Process Biochemistry 41 (2006) 15211528 www.elsevier.

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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 identied by sequencing fragments of their bacterial 16S rDNA. Those were Comamonas sp., Pseudomonas putida, Acinetobacter sp, Acinetobacter lwofi 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 inuence of 2,4-D concentration on instantaneous cell growth yield was quantied. 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 efciency 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, benecial insects, soil annelids and non-target plants, it also negatively impacts aquatic life, affecting algae, small invertebrates, amphibians, and shes, 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 coefcient 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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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, efuents 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 efuents 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 efuent quality is frequently reected in sludge compactness, inhibition of the nitrifying microorganisms in sludge or inhibition of microorganisms along the trophic chain [33]. A fast and efcient 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 efuent 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 efciently 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 L1); KH2PO4 (1.36), Na2HPO4 (1.40), (NH4)2SO4 (0.30); MgSO47H2O (0.05). The micronutrients added were (mg L1); 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 L1); 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 ltering in Whatman GF/F, 0.7 mm lters. Alternatively, the cell growth was quantied 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 L1 OD1 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 L1.

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 ask containing basal medium with 2,4-D as the sole carbon and energy source (200 mg L1). The ask was incubated for 96 h in agitation at 28 8C. Supernatant aliquots were used to successively inoculate (1% in volume) asks containing 2,4-D (200 mg L1), 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 L1) as the sole carbon and energy source. A frozen vial of the enriched mixed bacterial culture was used to inoculate a pre-culture ask (200 mL BM containing 200 mg L1 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 L1) and herbicide concentration c (g 2,4-D L1), 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 tting 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-tting and subsequent curve-analysis, the experimental batch data of x and c were tted to several empirical models [3841], most based on variations of the classical Verlhust logistic equation. Supported on the best tting results, two modied 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 and ct c0 a g1 ebkt v (2) a ; 1 ebct n (1)

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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 ow rate F o (22 0.5 mL h1) maintaining a dilution rate Do of 0.018 h1. 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 L1 of 2,4-D, maintaining the same ow 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 L1 of 2,4-D. After 24 h of batch culture, the reactor was fed with the same basal medium at a ow rate F (10 0.5 mL h1). When the reactors efuent 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 L1 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 h1. This time was considered the beginning of the toxic shock in the bioaugmented system. Efuent 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 reux 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 specic 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 eDi t . This equation describes the accumulation of 2,4-D in a continuous system operating at a dilution rate Di.

Using Mathematica 5.0 (Wolfram Research Inc.), both logistic equations were derived to evaluate the instantaneous changes in growth rx dx and biodegradt dation rc dc rates, which were used to obtain the transient variation of the dt rx instantaneous cell growth yield Yxc rc along the batch cultures. From the initial and nal values of biomass and substrate concentrations, the global cell growth yield at the end of each culture was calculated as Y xc xf0x0 : c 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 specic 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 min1. 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 L1 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 ow rate of 10 mL h1. The ow rate was periodically measured and adjusted when necessary, in order to maintain the dilution rate D at 0.014 0.001 h1. The herbicide concentration in the feeding line started at 75 mg 2,4-D L1. 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 L1 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 efciency h cicc, and i cell growth yield Yxc ci x were calculated in the continuous reactor at the c herbicide concentrations used.

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, ve strains able to use the herbicide as the only carbon and energy source were isolated and identied (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,

2.6. Identication 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 L1 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 fragmentamplication-procedure was used. Primers 8FPL and 1492RPL [42] were used to amplify the 16S rDNA present in the DNA extracted and puried from each one of the bacterial strains isolated. The purity of each strain was veried by gel electrophoresis of their 16S rDNA fragments amplied by PCR. Amplicons, of

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Table 1 Identication, 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 C1 C2 C3 C6 C7 Genbank access number U78183 AF532869 AY176770 AS244765 AF509331 Sequence homology (%) 96 89 98 98 98 Closest relative strain Klebsiella oxytoca Comamonas sp. Acinetobacter lwoi Acinetobacter sp. Pseudomonas putida

we were unable to nd 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 identied as the closest relative strain Comamonas sp., but the low sequence homology of its 16S rDNA (89%) suggests that C2 could be an unidentied 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 L1), the inhibitory effect exerted by 2,4-D on the

microbial growth could be observed by measuring the specic 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 inuence 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 L1 or higher, the instantaneous cell growth yield was lower than the global cell growth yield (Table 2), reecting 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 overow) [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.

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Table 2 Effect of 2,4-D concentration on specic 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 L1) 50 100 150 200 250 300
a

Specic growth rate (initial) mi (h1) (r2 = 0.979) (r2 = 0.995) (r2 = 0.865) (r2 = 0.984) (r2 = 0.851) (r2 = 0.998)

Specic growth rate (maximum) mmax (h1) 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 g1-D ) 2;4 0.664 0.06 0.687 0.04 0.612 0.05 0.663 0.06 0.646 0.06 0.465 0.05

0.124 0.141 0.080 0.081 0.072 0.053

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

The specic 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 specic growth rate and cell yield on this substrate, were 0.09 h1, and 0.14 gcel g1-D , respectively. 2;4

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 efciency 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 h1 2,4-D concentration in the incoming stream ci (mg L1) 75 100 150 200 300 2,4-D concentration in the reactor efuent c (mg L1) 1.32 0.045 2.20 0.070 2.84 0.075 2.97 0.16 4.50 0.12 Cell concentration in the reactor efuent x (mg L1) 39.2 0.045 52.1 0.070 83.7 0.075 81.9 0.160 81.1 0.120 2,4-D removal efciency h (%) 98.25 0.06 97.8 0.07 98.11 0.05 98.52 0.08 98.50 0.04 Global cell yield Yxs (gcel g1-D ) 2;4 0.52 0.003 0.53 0.004 0.56 0.003 0.41 0.003 0.27 0.001

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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 L1) 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 efciency 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. 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 efciency diminished from 91.7% to less than 85% in the shocked system. The drop of efuent quality also was evidenced by the increase in efuent 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 efuent quality for long time. With a delay of about 120 h,

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 biomagnied wastewater treatment system.

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.

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relative to the native system, the volume of settled solids diminished, COD and pH values increased, and the 2,4-D nally 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. Efuent 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

efciency 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 lwoi, 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. References
[1] Cox C. 2,4-D: ecological effects. Herbicide factsheet. J Pestic Reform 1999;19(3):149. [2] Nitscke L, Schussler W. Surface pollution by herbicides from efuents of waste water treatment plants. Chemosphere 1998;36(1):3541. [3] Gerecke AC, Scharer M, Singer HP, Muller S. Sources of pesticides in surface waters in Switzerland: pesticide load trough waste water treatment plantscurrent situation and reduction potential. Chemosphere 2002;48(3):30715. [4] Schueler T. Urban pesticides: from the lawn to the stream. Watershed Protect Tech 1995;2(1):24753. [5] Howard PH. Handbook of environmental fate and exposure data for organic chemicals. Pesticides, vol. III Chelsea, MI: Lewis Publishers; 1991. [6] Cheah U-B, Kirkwood RC, Lum K-Y. Adsorption, desorption and mobility of four commonly used pesticides in Malaysian agricultural soils. Pestic Sci 1997;50:5363. [7] Bhat MA, Tsuda M, Horiike K, Nozaki M, Vaidyanathan CS, Nakazawa T. Identication and characterization of a new plasmid carrying genes for degradation of 2,4-dichlorophenoxyacetate from Pseudomonas cepacia CSV90. Appl Environ Microbiol 1994;60:30712. [8] Chaudhry GR, Huang GH. Isolation and characterization of a new plasmid from a Flavobacterium sp. which carries the genes for degradation of 2,4dichlorophenoxyacetate. J Bacteriol 1988;170:3897902. [9] Don RH, Pemberton JM. Properties of six pesticide degradation plasmids isolated from Alcaligenes paradoxus and Alcaligenes eutrophus. J Bacteriol 1981;145:6816. [10] Ka JO, Holben WE, Tiedje JM. Genetic and phenotypic diversity of 2,4dichlorophenoxyacetic acid (2,4-D)-degrading bacteria isolated from 2,4D-treated eld soils. Appl Environ Microbiol 1994;60:110615. [11] Suwa Y, Wright AD, Fukimori F, Nummy KA, Hausinger RP, Holben WE, et al. Characterization of a chromosomally encoded 2,4-dichlorophenox-

Fig. 6. Disturbance of the continuously biomagnied 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.

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E. Marron-Montiel et al. / Process Biochemistry 41 (2006) 15211528 yacetic acid-a-ketoglutarate dioxygenase from Burkholderia sp. strain RASC. Appl Environ Microbiol 1996;62:24649. Don RH, Pemberton JM. Genetic and physical map of the 2,4-dichlorophenoxyacetic acid-degradative plasmid pJP4. J Bacteriol 1985;161: 4668. Don RH, Weightman AJ, Knackmuss HJ, Timmis KN. Transposon mutagenesis and cloning analysis of the pathways for degradation of 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate in Alcaligenes eutrophus JMP134(pJP4). J Bacteriol 1985;161:8590. Harker AR, Olsen RH, Seidler RJ. Phenoxyacetic acid degradation by the 2,4-dichlorophenoxyacetic acid (TFD) pathway of plasmid pJP4: mapping and characterization of the TFD regulatory gene, tfdR. J Bacteriol 1989;171:31420. Laemmli CM, Leveau JH, Zehnder AJB, Van der Meer JR. Characterization of a second tfd gene cluster for chlorophenol and chlorocatechol metabolism on plasmid pJP4 in Ralstonia eutropha JMP134(pJP4). J Bacteriol 2000;182:416572. Leveau JH, Konig F, Fuchslin H, Werlen C, Van der Meer JR. Dynamics of multigene expression during catabolic adaptation of Ralstonia eutropha JMP134 (pJP4) to the herbicide 2,4-dichlorophenoxyacetate. Mol Microbiol 1999;33:396406. Muller RH, Kleinsteuber S, Babel W. Physiological and genetic characteristics of two bacterial strains utilizing phenoxypropionate and phenoxyacetate herbicides. Microbiol Res 2001;156(2):12131. Torang L, Nyholm N, Albrechtsen HJ. Shifts in biodegradation kinetics of the herbicides MCPP and 2,4-D at low concentrations in aerobic aquifer materials. Environ Sci Technol 2003;37(14):3095103. Kopytko M, Chalela G, Zauscher F. Biodegradation of two commercial herbicides (Gramoxone and Matancha) by the bacteria Pseudomonas putida. Electron J Biotechnol 2002;5(2):18295. Pattanasupong A, Nagase H, Inoue M, Hirata K, Tani K, Nasu M, et al. Ability of a microbial consortium to remove pesticide, carbendazim and 2,4-dichlorophenoxyacetic acid. World J Microbiol Biotechnol 2004;20(5):51722. Mangat SS, Elefsiniotis P. Biodegradation of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in sequencing batch reactors. Water Res 1999;33(3):8617. Chin H, Elefsiniotis P, Singhal N. Biodegradation of 2,4-dicholophenoxyacetic acid using an acidogenic anaerobic sequencing batch reactor. J Environ Eng Sci 2005;4(1):5763. Daugherty DD, Karel SF. Degradation of 2,4-dichlorophenoxyacetic acid by Pseudomonas cepacia DBO1(pRO101) in a dual-substrate chemostat. Appl Environ Microbiol 1994;60(9):32617. Clarkson WW, Yangi C-P, Harker AR. 2,4-D degradation in monoculture biolm reactors. Water Res 1993;27(8):127584. Hinteregger C, Streichsbier F. Continuous biodegradation of phenoxyalkanoate herbicides by Sphingomonas herbicidovorans MH in a PU-supplied bubble reactor. Acta Biotechnol 1999;19(4):27992. Fradette S, Rho D, Samson R, Leduy A. Biodegradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by Pseudomonas cepacia: stoichiometric study. Can J Chem Eng 1994;72(3):497503. Santacruz G, Bandala ER, Torres LG. Chlorinated pesticides (2,4-D and DDT) biodegradation at high concentrations using immobilized Pseudomonas uorescens. J Environ Sci HealthPart B Pestic Food Contam Agric Wastes 2005;40(4):57183. Fuchslin HP, Ruegg I, Van der Meer JR, Egli T. Effect of integration of a GFP reporter gene on tness of Ralstonia eutropha during growth with 2,4-dichlorophenoxyacetic acid. Environ Microbiol 2003;5(10): 87887. Blanchoud H, Farrugia F, Mouchel JM. Pesticide uses and transfers in urbanised catchments. Chemosphere 2004;55(6):90513. Beccari M. Distribution and fate of xenobiotic compounds in wastewater treatment plants. In: Proceedings of the COST 624 WG4 Meeting on Biodegradation of toxic and biorefractory compounds and their impact on wastewater treatment plants; 2001. [31] Buenrostro-Zagal JF, Ramrez-Oliva A, Caffarel-Mendez S, Schettino Bermudez B, Poggi-Varaldo HM. Treatment of a 2,4-dichlorophenoxyacetic acid (2,4-D) contaminated wastewater in a membrane bioreactor. Water Sci Technol 2000;42(56):18592. [32] Meric S, Eremetkar G, Ciner F, Tunay O. An OUR based approach to determine the toxic effects of 2,4-dichlorophenoxyacetic acid in activated sludge. J Hazard Mater 2003;101(2):14755. [33] Boon N, Top EM, Verstraete W, Siciliano SD. Bioaugmentation as a tool to protect the structure and function of an activated-sludge microbial community against a 3-chloroaniline shock load. Appl Environ Microbiol 2003;69(3):151120. [34] Jianlong W, Xianchung Q, Libo U, Yi Q, Hegemann W. Bioaugmentation as a tool to enhance the removal of refractory compound in coke plant wastewater. Process Biochem 2002;38(5):77781. [35] Babcock RW, Ro KS, Hsieh C-C, Stenstrom MK. Development of an offline enricher-reactor process for activated sludge degradation of hazardous wastes. Water Environ Res 1992;64(6):78291. [36] Kelly MP, Hallberg KB, Tuovinen OH. Biological degradation of 2,4dichlorophenoxyacetic acid: chloride mass balance in stirred tank reactors. Appl Environ Microbiol 1989;55(10):27179. [37] Kwan CY, Chu W. Photodegradation of 2,4-dichlorophenoxyacetic acid in various iron-mediated oxidation systems. Water Res 2003;37:440512. [38] Zwietering MH, Jongenburger I, Rombouts FM, Vant Riet K. Modeling of the bacterial growth curve. Appl Environ Microbiol 1990;56(6):187581. [39] Birch CPD. A new generalized logistic sigmoid growth equation compared with the Richards growth equation. Ann Bot 1999;83:71323. [40] Annadurai G, Rajesh-Babu S, Srinivasamoorthy VR. Development of mathematical models (Logistic, Gompertz and Richards models) describing the growth pattern of Pseudomonas putida (NICM 2174). Bioprocess Eng 2000;23:60712. [41] Tsoularis A, Wallace J. Analysis of logistic growth models. Math Biosci 2002;179(1):2155. [42] Relman DA. Universal bacterial 16S rRNA amplication and sequencing. In: Persing DH, Smith TF, Tenover FC, White TJ, editors. Diagnostic molecular microbiology: principles and applications. Washington, DC: American Society for Microbiology; 1993. p. 48995. [43] Chemical oxygen demand. Reactor digestion method. In: Hach water analysis handbook. Colorado, USA: Hach Company; 1997. p. 4945. [44] Greer LA, Robinson JA, Shelton DR. Kinetic comparison of seven strains of 2,4-dichlorophenoxyacetic acid-degrading bacteria. Appl Environ Microbiol 1994;58(3):102730. [45] Daugherty DD, Karel SF. Degradation of 2,4-dichlorophenoxyacetic acid by Pseudomonas cepacia DB1(pRO101) in a dual-substrate chemostat. Appl Environ Microbiol 1994;60(9):32617. [46] Davison J. Genetic exchange between bacteria in the environment. Plasmid 1999;42:7391. [47] Ka JO, Tiedje JM. Integration and excision of a 2,4-dichlorophenoxyacetic acid-degradative plasmid in Alcaligenes paradoxus and evidence of its natural intergeneric transfer. J Bacteriol 1994;176:52849. [48] Muller RH, Simon D, Grosse HJ, Babel W. Substrate inhibition under stationary growth conditionsnutristat experiments with Ralstonia eutropha JMP 134 during growth on phenol and 2,4-dichlorophenoxyacetate. Appl Microbiol Biotechnol 1997;48(5):64855. [49] Loffhagen N, Hartig C, Babel W. Energization of Comamonas testosteroni ATCC 17454 for indicating toxic effects of chlorophenoxy herbicides. Arch Environ Contam Toxicol 2003;45(3):31723. [50] Liu Y, Tay JH. A kinetic model for energy spilling-associated product formation in substrate-sufcient continuous culture. J Appl Microbiol 2000;88(4):63863. [51] Muller RH, Babel W. Measurement of growth at very low rates (m ! 0), an approach to study the energy requirement for the survival of Alcaligenes eutrophus JMP 134. Appl Environ Biotechnol 1996;62(1):14751. [52] Shaler TA, Klecka GM. Effects of dissolved oxygen concentration on biodegradation of 2,4-dichlorophenoxyacetic acid. Appl Environ Microbiol 1986;51(5):9505.

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24] [25]

[26]

[27]

[28]

[29] [30]