Degradation and detoxification of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) by an indigenous Delftia sp. strain in batch and continuous systems

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Abstract

An indigenous bacterial strain capable of utilizing 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon and energy source was isolated from a polluted river in Buenos Aires, Argentina. The strain was identified as Delftia sp. by the 16S rRNA gene sequencing. Aerobic biodegradation assays in batch process were performed using a 2 L volume microfermentor at 28 °C with agitation (200 rpm). The continuous-flow system employed consists in a down-flow fixed-bed reactor, using polyurethane foam cubes as a support for immobilizing bacterial cells. Degradation of 2,4-D was evaluated by UV spectrophotometry, chemical oxygen demand (COD) and gas chromatography (GC). In the batch reactor microbial growth was measured by the plate count method. Biofilm development on polyurethane foam was observed by scanning electronic microscopy. Detoxification was assessed by using seeds toxicity tests. Under batch conditions Delftia sp. was able to degrade 100 and 200 mg L−1 of 2,4-D in 24 and 28 h respectively. Compound and COD removals were 99.9% and 91.3% respectively at an initial 2,4-D concentration of 100 mg L−1, and 99.0% and 89.4% when 2,4-D concentration was increased to 200 mg L−1. When assays were performed in the continuous-flow reactor under operating conditions 2,4-D and COD removals of 99.6% and 90.8% were achieved, respectively. Toxicity was neither detected at the end of the batch process nor in the continuous-flow reactor effluent.

Highlights

► This paper focuses on the biodegradation of the herbicide 2,4-D by Delftia sp. ► The strain is able to degrade and detoxify the compound. ► The continuous reactor constructed has high efficiency in compound removal. ► Its low cost allows the transfer of results to their use in bioremediation processes.

Introduction

2,4-Dichlorophenoxyacetic acid (2,4-D) is a phenoxy herbicide widely used for post-emergence control of annual and perennial broad-leaved weeds in cereal cropland, pastures, forests and in non-cropland, including areas adjacent to water. It is also used to control broad-leaved aquatic weeds (WHO, 2003). Phenoxyalkanoic herbicides like 2,4-D are highly water-soluble acids (pKa = 3.11) and have a low tendency to accumulate in organic matter (El-Bestawy and Hans-Jorgen, 2007), therefore they can enter as contaminants into streams, rivers or lakes directly from drainage of agricultural lands (Laganá et al., 2002). Moreover, contamination of the aqueous environment could be the result of pesticides discharges from manufacturing plants, storage sites and accidental spills (Celis et al., 2008). Once 2,4-D reaches surface water its half-life ranges from 1 to several weeks under aerobic conditions and can exceed 120 days under anaerobic conditions (WHO, 2003). Although the application of this herbicide increases agricultural productivity, it exhibits serious ecological effects. It causes toxicity in receiving waters and inhibition of biological treatment systems even at low concentrations (Marrón Montiel et al., 2006).

In Argentina, the National Act on Hazardous Wastes (Act 24051/92) recommends restricting 2,4-D concentrations in surface waters to 4 μg L−1 in order to protect the aquatic wildlife. However, it is among the most used herbicides in cultivable lands (Merini et al., 2007).

Biodegradation is the main process involved in the breakdown of 2,4-D in soil and water. Therefore, the use of native 2,4-D degrading microorganisms is a feasible alternative for the remediation of polluted sites and the treatment of wastewaters containing 2,4-D. Several bacterial strains able to use 2,4-D as the sole carbon and energy source in mixed or pure cultures have been described (Vallaeys et al., 1998, Marrón Montiel et al., 2006, Baelum et al., 2010). Among these bacteria, some Delftia strains exhibit high potential for the degradation of the herbicide (Maltseva et al., 1996, Müller and Babel, 2004). Although the biodegradation of 2,4-D is well documented, the bulk of the experimental research has been carried out in batch and sequential batch reactors (Celis et al., 2008, Quan et al., 2010, Quan et al., 2011), whereas the information about the feasibility of continuously running systems for the treatment of concentrated liquid wastes and the assessment of detoxification is still scarce (Mangat and Elefsiniotis, 1999, Chong and Chen, 2007).

The aims of this investigation were: (a) to isolate an indigenous bacterial strain able to use 2,4-D as the sole carbon and energy source and (b) to assess the ability of the strain to degrade and detoxify 2,4-D in synthetic wastewater in both batch and continuous-flow fixed-bed aerobic reactors.

Section snippets

Selection and identification of 2,4-D degrading bacteria

Enrichment was performed by successive subculturing of samples from a Buenos Aires polluted river, in a synthetic minimal medium (Korol et al., 1989) containing 2,4-D (50 mg L−1) as the sole carbon and energy source. The composition of the synthetic minimal medium was (L−1): 1.73 g K2HPO4; 0.68 g KH2PO4; 0.83 g (NH4)2SO4 and 0.1 g MgSO4.7H2O (final pH 7.4), 0.5 mL of a trace elements solution and 1 mL of a stock vitamin solution which were sterilized separately by filtration and added aseptically to

Selection and identification of 2,4-D degrading bacteria

An indigenous bacterial strain capable of degrading 2,4-D was isolated from a polluted river in Buenos Aires, Argentina. The strain was a Gram-negative, non fermenter, aerobic and motile road. It was identified as Delftia acidovorans with a 97% of probability using the API 20 NE system. The partial 16S rRNA gene sequence determined showed too that the isolated strain was closely related to the genus Delftia with 99% identity. Several organic compounds were tested as the sole carbon source for

Discussion

In this study we isolated an indigenous bacterial strain capable of utilizing 2,4-D as the sole carbon source. The strain was isolated from water samples of a polluted river located in Buenos Aires, Argentina. Several authors have reported the isolation of microorganisms able to degrade pesticides from polluted environments, like agricultural soils, wastewater or sediment samples, where microorganisms are previously exposed to these compounds (Smejkal et al., 2001, Widehem et al., 2002,

Acknowledgements

We thank the University of Buenos Aires for the grant given for this study, supported by UBACYT Program-Projects B125-B022.

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