Elsevier

Journal of Hazardous Materials

Volume 361, 5 January 2019, Pages 10-18
Journal of Hazardous Materials

Biodegradation of 2,6-dibromo-4-nitrophenol by Cupriavidus sp. strain CNP-8: Kinetics, pathway, genetic and biochemical characterization

https://doi.org/10.1016/j.jhazmat.2018.08.063Get rights and content

Highlights

  • The microbial degradation process of 2,6-DBNP was revealed for the first time.

  • The degradation kinetics of 2,6-DBNP by strain CNP-8 was investigated.

  • The genes responsible for 2,6-DBNP catabolism was identified and characterized.

  • This study enhance our understanding of the environmental fate of 2,6-DBNP.

Abstract

Compound 2,6-dibromo-4-nitrophenol (2,6-DBNP) with high cytotoxicity and genotoxicity has been recently identified as an emerging brominated disinfection by-product during chloramination and chlorination of water, and its environmental fate is of great concern. To date, the biodegradation process of 2,6-DBNP is unknown. Herein, Cupriavidus sp. strain CNP-8 was reported to be able to utilize 2,6-DBNP as a sole source of carbon, nitrogen and energy. It degraded 2,6-DBNP in concentrations up to 0.7 mM, and the degradation of 2,6-DBNP conformed to Haldane inhibition model with μmax of 0.096 h−1, Ks of 0.05 mM and Ki of 0.31 mM. Comparative transcriptome and real-time quantitative PCR analyses suggested that the hnp gene cluster was likely responsible for 2,6-DBNP catabolism. Three Hnp proteins were purified and functionally verified. HnpA, a FADH2-dependent monooxygenase, was found to catalyze the sequential denitration and debromination of 2,6-DBNP to 6-bromohydroxyquinol (6-BHQ) in the presence of the flavin reductase HnpB. Gene knockout and complementation revealed that hnpA is essential for strain CNP-8 to utiluze 2,6-DBNP. HnpC, a 6-BHQ 1,2-dioxygenase was proposed to catalyze the ring-cleavage of 6-BHQ during 2,6-DBNP catabolism. These results fill a gap in the understanding of the microbial degradation process and mechanism of 2,6-DBNP.

Introduction

Halogenated nitrophenols are widely used in the syntheses of agricultural and industrial chemicals and are common aromatic halogenated disinfection by-products (DBPs) during chloramination and chlorination of water [1,2]. Due to their mutagenic, teratogenic and carcinogenic potential, halogenated nitrophenols in the environment have caused great problems for human health and ecosystems security. As an emerging aromatic halogenated DBP, 2,6-dibromo-4-nitrophenol (2,6-DBNP) has been identified in drinking water [3], swimming pool water [2] and saline wastewater [4]. Previous toxicological studies has demonstrated that 2,6-DBNP was dozens to hundreds of times more cytotoxic than several regulated DBPs by the U.S. Environmental Protection Agency (EPA) [5,6]. Recently, 2,6-DBNP has also been detected in various mollusks from Bohai Sea, China [7], indicating its potential risk to human by bioaccumulation. Therefore, considering the high toxicity of 2,6-DBNP and its wide distribution, removal of this halogenated nitrophenol from the environment is very meaningful.

Although several physico-chemical methods have been applied for halogenated nitrophenols degradation [1,8], they are not cost-effective and are unable to mineralize the pollutants completely. Microorganisms play key roles in the degradation of halogenated nitrophenols in the environment. Currently, bioremediation was considered to be the most cost-effective and environmental-friendly means for pollution abatement, and it has been sucessfully used in treatment of numerous aromatics-contaminated environments [[9], [10], [11], [12], [13], [14], [15]]. Therefore, in order to eliminate 2,6-DBNP from the environment by bioremediation, the essential prerequisite is to obtain a microorganism with the ability to degrade this toxicant.

Brominated nitrophenols (BNPs) are structurally analogues of chlorinated nitrophenols (CNPs) which are the most common halogenated nitrophenols. To data, many microorganisms have evolved their capacity to degrade various isomers of mono-chlorinated nitrophenols (MCNPs) including 2-chloro-4-nitrophenol (2C4NP) [[16], [17], [18], [19]], 4-chloro-2-nitrophenol [20], 4-chloro-3-nitrophenol [21] and 2-chloro-5-nitrophenol [22]. Noteworthily, the relative position of nitro and chloro significantly affected MCNPs degradation by microorganisms because of electron delocalization on the aromatic ring. Therefore, the enzyme(s) involved in 2C4NP degradation were not able to catalyze the transformation of other MCNP isomers [16]. Moreover, even against the same MCNP substrate, microorganisms have evolved different metabolic pathways [1]. The MCNPs-utilizers may also degrade the corresponding mono-brominated nitrophenols (MBNPs), but there is no experimental evidence available. Further, in contrast to MBNPs, dibrominated nitrophenols (DBNPs) are more toxic and more resistant to microbial degradation because of an additional bromine on the benzene ring. Thus, to date there is no data available on the microbial degradation of DBNPs.

Herein, Cupriavidus sp. strain CNP-8 was proved to be able to completely mineralize 2,6-DBNP. The degradation kinetics of 2,6-DBNP by this strain was investigated. Moreover, the genes responsible for 2,6-DBNP catabolism was identified by comparative transcriptome assay and biological experimental validation. This is the first report of microbiol degradation of 2,6-DBNP and enhance our understanding of the environmental fate of this pollutant.

Section snippets

Strains, plasmids, primers and culture conditions

The bacterial strains and plasmids are described in Table 1, and the primers are listed in Table S1. Cupriavidus sp. strain CNP-8 isolated previously [22] has been deposited in the China Center for Type Culture Collection (accession number: M 2017546). Escherichia coli strains were grown in lysogeny broth (LB) (tryptone: 10 g L−1, NaCl: 10 g L−1 and yeast extract: 5 g L−1) at 37 °C. Cupriavidus strains were grown in minimal medium [23] (MM, without CaCl2) at 30 °C supplemented with 2,6-DBNP.

Degradation of 2,6-DBNP by strain CNP-8

As an emerging aromatic halogenated DBP, 2,6-DBNP has been recently identified in various water environment, especially in China [[2], [3], [4],7]. However, very little is known about the biodegradation process of 2,6-DBNP to data. The time course assay of 2,6-DBNP degradation, together with the growth of CNP-8 and accumulation of nitrite and bromide ions is shown in Fig. 1A. After a delay period of approximately 10 h, strain CNP-8 rapidly degraded 0.3 mM 2,6-DBNP to undetectable levels within

Conclusions

Cupriavidus sp. strain CNP-8 was the first microorganism capable to utilize 2,6-DBNP. It could degrade 2,6-DBNP in concentrations as high as 0.7 mM, with μmax = 0.096 h−1, Ks = 0.05 mM and Ki = 0.31 mM. The hnp gene cluster responsible for 2,6-DBNP degradation was identified, and three enzymes were purified and functionally verified. HnpA could catalyze the sequential denitration and debromination of 2,6-DBNP to 6-BHQ in the presence of HnpB. HnpC likely catalyzed the ring-cleavage of 6-BHQ

Conflict of interests

The authors declare no competing financial interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 31600085), the State’s Key Project of Research and Development Plan (2016YFC1402300), the Foreword Key Priority Research Program of Chinese Academy of Sciences (QYZDB-SSW-DQC013), the State Key Laboratory of Microbial Metabolism, Shanghai JiaoTong University (MMLKF17-04), and the Yantai Science and Technology Project (2017ZH092).

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