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Process Biochemistry 41 (2006) 1521–1528 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-Ramırez, C.J. Galındez-Mayer *
´ ´ ´ Departamento de IngenierıaBioquımica, 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 microorganismswere 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 ofherbicide concentration on consortium’s 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 culture’s 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 inagriculture 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 systemseven at low concentrations [2–4]. 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. Galındez-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-adsorptioncoefficient 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[12–16]. 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 [17–28] exists, however, to understand the microbial response to...
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