Biodegradation pathways of chloroanilines by Acinetobacter baylyi strain GFJ2

Abstract

The Acinetobacter baylyi strain GFJ2 was isolated from soil that was potentially contaminated with herbicides. It exhibited complete biodegradations of 4-chlroaniline (4CA) and 3,4-dichloroaniline (34DCA), a wide range of monohalogenated anilines (chloro-, bromo-, and fluoro-anilines) and other dichloroanilines. An in-depth investigation of the biodegradation pathway revealed that a dechlorination reaction may be involved in 34DCA biodegradation, which forms 4CA as the first intermediate. By detecting the transient intermediates and characterizing the relevant enzymes, this investigation is also the first to report that A. baylyi strain GFJ2 has two distinct 4CA degradation pathways that yield 4-chlorocatechol (4CC) and aniline as the first intermediate in each route, which are further metabolized through an ortho-cleavage pathway. Analysis of biodegradation kinetics analysis illustrated that A. baylyi GFJ2 utilized aniline and 4CC at significantly slower rates than it used 4CA, suggesting that the transformations of aniline and 4CC were probably the limiting steps during 4CA biodegradation. Our results suggest the potential application of A. baylyi strain GFJ2 in bioremediation and waste treatment, and the kinetic data provide the insights into the degradation mechanism, dynamics and possible limitations of the biodegradation which include substrate and product inhibitions.

Introduction

Chloroanilines (CAs) are a group of chlorinated aromatic amines which are originated from the biotransformation of herbicides, for example phenylcarbamate, phenylurea, or acylanilides [1]. In addition, they are widely used as intermediate compounds in the production of dyes, polyurethanes, pesticides, and pharmaceutical products. As a consequence of intensive applications in agriculture and industries, chloroanilines, especially 4-chloroaniline (4CA) and 3,4-dichloroaniline (34DCA), have been ubiquitous and accumulated in the environment including agricultural soil/water, industrial wastewater and sludge. Due to their toxicity and recalcitrant properties, they have been considered as the important environmental pollutants and are subject to legislative control by the environmental protection agency of United States and Europe [2]. To dissimilate the environmental contaminated aniline and chloroanilines, bioremediation has been noted as a primary treatment technique in which a detoxification process is depending on the microbial biodegradability. Since the resistance of microbial biodegradation and toxicity of chloroanilines mainly depend on the number and position of chlorine atoms on the aromatic ring, there are many more reports of aniline-degrading bacteria than those of monochloroaniline (MCA)- or dichloroaniline (DCA)-metabolizing bacteria, some of which strictly required aniline as an inducer for cometabolic biodegradation [3]. In addition, while there are a number of publications describing microbial degradation of MCAs and DCAs under aerobic conditions, there are only a few reports on bacteria able to effectively degrade both chemical types. Pseudomonas diminuta was first bacterium described for its capability of degrading 3CA, 4CA and 34DCA (up to 0.3 mM), but not aniline [4], while Alcaligenes faecalis was reported for its ability to convert 34DCA to 4,5-dichloropyrocatechol before further metabolized [5]. Since the presence of MCAs and DCAs contaminated in the environment generally appears as co-contaminants, therefore to improve the biological remediation, microorganism with biodegradability of both MCAs and DCAs is importantly required. Moreover, in-depth information of its biodegradation pathway as well as biodegradation kinetics is necessary in order to have thorough understanding of biotransformation process which will further lead to effective control of bioremediation system.

In the present study, we successfully isolated and characterized 4CA-degrading bacterium, Acinetobacter baylyi strain GFJ2, which has a broad range biodegradability towards various halogenated anilines. Investigation of biodegradation kinetics and biodegradation inhibition profiles were also carried out for single substrates and mixed substrates. Interestingly, transient intermediate detected during biodegradation of 34DCA suggested a novel reaction involving dechlorination of 34DCA, while those detected during 4CA degradation leaded us to propose that two different biodegradation pathways of 4CA are existed in A. baylyi strain GFJ2. These results not only illustrated in-depth information of biodegradation mechanism of 34DCA and 4CA in A. baylyi strain GFJ2, but its effective biodegradability also demonstrates its potential application for bioaugmentation in the bioremediation treatment of contaminated site.