bed reactor with no maltose supplementation, which showed the lowest efficiencies and

Table 2 Average removal efficiencies () and specific unit net removal efficiencies (SUN) of the aerobic
reactor packed with Trametes versicolor immobilised on wood cubes

Hydraulic retention time (days)

5 2.5
Parameter COD Colour Ligninoids COD Colour Ligninoids

(%) 30.22 4.96 68.59 3.91 52.25 3.29 32.08 4.26 69.22 3.88 54.52 2.93
SUNa 4.4104 4.8104 3.7104 3.7104 4.4104 4.7104
6.9105 6.1105 4.8105 3.4105 5.6105 3.9105
Notes: a: units are mg O2/(mg O2*mg biomass), %colour/(%colour*mg biomass) and mg lig/(mg lig*mg
152 biomass) for COD, colour and ligninoids removals respectively
 3

ABS (@ 280 nm)

2

A. Ortega-Clemente et al.
0
0 10 20 30 40
ELUTION VOLUME (mL)
DL EAn Le/Alg-1thC Le/Alg-2ndC
Figure 6 Gel permeation chromatographic analysis of weak black liquor (WBL), anaerobic effluent (EAn)
and post-treated effluent (EAe). The EAe effluent corresponded to semi-continuous post-treatment of the
EAn with immobilised ligninolytic fungi (1C: first cycle; 2C: second cycle; each cycle of 7 days)

removal activity was sustained for only 14 days. In another work, Van Driessel and
Christov (2001) observed decolourization efficiencies of 5373% at 23 h HRT during the
treatment of bleach plant effluents in a rotating biological contactor reactor with immo-
bilised T. versicolor. Addition of glucose to the media stimulated color removal; Mn-P and
laccase were detected in the treated effluent.

Series anaerobic/aerobic performance

Combining the efficiencies of the anaerobic- and the aerobic-stage (Eq. (4)), we obtain the
overall removal efficiencies of 78.24; 78.67 and 73.26% for organic matter as COD, Colour
and Ligninoids respectively. Regarding the COD parameter, the anaerobic stage removes
the largest portion (ca. 68% at 1-day HRT), whereas the fungal post-treatment stage
contributes with only 10% more (absolute basis; 32% on stage basis). The contribution of
the fungal post-treatment to removal of colour and ligninoids is more important.

Gel permeation chromatographic determinations

The gel permeation chromatographic runs showed the presence of two peaks of maximum
UV absorbance (Figure 6), one in a region of high molecular weight [MW] (approximately
20 kDa; 10 mL of elution volume) and the second at low MW (200 Da; 30 mL of elution
volume). Similar elution curves were obtained for colour and COD (data not shown here).
The anaerobic treatment seemed to decrease the UV absorbance of the two peaks, consis-
tent with the findings in Table 1. In all cases (UV, VIS and COD curves) reduction of peaks
was observed when the HRT increased. Our results were in agreement with those reported
by Sierra-Alvarez et al. (1990) for soda pulp liquors of pine, spruce and wheat straw treated
in UASB reactors where they determined two absorbance peaks. The fungal post-treatment
of the anaerobic effluent in our work effected an important removal of the peak of high MW
which agrees with average results shown in Table 2. Interestingly, a light increase of the
peaks of low MW was noticed. This low MW peak increase may be the result of lignins
degradation, which produces lignin derivatives that are small fragments of lignin.

Conclusions
The following conclusions can be drawn from this work.
The continuous anaerobic treatment (RANLEF) of wastewater contaminated with weak
black liquor (WBL) was feasible at low to medium loading rates (1 to 10 g
COD/[LFB*day]) with a total COD removal of 8048% and biodegradable COD 153
 reductions of 9687% (at the highest loading rate). The RANLEF was able to operate in
a stable methanogenic regime despite indications that toxicity of WBL to the
methanogenic consortium imposed sub-optimal conditions. Colour and ligninoid
removals also decreased with decreasing HRT. Although the methanogenic fluidised
bed reactor provided an effective treatment for the degradable organic matter, important
concentrations of recalcitrant organic matter and colour still remained in the anaerobic
effluent.
A. Ortega-Clemente et al.

An aerobic reactor, packed with Trametes versicolor immobilised on wood cubes was
fed with the anaerobic effluent from the RANLEF. The reactor averaged organic matter
removals of 30 and 32% COD basis, during an experimental run of 60 days at 5-day HRT
and 35 days at 2.5-d HRT, respectively. Colour and ligninoids contents were removed in
higher percentages (69% and 54% respectively). There was no significant difference in
reactor performance at 5- and 2.5-day HRT. A positive correlation between pollutant
removal efficiencies and Laccase activity in crude extracts of the reactor liquor was
found. No supplemental soluble carbohydrate was required to sustain the fungus activi-
ty and the consistent reactor performance for 95 days.
Overall, the two-stage treatment achieved approximately a 78% removal of the original
organic matter of the WBL (COD basis). The process seems to be a step towards the
development of a full biological alternative to complete treatment of recalcitrant efflu-
ents from the pulp and paper industry.

Acknowledgements
Support from the Mexican Council of Science and Technology (CONACYT) as graduate
scholarships to CE-V and AO-C is gratefully acknowledged. This work was partially fund-
ed by the ICAITI (Guatemala)-AID; CINVESTAV and COSNET-TESE-SEP, Mxico. Dr.
Ian Reid from PAPRICAN kindly supplied the strain of Trametes versicolor. NR-S appre-
ciates support from COFAA-IPN. The authors thank Design-Ease Inc. for granting a free
license of the software Design-Expert 6.0.

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