Process Biochemistry 41 (2006) 15211528

www.elsevier.com/locate/procbio

2,4-D-degrading bacterial consortium

Isolation, kinetic characterization in batch and continuous
culture and application for bioaugmenting an activated
sludge microbial community
E. Marron-Montiel, N. Ruiz-Ordaz, C. Rubio-Granados,
C. Juarez-Ramrez, C.J. Galndez-Mayer *
Departamento de Ingeniera Bioqumica, Escuela Nacional de Ciencias Biologicas,
IPN, Carpio y Plan de Ayala, Colonia Santo Tomas, s/n CP 11340, D.F., Mexico
Received 28 October 2005; received in revised form 7 February 2006; accepted 21 February 2006

Abstract
Soil samples collected from the central region of Mexico were used as a source of microorganisms able to degrade 2,4-dichlorophenoxyacetic
acid. These microorganisms were enriched by successive transfer of microbial cells batch cultivated in basal medium to which 2,4-D was added as
the sole carbon and energy source. Five bacterial strains able to grow on 2,4-D were isolated and identified by sequencing fragments of their
bacterial 16S rDNA. Those were Comamonas sp., Pseudomonas putida, Acinetobacter sp, Acinetobacter lwoffii and Klebsiella oxytoca. The effect
of herbicide concentration on consortiums growth and 2,4-D degradation kinetics was studied in batch culture. By differential analysis of cell
growth and 2,4-D depletion curves, the influence of 2,4-D concentration on instantaneous cell growth yield was quantified. Low growth yields in
the cultures early phase could be attributed to metabolic uncoupling caused by the herbicide. In chemostat culture, 2,4-D removal efficiency was
higher than 97% and global cell growth yields were lesser than those obtained in batch. Finally, in order to prevent a toxic shock provoked by the
herbicide present in synthetic wastewater, the bacterial consortium was inoculated in a bench-scale wastewater treatment plant (WTP). However,
the system was only temporally protected from an upset caused by 2,4-D. Hence, it was designed a system for continuous bacterial inoculation,
allowing an undisturbed operation of the bench scale WTP.
# 2006 Elsevier Ltd. All rights reserved.
Keywords: 2,4-D biodegradation; Growth kinetics; Chemostat selection; Bioaugmentation; Activated sludge; Wastewater

1. Introduction
2,4-Dichlorophenoxyacetic acid (2,4-D) is one of the most
commonly used phenoxy acid herbicides in agriculture and
gardening, and it exhibits serious ecological effects. Regardless of its toxic effects on birds, beneficial insects, soil
annelids and non-target plants, it also negatively impacts
aquatic life, affecting algae, small invertebrates, amphibians,
and fishes, particularly in their juvenile stages [1]. It causes
toxicity in receiving waters and inhibition of biological
treatment systems even at low concentrations [24]. Contamination of groundwater with pesticides occurs frequently.

* Corresponding author. Tel.: +52 5729 6000x62352; fax: +52 5396 3503.
E-mail address: cmayer@encb.ipn.mx (C.J. Galndez-Mayer).
1359-5113/$ see front matter # 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2006.02.012

Relatively high water solubility and low soil-adsorption

coefficient of 2,4-D free acid, suggest that it has a high
potential to permeate soil. So, it probably moves to groundwater with percolating water [5,6].
Many 2,4-D-degrading microorganisms have been isolated
from agricultural, urban, and industrial soils and sediments [7
11], and the catabolic pathway of 2,4-D mineralization in
Ralstonia eutropha JMP134 (pJP4) is probably the best
investigated [1216]. In this bacterial strain, the catabolic
pathway involve initial ether bond cleavage to form 2,4dichlorophenol followed by hydroxylation to form 3,5dichlorocatechol, before intradiol ring cleavage [16].
Some raw information about kinetics of cell growth and
biodegradation of 2,4-D of microorganisms growing on this
herbicide [1728] exists, however, to understand the
microbial response to environmental variables such as toxic

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E. Marron-Montiel et al. / Process Biochemistry 41 (2006) 15211528

compound concentration, a thorough kinetic analysis

of raw data obtained from batch or continuous cultures is
required.
In addition to water contamination caused by the agricultural
use of pesticides, effluents from wastewater treatment plants
greatly contribute to the contamination of surface waters with
these compounds [2,3]. Due to rainfall, pesticides applied in
urban areas are transported to the sewer system and
subsequently to wastewater [4]. In some regions, urban uses
of pesticides exceed those in agriculture [29] and, frequently, a
conventional wastewater treatment process is not enough to
degrade it, so effluents from treatment plants have the potential
to contribute to the contamination of surface waters by nondegraded toxic compounds [2,3].
Toxic and bio-refractory compounds could affect wastewater treatment plants when streams of industrial and
municipal wastewater are mixed together [30] as occurs with
2,4-dichlorophenoxyacetic acid in the wastewater treatment
plant of Ecatepec, Mexico [31].
The continuous or intermittent presence of toxic compounds
in a wastewater treatment plant negatively affects the quality of
the treated wastewater [32]. The drop in effluent quality is
frequently reflected in sludge compactness, inhibition of the
nitrifying microorganisms in sludge or inhibition of microorganisms along the trophic chain [33].
A fast and efficient removal of toxic compounds present in
wastewater is necessary in order to prevent a system fail.
Bioaugmentation of a wastewater treatment system with
exogenous microorganisms able to degrade a toxic compound
from the initial stages could contribute to prevent the system
from undergoing a toxic shock [34]. However, not all cases of
bioaugmentation are effective. Several cases of wastewater
treatment processes, showing either successful or unsuccessful
persistence of inoculated microorganisms are summarized by
Babcock et al. [35].
We explored the feasibility of using mixed cultures for 2,4D degradation, with the ultimate aim of application for
effluent treatment. In this work, a mixed microbial population
able to use 2,4-dichlorophenoxyacetic acid as the sole carbon
and energy source was selected from soil samples obtained
from the central region of Mexico. One objective of the
present work was to study the kinetics of cell growth and 2,4D degradation by the microbial population in batch and
continuous cultures.
The ultimate aim of this work was the enrichment of the
activated sludge of a bench scale wastewater treatment plant
with the selected consortium, to avoid or reduce the toxic shock
caused to the native biota by the 2,4-D and remove it efficiently
from synthetic wastewater.
2. Material and methods
2.1. Chemicals
2,4-Dichlorophenoxyacetic acid was from SigmaAldrich Co. (St. Louis,
Missouri, USA) with purity higher than 98%. Gelatin peptone, and beef extract
were from Bioxon (Mexico). The other products were from Merck Co.
(Darmstadt, Germany).

2.2. Microorganisms and culture media

2.2.1. Basal medium (BM)
The inorganic salt medium used for the isolation and growth of the
microorganisms used throughout this study consisted of (g L
1); KH2PO4
(1.36), Na2HPO4 (1.40), (NH4)2SO4 (0.30); MgSO47H2O (0.05). The micronutrients added were (mg L
1); CaCl2H2O (5.8); FeSO47H2O (2.75);
ZnSO47H2O (1.15); MnSO47H2O (1.7); CoCl2 (0.325); CuSO45H2O
(0.235); Na2MoO42H2O (0.17). After autoclaving the basal medium at
121 8C for 25 min, 2,4-dichlorophenoxyacetic acid was added to reach the
required concentrations.
2.2.2. Synthetic wastewater (SWW)
The synthetic wastewater used to operate the wastewater treatment reactor
consisted of (g L
1); peptone (0.64); beef extract (0.44); urea (0.12); NaCl
(0.028); CaCl2 (0.0124); K2HPO4 (0.087), KH2PO4 (0.034), Na2HPO47H2O
(0.252), MgSO47H2O (0.008); NH4Cl (0.0068). The synthetic wastewater was
autoclaved at 121 8C for 25 min. The sludge used to operate this system came
from the Cerro de la Estrella wastewater treatment plant at Ixtapalapa, D.F.,
Mexico.

2.3. Analytical methods

The cell concentration in batch and chemostat cultures was determined by
weighting the cells retained alter sample filtering in Whatman GF/F, 0.7 mm
filters. Alternatively, the cell growth was quantified by measuring the sample
absorbance at 600 nm in a Beckman DU650 spectrophotometer. In this case, to
calculate the cell concentration, a conversion factor of 2.32 g cell L
1 OD
1
600
was used.
2,4-D was analyzed by liquid chromatography (HPLC) using a Beckman
HPLC System equipped with a Lichrosorb C18 reverse-phase column, together
with a diode-array detector (UV 235 nm). The mobile phase consisted of 0.12 M
phosphate buffer:acetonitrile (1:1), pH 3.0.
Alternatively, in batch experiments, the 2,4-D concentration was determined by measuring the sample absorbance at an absorption peak of 283 nm
[36,37] in a Beckman DU650 spectrophotometer. Both methods had a detection
limit of about 0.5 mg L
1.

2.4. Enrichment of microorganisms by successive transfers in batch

reactor
Microorganisms able to degrade pesticides were isolated from soil samples
obtained from several agricultural regions of Mexico (Cuamio, Mich., Ojitlan,
Ver., and Orizaba Ver.), where regularly 2,4-D is used. Sample soils were mixed
and suspended (2.5% dry weight) in an Erlenmeyer flask containing basal
medium with 2,4-D as the sole carbon and energy source (200 mg L
1). The
flask was incubated for 96 h in agitation at 28 8C. Supernatant aliquots were
used to successively inoculate (1% in volume) flasks containing 2,4-D
(200 mg L
1), which were incubated in the abovementioned conditions until
2,4-D degradation was evident by measuring OD283 of centrifuged aliquots.
After eighteen successive transfers, the time required to remove the herbicide
diminished. At the last transfer, the time for 2,4-D depletion was about 24 h.
Finally, the culture was harvested, distributed in 1.5 mL Eppendorf tubes, and
centrifuged. For further use, the cell pellets obtained were suspended in glycerol
and frozen at
20 8C.

2.5. Kinetic evaluation of the microbial mixed population

2.5.1. Batch culture
The kinetic evaluation of the mixed population was made in batch culture
using basal medium with 2,4-D (50300 mg L
1) as the sole carbon and energy
source. A frozen vial of the enriched mixed bacterial culture was used to
inoculate a pre-culture flask (200 mL BM containing 200 mg L
1 of 2,4-D).
The cells were grown for 24 h at 28 8C, harvested, distributed in 50 mL sterile
Falcon tubes, and centrifuged. The cell pellets obtained were suspended in a
small volume of BM and aliquots were used to inoculate each batch (200 mL).
The cell growth x (g cell L
1) and herbicide concentration c (g 2,4-D L
1), was

E. Marron-Montiel et al. / Process Biochemistry 41 (2006) 15211528

determined every two hours until 2,4-D was exhausted. Each batch culture was
sampled to estimate cell growth and residual 2,4-D by measuring, respectively,
the absorbance of the cell suspension at 600 nm (transformed to cell mass by a
conversion factor obtained beforehand) and the absorbance at 283 nm of the
supernatant after sample centrifugation.
Mathematical differentiation is a powerful tool for curve-analysis but
requires a mathematical model, which is usually obtained by fitting the
experimental data to a selected function. In this case, the model is viewed
as a purely empirical function, meaning that the equation parameters lack of
biological or physical implications. Under these circumstances, the model is
used only as a differentiable intermediate function. For curve-fitting and
subsequent curve-analysis, the experimental batch data of x and c were fitted
to several empirical models [3841], most based on variations of the classical
Verlhust logistic equation. Supported on the best fitting results, two modified
sigmoid models representing the transient behavior of cell growth x[t] and 2,4-D
concentration c[t] were chosen. Those models were:
xt x0

a
;
1 eb
ct n

(1)

a
g1 eb
kt v

(2)

and
ct c0

Using Mathematica 5.0 (Wolfram Research Inc.), both logistic equations were
derived to evaluate the instantaneous changes in growth rx dx
dt and biodegradation rc dc
dt rates, which were used to obtain the transient variation of the
rx
instantaneous cell growth yield Yxc
r
along the batch cultures. From the
c
initial and final values of biomass and substrate concentrations, the global cell
0
growth yield at the end of each culture was calculated as Y xc xcf0
x

cf :
Plotting against time the logarithm of the biomass data obtained in the early
growth phase and evaluating the slope of the linear segment, It was determined
the maximum specific growth rate mmax of the mixed culture corresponding to
each one of the initial 2,4-D concentrations used in batch cultures.
2.5.2. Continuous culture
The magnetically stirred chemostat (0.7 L) was operated at room temperature. An aerobic environment was maintained bubbling air at
0.35  0.03 L min
1. To assure that that the culture was not limited by O2,
the dissolved oxygen was periodically measured using a YSI-55 DO probe (YSI
Inc., USA). The reactor, containing BM with 75 mg L
1 of 2,4-D as the sole
carbon and energy source was inoculated with a cell suspension obtained from a
frozen vial of the bacterial consortium. After that, it was batch cultivated for
24 h. Subsequently, the reactor was fed with the same medium at a flow rate of
10 mL h
1. The flow rate was periodically measured and adjusted when
necessary, in order to maintain the dilution rate D at 0.014  0.001 h
1. The
herbicide concentration in the feeding line started at 75 mg 2,4-D L
1. The
chemostat was periodically sampled. When no appreciable variation in OD600
and OD283 values was observed, it was considered that the steady state was
reached. Then, the herbicide concentration was increased in the reservoir tank.
This procedure was repeated until a 2,4-D concentration up to 300 mg L
1 was
reached in the basal medium fed to the reactor. The samples obtained at each
steady state were used to estimate 2,4-D concentration by HPLC and cell growth
as previously described. With these data, the removal efficiency h cic
c
, and
i
cell growth yield Yxc ci x
c were calculated in the continuous reactor at the
herbicide concentrations used.

2.6. Identification of microorganisms

From frozen vials containing the enriched culture, the strains present in the
mixed microbial population were isolated. Appropriate dilutions of each sample
was poured in Agar-BM plates containing 200 mg L
1 of 2,4-D. Strains that
grew on 2,4-D as the sole carbon and energy source and showed differences in
colonial morphology, were isolated and conserved in BM-2,4-D-agar slants. To
identify the microorganisms isolated in this study, a 16S rDNA fragmentamplification-procedure was used. Primers 8FPL and 1492RPL [42] were used
to amplify the 16S rDNA present in the DNA extracted and purified from each
one of the bacterial strains isolated. The purity of each strain was verified by gel
electrophoresis of their 16S rDNA fragments amplified by PCR. Amplicons, of

1523

about 1500 bp, were sequenced in the Instituto de Biologa at the Universidad
Nacional Autonoma de Mexico. The data from amplicons sequencing were
compared with known sequences of 16S rDNA in the National Center for
Biotechnology Information GenBank by using the Basic Local Alignment
Search Tool (BLAST) algorithm. The reported species with the best similarity
match were regarded as the isolated species.

2.7. Toxic shock of the wastewater treatment system

The toxic shock of the wastewater treatment system was carried out as
shown in Fig. 4. The wastewater treatment reactor (WWTR) containing 1.2 L of
synthetic wastewater (SWW) was inoculated with 25 mL of activated sludge.
The reactor was operated in batch mode during 24 h, then, it was fed with
synthetic wastewater; stream (A) at a flow rate F o (22  0.5 mL h
1) maintaining a dilution rate Do of 0.018 h
1. After three changes of the reactor
volume and when any appreciable variation in the COD concentration was
observed, it was considered that the reactor steady state was reached (this
system was denominated native system). At this moment the SWW stream (A)
entering the reactor was changed for a stream B of SWW containing 60 mg L
1
of 2,4-D, maintaining the same flow and dilution rate aforesaid. This time was
considered the beginning of the toxic shock.
When the toxic shock was carried out in the bioaugmented system, the
procedure was slightly different. The bacterial consortium conserved in a frozen
vial was suspended in 500 mL of BM and inoculated in the seed reactor (SR)
containing BM plus 200 mg L
1 of 2,4-D. After 24 h of batch culture, the
reactor was fed with the same basal medium at a flow rate F (10  0.5 mL h
1).
When the reactors effluent showed no appreciable variation in OD600 and
OD283 lectures, the culture was considered stable. Once stabilized both reactors
(WWTR and SR), the SWW stream was changed for the stream B which
contains 60 mg L
1 of 2,4-D and simultaneously the stream C from SR,
containing the mixed microbial culture x1 was connected to the WWT reactor.
The sum of both streams increased the dilution rate Di to 0.027 h
1. This time
was considered the beginning of the toxic shock in the bioaugmented system.
Effluent quality was evaluated by measuring the volume of solids settled
after 1 h in a 50 mL graduated conical tube, supernatant turbidity at 600 nm
(As600), supernatant pH, COD and herbicide concentrations, as described
above.
The COD of the synthetic wastewater entering and leaving the chemostat
was determined by a closed reflux method in a Hach reactor [43]. The 2,4-D
accumulated in the WWTR were evaluated by liquid chromatography of the
centrifuged samples, as described earlier. The actual 2,4-D accumulation in
the WWTR determined by HPLC was compared with a theoretical one,
which was calculated assuming that the cell mass x in the chemostat was
unable to degrade the herbicide fed to the reactor at a concentration ci and
dilution rate Di. In this case, the specific degradation rate of the compound
(qc) would be 0 and the balance equation for c in the reactor
dc
dt Dici
c
qc x; could be easily solved in transient state, resulting in
c ci 1
e
Di t . This equation describes the accumulation of 2,4-D in a
continuous system operating at a dilution rate Di.

3. Results and discussion

3.1. Enrichment of bacterial mixed culture able to grow on
2,4-D
Using the technique of enrichment by successive transferences in BM medium with 2,4-D as the only carbon and energy
source, a bacterial mixed culture able to degrade the herbicide
was obtained from soil samples obtained from the regions of
Cuamio, Mich., Ojitlan Ver, and Orizaba Ver., Mexico, with
past history of 2,4-D application. From this culture, five strains
able to use the herbicide as the only carbon and energy source
were isolated and identified (Table 1). Several species of
Pseudomonas and Comamonas (Delftia) have been described
as bacteria able to degrade 2,4-D [17,44,45]. On the contrary,

E. Marron-Montiel et al. / Process Biochemistry 41 (2006) 15211528

1524

Table 1
Identification, by BLAST homology of 16S rDNA genes, of soil-isolated
bacterial strains able to grow on 2,4-D as the only carbon and energy source
Isolated
strain

Genbank access
number

Sequence
homology (%)

Closest relative
strain

C1
C2
C3
C6
C7

U78183
AF532869
AY176770
AS244765
AF509331

96
89
98
98
98

Klebsiella oxytoca
Comamonas sp.
Acinetobacter lwofii
Acinetobacter sp.
Pseudomonas putida

we were unable to find references relating Acinetobacter or

Klebsiella with biodegradation of the herbicide. However,
bearing in mind that genes encoding 2,4-D degradation are
generally plasmid borne [46] and considering evidences
pointing to intergeneric gene transfer of a 2,4-D-degradative
plasmid [47], it could be possible that these strains harbored a
2,4-D-degradative plasmid, horizontally transferred.
Another isolated strain, C2, was identified as the closest
relative strain Comamonas sp., but the low sequence homology
of its 16S rDNA (89%) suggests that C2 could be an unidentified
strain not registered in GENBANK.
3.2. Batch cultivation of the bacterial mixed culture
in 2,4-D
The cell growth and 2,4-D degradation curves obtained in
batch at different concentrations of the herbicide are shown in
Fig. 1. Except for the lowest 2,4-D concentrations (50 and
100 mg L
1), the inhibitory effect exerted by 2,4-D on the

microbial growth could be observed by measuring the specific

growth rates in the early growth phase of the batch cultures
(Table 2). Particularly at the highest herbicide concentrations,
the depletion curves of the 2,4-D revealed that the degradation
rate of the compound was less affected than the cell growth rate
(Fig. 1). In order to quantify the influence that 2,4-D
concentration has on the instantaneous cell growth yield
Yxc, a differential analysis of cell growth and 2,4-D
biodegradation curves was done. From results shown in
Fig. 2, it could be observed that in the early phase of the culture,
at 2,4-D concentrations of 200 mg L
1 or higher, the
instantaneous cell growth yield was lower than the global
cell growth yield (Table 2), reflecting an over-consumption of
2,4-D, not proportional to the cell mass produced. The
notorious effect of 2,4-D concentration on cell yield decrease
also has been observed with R. eutropha JMP 134 (formerly
Alcaligenes euthrophus) in continuous culture [48] and could
be related to the metabolic uncoupling caused by chlorophenoxy herbicides [49]. Some authors have associated the
decrease in cell growth yield caused by energy spilling to byproduct formation (metabolic overflow) [50]. However, when
Alcaligenes eutrophus was grown on 2,4-D, it was observed
that the uncoupling herbicide caused a vast energy spilling
without by-product accumulation [51]. Therefore, the transient
behavior of the instantaneous cell growth yield of the mixed
culture could be explained either, by consumption of nonaromatic by-products accumulated in the early phase of batch
cultures or by the decrease in herbicide concentration at the end
of the culture, with the consequent reduction in the uncoupling
effect of 2,4-D.

Fig. 1. Effect of 2,4-D concentration in cell growth (*) and 2,4-D ( ) depletion kinetics in batch cultures of a mixed microbial population.

E. Marron-Montiel et al. / Process Biochemistry 41 (2006) 15211528

1525

Table 2
Effect of 2,4-D concentration on specific growth rates and global cell yields of a mixed population batch cultured in basal medium with 2,4-D as the sole carbon and
energy source
Initial 2,4-D concentration
co (mg L
1)

50
100
150
200
250
300

0.124
0.141
0.080
0.081
0.072
0.053

Specific growth rate

(initial) mi (h
1)

Specific growth rate

(maximum) mmax (h
1)

(r2 = 0.979)
(r2 = 0.995)
(r2 = 0.865)
(r2 = 0.984)
(r2 = 0.851)
(r2 = 0.998)

0.132
0.167
0.173
0.194
0.207
0.249

(r2 = 0.982)
(r2 = 0.996)
(r2 = 0.988)
(r2 = 0.988)
(r2 = 0.993)
(r2 = 0.990)

Global cell yield

Yxc (gcel g
1
2;4-D )
0.664  0.06
0.687  0.04
0.612  0.05
0.663  0.06
0.646  0.06
0.465  0.05

Fig. 2. Effect of 2,4-D concentration in instantaneous cell growth yield behavior in batch cultures of a mixed microbial population.

The specific growth rate and cell growth yield (Table 2)

obtained with the microbial mixed population in batch cultures
were superior to that reported by Shaler and Klecka [52] for a
bacterial population isolated from industrial sewage growing on
2,4-D. The values obtained by these authors for the maximum
specific growth rate and cell yield on this substrate, were
0.09 h
1, and 0.14 gcel g
1
2;4-D , respectively.

3.3. Continuous cultivation of the bacterial consortium

Table 3 summarizes kinetic parameters obtained in steady
state continuous culture and Fig. 3 shows the kinetic behavior
of the consortium growing on 2,4-D in a chemostat for almost 3
months. With all the 2,4-D concentrations used, the removal
efficiency h was superior to 97%, however the cell mass

Table 3
Kinetic behavior of a mixed population affected by the 2,4-D concentration in the input medium of a steady state chemostat operating at a dilution rate D = 0.014 h
1
2,4-D concentration in the
incoming stream ci (mg L
1)

2,4-D concentration in the

reactor effluent c (mg L
1)

Cell concentration in the

reactor effluent x (mg L
1)

2,4-D removal
efficiency h (%)

Global cell yield

Yxs (gcel g
1
2;4-D )

75
100
150
200
300

1.32  0.045
2.20  0.070
2.84  0.075
2.97  0.16
4.50  0.12

39.2  0.045
52.1  0.070
83.7  0.075
81.9  0.160
81.1  0.120

98.25  0.06
97.8  0.07
98.11  0.05
98.52  0.08
98.50  0.04

0.52  0.003
0.53  0.004
0.56  0.003
0.41  0.003
0.27  0.001

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E. Marron-Montiel et al. / Process Biochemistry 41 (2006) 15211528

Fig. 3. Kinetic behavior of the mixed microbial population in the continuous

selector. Incoming 2,4-D concentration; solid line; cell concentration (^);
output 2,4-D concentration (^).

increased proportionally only at low 2,4-D concentrations in the

feeding medium (75150 ppm). For larger values (200 and
300 ppm), although most of the 2,4-D fed to the reactor was
degraded, there was no further transformation of substrate into
biomass and aromatic by-products accumulation was not
detected by HPLC. At these concentrations, the cell yield Yxc
decreased, suggesting again phosphorylative uncoupling
caused by 2,4-D [49]. Although, this decrease also could be
attributed to differences in the consortium dynamics caused by
the distinct selective pressures applied (increased loading rates
of 2,4-D). Cell yield decrease could not be explained by oxygen
limitation (dissolved O2 in the chemostat 3 mg O2 L
1) or
limitation by other nutrients, since the culture medium was
formulated to assure that growth was carbon limited. Despite
the cell yield diminution observed at the aforesaid 2,4-D
concentrations, the removal efficiency was always greater than
97%. Although smaller than cell global cell growth yields
obtained in batch culture, the relatively high cell yields
obtained in chemostat suggests an elevated incorporation of
carbon substrate to cell carbon, without HPLC-detectable byproduct accumulation.

Fig. 4. Strategy for a toxic shock of a bench scale wastewater treatment system.
(A) Normal operation of the system, (B) 2,4-D shock of the native wastewater
treatment system and (C) 2,4-D shock of the continuously biomagnified
wastewater treatment system.

3.4. Toxic shock of the wastewater treatment system

When the autochthonous microorganisms present in the
activated sludge were subject to a 2,4-D toxic shock, a
disturbance in the behavior of the native system was observed
(Fig. 5). The COD removal efficiency diminished from 91.7%
to less than 85% in the shocked system. The drop of effluent
quality also was evidenced by the increase in effluent turbidity,
a drastic decrease in settling solids volume and, pH alteration.
In Fig. 5C, the herbicide accumulation in the reactor can be
compared with a theoretical accumulation curve, assuming no
degradation of 2,4-D. After 400 h of continuous operation, the
experimental value reached the theoretical maximum, meaning
that the native population was unable to degrade 2,4-D and
herbicide was not adsorbed onto biomass.
When a toxic shock with 2,4-D was imposed to the
wastewater treatment system bioaugmented with the mixed
microbial population, it was not possible to maintain the
effluent quality for long time. With a delay of about 120 h,

Fig. 5. Disturbance of the native wastewater treatment system by a 2,4-D toxic

shock. (A) Change in settled solids volume (&) and supernatant turbidity (&);
(B) pH (*) and COD (*) change in the output stream; (C) actual (^) and
theoretical (solid line) accumulation of 2,4-D in the output stream.

E. Marron-Montiel et al. / Process Biochemistry 41 (2006) 15211528

relative to the native system, the volume of settled solids

diminished, COD and pH values increased, and the 2,4-D
finally accumulated in the reactor (data not shown).
In order to maintain the mixed microbial population in the
wastewater treatment system, it was separately propagated in a
chemostat, and supplied continuously to the system, according
to the scheme shown in Fig. 4.
Fig. 6 shows the behavior of the wastewater treatment
system enriched with the mixed microbial population
propagated in the seed reactor. It was evident that the
continuously bioaugmented system demonstrated higher
capacity than the native system to cope with an herbicide
shock loading. Effluent quality was not negatively affected,
since the supernatant turbidity remained low, whereas the
settling solids remained adequately high. pH remained almost
constant, COD values decreased even more than the native
system, and there was no 2,4-D accumulation in the reactor
(Fig. 6), indicating that the herbicide was removed by the mixed
microbial population added to the system. The COD removal

1527

efficiency increased from 91.7 %, in the native system to 96%

when it was continuously bioaugmented. These results shown
that independently of the systems hostile environment towards
the introduced population, it was effective along the shock
loading. Therefore, a continuous bioaugmentation procedure
could be a useful alternative for wastewater treatment plants
that frequently receives toxic or recalcitrant compounds
altering the normal plant operation.
4. Conclusions
Strains of Pseudomonas putida, Comamonas sp, Klebsiella
oxytoca Acinetobacter sp. and Acinetobacter lwofii, able to use
2,4-D as the only carbon and energy source, were isolated from
agricultural lands of Mexico.
The mineralization of 2,4-dichlorophenoxyacetic acid by a
microbial consortium was demonstrated in batch and
continuous culture by measuring the cell growth yields
obtained when 2,4-D was used as the only carbon and energy
source. The diminution in the cell growth yield observed when
increasing the supply of 2,4-D to batch or continuous culture
could be attributed to an uncoupling effect exerted by this
compound.
Bioaugmenting the activated sludge with the selected 2,4-Ddegrading microorganisms did not permanently protect the
system from an herbicide shock loading. However, it was
evidenced that a continuously inoculated system was capable to
cope with a toxic shock produced by 2,4-D.
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Fig. 6. Disturbance of the continuously biomagnified wastewater treatment

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supernatant turbidity (&); (b) pH (*) and COD (*) change in the output
stream; (C) actual (^) and theoretical (solid line) accumulation of 2,4-D in the
output stream.

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